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•Ligands Synthesis and Confonnational Studies for the Investigation of Opiate and

Protease Receptor sites

by

Gérald Blaise Villeneuve

Department of Chemistry

McGill University

Montréal

September 1994

A Thesis submitted to the faculty of Graduate Studies and Research in partial

fulfillment of the requirement of the degree of Doctor in Philosophy (Ph.D)

@ Gérald Blaise Villeneuve 1994

1+1 National Ubraryof Canada

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L'AUTEUR CONSERVE LA PROPRIETEDU DROIT D'AUTEUR QUI PROTEGESA THESE. NI LA THESE NI DESEXTRAITS SUBSTANTIELS DE CELLE­CI NE DOIVENT ETRE IMPRIMES OUAUTREMENT REPRODUITS SANS SONAUTORISATION.

ISBN 0-612-00143-1

Canad~

Short title for the thesis of Gérald Blaise ViHeneuve

Investigation (lf Opiate and Protease Receptor sites

•A mon père et à ma mère

ii

Hi

Table of Contents.

Table of contents HiThesis format viiAcknowledgments viiiRésumé ix

Abstract xUst of abbreviations xiChapter 1 Introduction

1.1 Receptor sites investigation: the receptors structure 11.2 Receptor sites investigation: the ligands structure 21.3 Recent progress in the study of opiates receptor site 3

1.3.1 Receptor selectivity of endogenous opiates a..~d opioid peptidesfrom other sources 31.3.2 Concepts in the development of new potent and selective opiates 51.3.3 Opiate receptor genes cloning 10

1.4 References and notes IlChapter 2 A proposai for the molecular basis of II. and Il opiate receptor

differentiation based on modeling of two types of cyclic enkephalins and a narcoticalkaioid 15

Authors' contribution and notes 15Summary 16

Introduction 17Materiais and Methods 19

Molecular model and force field 19Ring searching and ring closure for the cyclopeptides 19Vector map for PEO 20Superposition of pharmacophore 22

Results and discussion 23Characteristics of the bimolecular superposition: 27Comparison with other receptor models 28

Conclusion 30References 31Additionai comments 33Supplementary references 36

Chapter 3 Structural study of the modelll opiate selective cyclic enkephalinNaCbz-c[(D)A28u-Gly-Phe-Leu] 37

3.1 Preparation of NaCbz-c[(D)A2Bu-Gly-Phe-Leu] 383.2. NMR solution study of NaCbz-c[(D)A2Bu-Gly-Phe-Leu] 3.1 40

3.2.1 Similitude in NMR data between 3.1 and 3.28 433.2.2 NOE data, relative mobility, and internuclei distances 443.2.3 Hydrogen-bond acceptors and consequences on the macrocycleconformation 493.2.4 Computer molecular modeling and molecular dynamic simulationof CY(;:Jpeptide conformation 523.2.5 Interresidue interactions 553.2.6 Conformation of amino acids 57

3.3 X-Ray crystallography 613.4 Consequence on opiate receptor binding conformation 623.5 Conclusion and summary 663.6 Experimental section 66

3.6.1 Synthesi. 663.6.2 NMR solution study 723.6.3 Molecular dynamic simulation 73

3.7 References and notes. 74Chapter 4 Preparing ~-phenylcysteine: F'irst approaches 78

4.1 Introduction 784.2 Using N·benzoyl and S-benzyl protecting groups 78

4.2.1 Synthetic method 784.2.2 Separation 0f diastereoisomers 794,2.3 Enzymatic resolution and comments on a-chymotrypsin pocket 80

4.2.4 Removing the benzyl protecting group on sulfur 824.3 Using N-benzoyl and S-a·naphthylmethyl protecting groups 91

4.3.1 Synthetic method 924.3.2 Separation of diastereoisomers 924.3.3 Removing the a-naphthylmethyl group on sulfur. 93

4.3.4 Enzymatic resolution and comments on carboxypeptidase Apockets 93

4.4 Using N·benzoyl and S·p-NO, benzyl protecting groups 9S

4.S Conclusion and summary 9S4.6 Experimental 96

iv

•v

4.6 References and notes 104Chapter 5 S-p-Methylbenzyl-p-phenylcysteine: A Potential Tooi for ProbingReceptor Topologies. 107

Introductory comments 107Abstract 108Introduction 108Results 108

Synthesis and resolution 108X-Ray structure and conformational analysis 110

Discussion 111Experimental 111References 114Further comments 116Additional references 118

Chapter 6 Insertion of methylene-m,y as a substitute to amide bond in t-Boc-Val­Leu-OH: X-Ray Crystal Structures, Solution Conformations and MolecularModeling Studies 119

Authors' contribution 119Summary 120Introduction 120Experimental 120Results 124

X-Ray molecular conformations 124X-Ray molecular packings 125Solution conformation 125Conformational mimicry 128

Discussion 128References 128

Chapter 7 Conclusion 130Contribution to original knowledge 132Supplementary material AlH 300 MHz spectra of 3.1 AllH 300 MHz 2D NOE and of 3.1 A2Temperature dependance study of lH NMR NH signal of 3.1 A3lH 500 MHz spectra of 3.1 A7lH 500 MHz selective irradiation spectra of 3.1 A10

e

IH 500 MHz simulated spectra of 3.1 A14

IH 500 MHz selected area of 2D NOE speetra of 3.1 A20

The rule of thumb of Truce~. A25Observed and calculated structure factors of threo-N-acetyl-S-p-methyl·benzyl-p­

phenylcysteine methyl ester A26Effect of chemical shift reagent on resol'led and unresolved trifiuoroacetamidesA32

Observed and calculated structure factor of t-Boc-VaI-Leu-OH A33

Observed and calculated structure factor of t-Boc-VaI-1jI[C~-O]-Leu-OH A38

10 and 2D NOE speetra of t-Boc-VaI-Leu-OH and t-Boc-VaI-1jI[CH2-O]-Leu

-OH A43

vi

Thesis fonnat

Manuscripts and Authorships

Candidates have the option, subject to the approval of their department, ofincluding, as part of their thesis, copies of the text of a paper(s) submitted forpublication, or the clearly duplicated text of a published paper(s), provided thatthese copies are bound as an integral part of the thesis.

·If this option is chosen, connecting texts, providing logical bridges between thedifferent papers, are mandatory.-The thesis must still confonn to all other requirements of the "Guidelines

Concerning Thesis Preparation" and should be in literary fonn that is more a merecollection of manuscripts published or to be published. The thesis must include,as separate chapters or sections: (1) a Table of Contents; (2) a general abstract inEngiish and French, (3) an introduction which clearly states the rationale andobjectives of the study, (4) a comprehensive general review of the backgroundliterature to the subject of the thesis, when this review is appropriate, and (5) a

overall conclusion and lor summary.-Additional material· '(procedural and design data, as weil as description ofequipment used) must be provided where appropriate and in sufficient detail (e.g

in appendices) to allow a clear and precise judgement to be made of theimportance and originality of the research reported in the thesis.

-In the case a manuscripts co-authored by the candidate and others, the candidate

is required to make an explicit statement in the thesis of whom contributed to such

work and to what extend; supervisors must attest to the accuracy of such daims at

the Ph.D Oral Defense. Since the task of the examiners is made more difficuit inthese cases, it is in the candidate's interest to make perfectiy clear the

responsibilities of the different authors of co-authored papers.

vii

Acknowledgments

1 would like to acknowledge first my thesis supervisors, Pro André Michel

and Pro Tak Hang Chan for judicious advices and encouragements during the

process of my work. Helpful and stimulating discussion with Dr. John DiMaio isalso acknowledged.

Assistances provided by Mr. Marc Drouin for X-Ray diffraction studies, Dr.

Françoise Sauriol for NMR and Dr. Orval Mammer, Dr. John Finkenbine, and

Mr. Gaston Boulll.y for mass spectrometry are particularly acknowledged.

1would like to thank ail my colleagues in room 25 for aIl sort of helps, and

especiaIly Dr. Denis Labrecque. 1 like also to thank the department officemembers and particularly Mrs. Renée Charron.

Finally, we are indebted to Natural Sciences and Engineering Research

Council of Canada for financial support.

viii

• Résumé

Un modèle topochimique a été formulé pour rendre compte de la

sélectivité divergente de peptides opioides rigidifiés reliés à la séquence del'enképhaline ainsi que d'alkaloides narcotique~ envers les sous classes derécepteurs opiacés Il et li. Des efforts ont par la suite été accomplis dans le but de

confirmer ou d'infirmer ce modèle. Le modèle repose sur une étude par

modélisation moléculaire sur ordinateur utilisant le pharmacophore opiacé

comme préalable minimum à l'alignement des molécules ainsi que l'information

concernant la structure des macrocycles peptidiques issue d'étude

spectroscopique en solution. Nous avons étudié les propriétés conformation­nelles par RMN du proton du peptide N"'Cbz-c[(D)AzBu-Gly-Phe-Leu] à l'aide de

mesure d'effet nucleaire Overhauser pour déterminer semi-quantitativement les

distance internucléaires. Un des éléments importants que faisait ressortir le

modèle topochimique était la possibilité qu'un éventuel substituent aryl sur lecarbone P de la cysteine sur le peptide HTyr-(D)Pen·Gly-Phe-Cys-NHz devrait

induire une augmentation de l'affinité de ce dernier envers la sous classe derécepteur opiacé li. En conséquence, nous avons préparé la p-phenylcysteine par

une voie de synthèse basée sur l'addition de mercaptan à une azlactone. Les

diastéréoisomères furent séparés, puis identifiés par diffraction des Rayons-X.

Les énantiomères ont par la suite été resolus grâce à l'enzyme carboxypeptidase A

Plusieurs difficultés furent rencontré durant cette préparation en ce qui a trait au

choix du groupement S protecteur de façon à ce que celui-ci puisse être retiré de

façon quantitative et compatible avec la synthèse peptidique. De plus, la taille de

ce groupment a due être minimisé de façon à permettre la résolution avec

l'enzyme, ce qui nous à permis entre autre de sonder la dimension de la cavité

hydrophobique de l'exopeptidase. Grâce aux informations structurales obtenuespar diffraction des rayons-X sur les divers p-phenylcysteines crystallisées, nous

avons déterminé à l'aide de la modélisation moléculaire sur ordinateur les

préférences conformationnelles de la chaine latérale des diastéréomères de cet

acide aminé souffré particulié puis réévalué la possibilité de l'introduire dans le

cyclopeptide. Finalement, dans un domaine d'activité un peu différent du sujet

principal de recherche, nO\ls avons évalué les cOnséquences conformationnelles

produites par l'insertion d'un substitut methylene-oxy en lieu de la liaison

peptidique sur un dipeptide modèle par diffraction des Rayons-x, RMN en

solution et modélisation moléculaire sur ordinateur.

ix

Abstract

A topochemical model ha~ been derived to account for the diverseselectivity of cyclic opioid peptides related to enkephalins and for narcoticalkaloids towards the Il. and cS opiate subclasses. Efforts toward demonstrating its

validity have later been accomplished. The model is based on a computermolecular modeling study using the opiate pharmacophore as a minimumprerequisite to align the molecules and benefit aIso of informations obtaineà fromspectroscopic study in solution concerning the macrocycles conformation. Theconformational properties of NaCbz-c[(D)A2Bu-Gly-Phe-Leu] were studied by

nuclear Overhauser effect which provided semi-quantitative internuclear

distances. One of the important elements that the model brings out is that theeventual presence of an aromatic ring on the beta carbon of Cys of the opioidpeptide HTyr-(D)Pen-Gly-Phe-Cys·NH2 should induce an increase in selectivitytoward the cS opiate subclass. Consequently, we engaged in preparing the special

amino acid, P-phenylcysteine, using a method based on the addition of mercaptan

to unsaturated azlactone. The diastereoisomers obtained were separated, andtheir relative configurations assigned by X-ray crystaIlography. The pure

enantiomers were obtained by resolution with the enzyme carboxypeptidase ASeveral difficulties were encountered during this preparation, the main concernbeing the S protecting group. This proteeting group should be chosen with the

property that it can be quantitatively removed when desired and compatible withthe conditions of peptides synthesis. Moreover, the size of the proteeting group

should be minimized in order to be able to reaIize the resolution with the enzyme.We explored, by the same token, the size of the hydrophobic poeket of the

exopeptidase. Using the structural information obtained from the X-Raydiffraction study of several of the P·phenycysteine, we have determined with the

help of computer molecular modeling, the side chain preference of thediastereomers of this particular amino-acid and reinvestigated on this basis its

introduction into the potential cyclopeptide. Finally, in a slightly different area, we

have investigated the consequence of inserting the methylene-oxy surrogate to theamide bond on a model dipeptide. X-Ray diffraction, 2D NOE IH NMR and

computer modeling studies were used in this respect.

x

Ust of abbreviations• Ar;O

t-BocBoc-ONBzl

Cbzc-ORNCSOc-PEN

DCCDCMDCUDNADIPEADMAPDMFDMSODPPAEtOAcEtOHFmOHHOAcHObt

HPLC

IRMDSMeMtlCNMeOHNEMNMRNOEPCRPen

PEO

Acetic anhydride

tert-butoxycarbonyl2(-t-butoxycarbonyl-oximino)-2-phenylacetonitrileBenzylBenzoxycarbonylTyr-c[D-Om-Gly-Phe-Leu]Common space occupationTyr-D.Pen-Gly-Phe·Cys-NH 2

DicyclohexylcarbodiimideDichloromethaneDicyclohexylureaDeoxyribonucleic acidDiisopropylethylamineDimethylaminopyridineDimethylformamideDimethylsulfoxideDiphenylphosphoryl azideEthyl acetateEthanolFluorenylmethylene alcoholAceticacidI-hydroxybenzotriazoleHigh performance liquid chromatography

InfraredMolecular dynamic simulation

MethylAcetonitrileMethanolN·ethylmorpholineNuclear magnetic resonanceNuclear Overhauser effeetPolymerase chain reaetionPenici1lamine (p,p-dimethylcysteine)

7a·[(lR)-1·methyl.l.hydroxy·3·phenylpropyl]-6,14·endoethenotetra·hydro oripavine

xi

Ph

ppb

PyrRT

SARSCESHE

TEAnIFTfAneUV

Phenyl

Part per billion

Pyridine

Room temperature

Structure activity relationship

Standard calomel eleetrode

Standard hydrogen eleetrode

Triethylamine

Tetrahydrofuran

Trifluoroacetic acid

Thin layer chromatography

Ultraviolet

xii

• Chapter 1

Introduction

Biological processes are govemed by interactions of macromolecules withsmall or relatively large ligands. The study of both entities remains at the basis ofthe understanding of physiologicai phenomena and eventually for thedevelopment of therapeuticai applications in case of disorder.

1.1 Receptor sites investigation: the receptors structure.

Fantastic progress have occurred at the molecular level in biologicalsciences in the last two decades. Amongst it, the isolation of site-specifieendonucieases 1 has opened the door to the techniques grouped under the name

of gene cioning which are now particulariy facilitated by the ingenious polymerasechain reaction (PCR).2 Rapid DNA sequencing3 of the so cioned genes has givenaccess to the putative arnino acid sequence of severai membrane bound proteins

(receptors) that could not be isolated otherwise. Moreover the cioned genes

could be expressed in cells that usuaily do not produce the protein receptors andprovide an ideal template for biochemicai and pharrnacological receptors studies.C

In theory, the primary sequence of arnino acids of a protein should be

sufficient information to derive the secondary and tertiary structure of the

protein. This is still a doubtful task. The knowledge of the accurate structure of amacromolecule is mainiy the contribution of X-Ray crystallography.6 However it is

not aiways easy to obtain crystais of protein that are suitable for X-Ray

crystailography and moreover this does not ensure structure elucidation. 8 Despite

that, in several cases, the homology of amino acid sequences between the newly

isolated protein and a structurally well characterized protein is sufficient that thetertiary structure of the new protein can be deduced. Computer molecular

modeling and molecular dynarnic are important tools in this domain.7 Finding outthe location of the active site is aiso not obvious when X-Ray diffraction study of

the macromolecuie with an inhibitor or an irreversible ligand is not available but

·information now provided by specifie arnino acid mutation is particularly useful.'The approa<:h of X-Ray crystailography and molecular-moc1eling has been

particularly fruitful in the design of new therapeutic drug targeting specifie

enzymes.9 Notwithstanding the tremendous progress of receptor structuralstudies, the study of active sites topologies remain an area weIl covered by thedesign of specific ligands (probe) where the information on the structure of thebinding sites are obtained from the mutual complementarity between the ligandsand the receptors.

1.2 Receptors sites investigations: the ligands structure.

Although the homology based modeling has the merlt of relying on theknown amine acid sequence of the receptor, numerous hypotheses must beassumed to elaborate the model when accurate receptor structure is not known.The exploration of sites topology remains, in this case, and even with theknowledge of the accurate structure of the receptor, an area weIl covered by therational design of small synthetic ligands and the study of their pharmacologicalproperties. Rational design of new ligands is now widely associated with molecularmodeling technique based on active analogs approach.l0 Recent progress in highfield 2D NMR has also permitted access to accurate conformational information insolution 11 and in synthetic membrane environment12 for natural and syntheticligands.

In this context, chapter 2 reports the results of our investigations of the ILand 8 opiate receptor subcla~ses based on the modeling of two previously

reported moderately selective cyclic enkephalins and a narcotic alkaloid. In

chapter 3, the results of chapter 2 are put one step forward in including results ofour own spectroscopic investigation concerning the conformation of one of the IL

specific cyclic enkephalin in solution. We were also particularly int~rested indesigning new specific ligand for the 8 opiate receptor subclass based on the

model given in Chapter 2. To do so we had to prepare a specific amino acid and

obtain it in an enantiomerlcally pure form. Specific enzymes were trled to realized

the resolution of the amino-acid and by the same token the active sites tolerancesof these enzymes were explored and are discussed in Chapters 4 and S. Finally, ina side project, we studied the conformational consequences of inserting a

methylene-oxy surrogate to the amide bond in a model hydrophobic dipeptide.This is described in Chapter 6.

2

1.3 Recent progress in the study of opiates receptor site.

Since most of our work has been concemed with the elucidation of thetopology of II. and Il opiate receptors we present now a brief review of the recent

development in this area. A review of opioid peptide analogues was published

recently by Schiller.13 Earlier developments can be found in part in my M.Sc

thesis. 14 A very complete review concerning opioids can be found in a recent issueof Handbook ofExperimentalPharmacology.16

1.3.1 Receptors selectivity of endogenous opiates and opioid peptides from other

sources.

The mammal endogenous opiates are constituted mainly by three peptides.

Two pentapeptides, Leu6 and Met6 enkephalin (Tyr-Gly-Gly-Phe·Leu/Met-OH)differ only by their fifth arnino acid.16 Both act similarly but have differentprecursor molecu\es. 17 They show slight preference for Il over II. opiate receptors.

Dynorphin, 18 a heptadecapeptide (Table 1.1) is an extended form of Leu6

enkephalin but shows selectivity for another class of opiate receptors called le.

Morphine and related narcotics are generally moderately selective to the II. opiate

receptor. The structure activity relationship (SAR) related to enkephalins can be

summarized as follow. 1D

1) The N,onu Tyr residue is an absolute prerequisite for activity at opiate

receptor and presumably mimics the tyramine moiety of morphine.

2) Only D-amino acids can be tolerated in replacement of Gly2.

3) GlYS can not be exchanged for any D or L amino acids without dramatic

loss of activity.

4) Phe4 is essential to opiate activity, although it can be substituted by other

aryJ or very lipophilic amino acids.

5) The fifth arnino acid is not very important and can be substituted for

almost any Lor D amino acids. It can even be completely removed without

dramatic loss of activity.

6) The terminal carboxylate can be amidated or reduced to an alcohol and

this dces not affect affinity, although it affects selectivity.

3

Table 1.1 Structural components of opioid peptides

4

°P10ld

pharma­cophoricresidue

Message

spacer Phanna­cophoricresidue

Address

enkephalin Tyrdeltorphin Tyr

deltorphin 1 Tyr

deltorphin n Tyr

dermorphin Tyrp-casomorphin Tyr

dynorphinl-13 a Tyr

Gly-GlyD-Met

D-AIa

D-AIa

D-AIa

Pro

Gly-Gly

PhePhe

Phe

Phe

Phe

Phe

Phe

Met/Leu-OHHis-Leu-Met-Asp-NH 2

Asp-Val-Val-Gly-NH2Glu-Val-Val-Gly-~

Gly-Tyr-Pro-Ser-NH 2

Pro-G1y-Pro-I1e-OH

Leu-Arg-Arg-I1e-Arg-Pro-Lys­

Leu-Lys-OH

a) Dynorphin 1-13 account for ail the properties of Dynorphin 1-17.

The isolation of four peptides from the skin of Phyllomedusa bicolor,20 a

south American frog, had led to serious revision of points 3 and 4 of SAR related

to enkephalins. Dermorphin21 (Table 1.1), as its name indicated, shows significantpreference for 11. (morphine) opiate receptors. On the other hand, deltorphin,22

deltorphin I,u and deltorphin n23 (Table 1.1) as their name indicated are veryselective for the Il opiate receptor. AIthough the four peptides possess the critical

T)'!" and a D-amino-acid in position 2, the GIf residue has been deleted and the

original Phe4 is now presumably occupying the position 3. This apparent

controversy with the SAI": of enkephalin has been put forward to develop new

conformationally constraint analogs (vide infra) and will receive further comments

at the end of chapter 3.

The screening of p-caseine hydrolysate has permitted isolation of a

heptapeptide, P-casomorphin 24 (Table 1.1), that shows moderate 11. opiate

selectivity and affinity. Refinement of the "lead structure" has led to a highlypotent 11. selective analog: H-Tyr-Pro-NMePhe-D-Pro-OH.25 Not only in this case

the phenylalanine occupies the third position in this peptide but the second

residue is of L-configuration which, according to point 2, should not be tolerated.This apparent discordance has received particular attention recently throughextensive molecular modeling based on active analog approach and confor­mational study in solution. 26 A special comment will be given in chapter 3concerning the conformation of H-Tyr-Pro-NMePhe-D-Pro-OH in relation to ourmodel of the opiate receptor.

1.3.2 Concepts in the development of new potent and selective opiates.

Although systematic amino acids substitution can provide SAR infor­mation, it is quite a materially and time consuming operation. In seekingunderstanding it is more appropriate to prepare analogs that are designed toverify a hypothesis. Sorne "concepts" have been used by several authors and wewill mention now the main ideas and sorne of the results obtained.

-Conformational restriction.

The poor selectivity of endogenous enkephalin had been associated to theirrelative conformational flexibility. Indeed, no consensus has been reachedconcerning the preferred conformation of Met/Leu enkephalins in solution by IHNMR, energy calcu\ation,27 or in solid state from X-Ray diffraction study.28 Folded

and extended conformations have been observed with ail techniques. One

important step toward the deciphering of opiate receptor topologies was realized

by DiMaio and Schiller29 who cyclized the side chain terminal amine of D­diaminobutyric acid in position two (D·~Bu) of an enkephalin analog with theterminal carboxylate. The resulting cyclic compound was still very potent at )1.

opiate receptor but shows significant reduction of affinity for l) opiate receptor.

This 14-membered ring has been the prototype for preparing several other

analogs with smaller or larger ring size,lIO insertion of retro-inverso amide bond,sland thioamide bond. S2 Most of these compounds show more or less increased Il

specificity.

The same principle was applied when the structure of dermorphin was

reported. A D-Orn2 was substituted for the D·Aia2 residue and an Asp·

substituted for Glyt. The two side chains were linked together and the resulting14·membered ring compound was found to be the most Il selective cyclic analog.N

5

This prototype has also been used to prepare compounds with reduced or

enlarged ring size or reversed amide structureM without getting significantimprovement of IL selectivity.

Conformationally constrained cS selective analogs were finally obtained by

Mosberg wLS6,S8in adapting informations from SAR of Iinear enkephalins to non

selective cyclic enkephalins. It has been generally observed in Iinear enkephalin

analogs that increasing steric bulk on the side chain of the D-amino-acid inposition 2 increases cS selectivity.1S On the other hand, the cyclic enkephalin analog

H-Tyr-c[D-Cys-G1y-Phe-Cys)-NH2 has very good affinity for both IL or cS opiate

receptors. S7 When Mosberg~ substituted a D-penicillamine (p-p-dimethyl

cysteine =Pen) in position 2 of this analogs, they observed a significant increase incS selectivity. The selectivity for the cS opiate receptor culminated when they further

added a D-Pen in position 5 and did not amidated the terminal carboxylate.

Recently, particular attention has been paid on the effect of a 2,6-dimethyl Tyr and

by using different amide bond surrogate between G1yS-Phe4 on the prototype bis

penicillamine analog.S8 To our knowledge the equivalent cyclopeptide based on

deltorphin sequen.;e (without G1yS) has not been reported yet, but H-Tyr-c[D-Cys­Phe-D·Pen)-OH has been reported to be quite cS selective with still a good

affinity.SG

Finally it is worth mentioning that ail efforts to prepared li: selective cyclic

analogs of dynorphill. have failed up to now. The compounds prepared turn out

generally to have IL opiate preference.1S

-Message-Address

The concept of message-address was proposed by Schwyzert° who

employed it to analyzed SAR of long Iinear peptide-hormones. We can state it by

analogy with a letter in the mail: peptide hormones (or neuropeptides) contain a

message (a part that transmit information) and an address (a part that tell the

hormone where to go). Bach part is constituted by an amino acid sequence within

the peptide-hormone (or neuropeptide), but it is not excluded that each fragment

may overlap. In Table 1.1 the opioid peptides sequence is presented according tothis view. The NI..... tri- or tetrapeptide constitutes the message part while the

6

remaining is presumably the address. Accordingly, Leu6 and Met6 enkephaiin arehaving an "incomplete address" and are poorly selective.

This concept was applied by Schwyzer to relate receptors selectivity not

solely on structurai aspects of the ligands but on specific ligand-membraneinteractions. The penetration of a ligand within lipidic membrane will depend on

the nature of the lipid membrane (negatively charged or neutrai) and will be

related to the effective charge, the arnphiphilic character, and the dipole momentof the address or the message compone!'.t of the ligand. These elements would be

of prior importance in the subclass receptor selection. The model of selectivity as

formulated by Schwyzer is based on spectroscopic measurements on the nature of

interactions of different peptide hormones with different synthetic membranes.

The results of this approach as applied to the opioid peptides can be summarized

as followS.41

1)Increased effective positive charge in the address component of theligands increases " selectivity.

2)Increased effective positive charge in the message component of theligands increases Il seleetivity.

3)Increased effective negative charge or electrostatic indifference in theaddress component of the ligands increases 6 selectivity.

This model is generaily in agreement with the sequence of peptides given in

Table U. Dynorphin 1-13 has an address with formai charge of +5 and it is weilestablished that the residues 7 and 11 are quite criticai for "selectivity. On the

other hand, deltorphins addresses are neutral or have a formai charge of -l, in

agreement with the proposed mode!. No conclusion can be drawn howeverconcerning the Il selective opiate peptides since the message component is highly

conserved, but sorne experiments have been done in this regard. Schiller ~42reported that the tetrapeptide H-Tyr-D-Arg-Phe-Lys-NH z (formai charge +3) is

much more selective for the Il receptor relative to the 6 opiate receptor, but

unfortunately no selectivity ratio is given regarding the " opiate receptor.

Sirnilarly Lazarus ~4S have modified the address component of deltorphin bysubstituting hydrophobic residues with Asp. No increase in the 6 selectivity over Il

opiate receptors was noticed and eleetrostatic indifference of the message

component in this case seems rather to be the rule.

7

• Portoghese~ used ingeniously the concept of message-address to create

hybrid molecules using a message component formed by a rigid narcotic alkaloid

and an address component from endogenous opiates sequence. Using naltrexone

la (Fig. 1.1) as a message component, they reacted the C6 ketone withHzNHNCO-Phe-Leu-OH or HzNHNCO-Phe-Leu-Arg-Arg-Ile-OMe which

resulted in the hydrazone derivatives l.lb, and l.le (Fig. 1.1).44 Compound l.1bwas about two times more selective for .s opiate reeeptors over Il. while compound

l.le was about 2 times more selective for II: opiate receptors over Il.. This may

seem rather small difference but it was already a great step since the equivalentcarbazone l.ld (Fig. 1.1) was about 10 times more selective for the Il. opiate

receptor over .s and 50 times more for Il. over 11:. The concept culminated with

naltrindole (1.2b) a .s selective antagonist with 200-300 time more affinity for .sMe-::N

Ua X =0l.1b X = NNHCO-Phe-Leu-OHl.le X = NNHCO-Phe-Leu-Arg-Arg-Ile-OMel.ld X = NNHCONH2

8

x

A.

Fisher IndoleSynthesis

o

Fig. 1.1 Chemieal formula of derivatives of oxyrnorphone (Ua) showing increased.s and II: affinity (1.1b, l.le); chemical formula :lf naltrindole (1.2b), a highlyspecifie.s antagonist and its chemical precursor naltrexone (1.2a).

• opiate receptors over 11. or K: receptors.45 This compound was prepared through

Fisher indole synthesis starting form naltrexone (Ua) and phenylhydrazine (Fig.1.1). According to Portoghese ~, the newly fused indole aromatic nuclei isplaying the role of the rnixed message-address of Phe4 in enkephalins. Ourinterpretation of this phenomenon is different and will be elaborated at the end ofchapter 2.

-Other concepts.

Tw" other concepts have been used successfully by Portoghese ~ inorder to design new selective ligand based on alkaloids structure. These are thebivalent ligands concept and the affinity ligand concept. We won't give hereextensive examples but the reader is referred to the excellent review article byPortoghese for more details.46 The bivalent ligand is a compound constituted by

two heads, each head being a normal ligand. The two ligands are linked togetherby a chemical arm of appropriate length. The goal is to realized the simultaneousbinding to two neighboring receptors. The ideas about this concept are thefollowing: 1) The distance between two neighporing receptors can be used as asupplementary recognition element and therefore may lead to inerease selectivity.2) Less rotational and translational entropy is lost upon the binding of onebivalent ligand compared to the binding of two monovalent ligands and therefore

this should lead to increased affinity (this can also be explained in term ofconfinement effects). Although bivalent ligands have had success in the case of bi­

alkaloid, relatively few examples have been obtained with bivalent ligands basedon endogenous opioids structure. 13

Affinity ligands are molecules that bear reactive functional groups (such asnitrogen mustard, chloromethylketone, etc) and are of course irreversible ligands.

When reactivity and positioning of the reactive groups are weil selected these

ligands can be selective and by the same token may provide information on the

location of reactive groups (usually nucleophilic) inside the receptor protein. Onthis basis, Portoghese ~ developed ~-funaltrexamine47 (1.3, Fig 1.2), an

irreversible II. antagonist. Owing to the normally mild reactivity of the Q-~

unsaturated ester, they proposed that a free SH group located appropriately inthe receptor might be the reactive nucleophilic species. Interestingly, the Z isomer

is not an irreversible Iigand.48

9

o1.3

oFig. 1.2 Chemical fonnula of ~-funaltrexamine.

1.3.3 Opiate receptor genes cloning.

Two independent teams have reported very recently the amino acidsequence of the putative Il opiate receptor.49,60 The gene was transfected in COS

cells51 and the expressed receptors exhibit phannacological responses similar tothose of the native receptor. The putative 371 amino acid sequence showssignificant homology with G-protein coupled receptors and particularly with thesomatostatin receptor. These results were expected since it was already knownthat enkephalins and narcotic alkaloids acted on the second messenger cyclicadenosine monophosphate (cAMP).62 On the other hand, some analogs ofsomatostatin have recently been shown to be powerful opiate antagonists63

indicating therefore parent phylogeny. Amongst the sequenced G-proteincoupled receptors which bind neuropeptides and neurotransmitters witb a freeterminal nitrogen, extremely bigh homology was found for the aspartate residues(and neigbboring residues) at positions 97 and 145 of the putative Il opiate

receptor sequence. These aspartate residues bave been proposed te he thenegative counterpart involved in the binding of the positively charged nitrogen of

the endogenous ligands.

10

G-protein coupled receptors enclosed most of the receptor sequenced up

to now and amongst them rhodopsin.54 Crystallographic data are available

concerning the structure of rhodopsin surrounded by a membrane55 and this has

been used in several cases to generate receptors structure based on homology.56

To our knowledge, none have yet been attempted for the cS opiate receptor.

References and notes

1. Instead of refering to a specifie reference we refer rather to J. Darnel1, H.

Lodish, and D. Baltimore in Molecu/ar CellBiology, 2 nd Ed. Scientific American

Book, W. H. Freeman and co, 1990, New-York.

2. K. Mullis, F. Faloona, Methods in Enzymology , 1987, 155, 335.3. F. Sanger, Science, 1981,214, 1205.4. For example see H. Singh, J. H. Lebowitz, A. S. Baldwin Jr., and P. A. Sharp,

Cel~ 1988, 52, 415.5. M. F. Perutz in Protein Structure: new Approach to Disease Therapy W. H.

Freeman, New-York, 1992.6. A. McPherson in Preparation and Analysis of Protein Crystals , John Wiley and

sons, New-York, 1982.7. J. Moult in American Crystallographic Association Annual Meeting, June 1988,

Workshop on Biological Science.

8. M. Smith, AM. Rev. Genet., 1985, 19, 423.9. See for example M. G. Rossmann, and M. A. Mclnlay, Infectious Agent and

Disease, 1993, 1,3; C. E. Bugg, W. M. Carson, and J. A. Montgomery, ScientijicAmerican, 1993, December, 92.

10. F. A. Momany, and H. Chuman, Methods in Enzymology, 1986, 124,3; P. Gund,

T. A. Halgren, and G. M. Smith, AM. Rep. Med. Chem., 1987,22,269.11. Several papers in NMR andX-ray Crystallography: Interfaces and Challenges, M.

C. Etter Ed., Trans. Amer. Cryst. Assoc., 1988, Vol 24.12. See for example Y. 0000, M. Segawa, H. Ohishi, M. Doi, K. Kitamura, T.

Ishida, and T. Iwashita, EUT. J. Biochem., 1993,212, 185.13. P. W. Schiller, in Progress in Medicinal Chemistry, edited by G. P. Ellis and G. B.

West, 1991, Elsevier Science Publisher, B. V pp 301·340.14. G. Villeneuve in Modélisation Moléculaire d~nalogues Cyclisés de l'Enképhaline:

Différentiationdes Récepteurs Opiacés pet 6 Faculté des Sciences, Université de

Sherbrooke, June 1987.

11

• 15. Several papers in HandbookofExperimentaIPhannacology, 1993,104 (Opioids).16. J. Hughes, T. W. Smith, H. W. Kosterlitz, L. A Fothergill, B. A Morgan, and

H. R. Morris, Nature, 1975, 179,577.17. U. Gubler, P. Seeberg, B. J. Hoffrnan, L. P. Gage, and S. Udenfriend, Nature,

1982,295,206; H. Kakidani, W. Fischili, H. Takahashi, M. Noda, Y. Morimoto,

T. Hirose, M. Asai, S. Inayama, S. Nakanishi, and S. Numa, Nature, 1982, 298,

245.18. A Goldstein, W. Fischili, L. 1., Lownwey, M. Hunkapiller, and L. Hood, Proc.

NalL Acad. Sei. USA, 1981, 78, 7219.

19. J. S. Morley,AM. Rev. PhannacoL ToxicoL, 1980,20,81.

20. Interestingiy this animal has also provided potent and selective analogs ofSubstance P: Pysalaemin and Phyllomedusin. V. Erspamer, A Anastasi, G.

Bertaccini, and J. M. Cei, Experientia, 1964, 20, 489; A Anastasi, and G.

Falconieri-Erspamer, Experientia, 1970,26, 866.

21. P. C. Montecucchi, R. DeCastigiione, S. Piani, L. Gozzinin, an': V. Erspamer,

[nt. J. Pept. Protein Res., 1981, 17,275.

22. G. Kreil, D. Barra, M. Simmaco, V. Espamer, G. Falconieri-Erspamers, L.Negri, C. Severini, R. Corsi, and P. Melchiorri, EUT. J. Pharm., 1989, 162, 123.

23. V. Espamer, P. Melehiorri, G. Falconieri-Erspamers, L. Negri, R. Corsi, C.

Severini, D. Barra, M. Simmaco, and G. Kreil, Proc. NalL Acad. Sei. USA, 1989,86,5188.

24. A. Henschen, F. Lottspeich, V. Brandt, and H. Tesehemacher, Hoppe-Seyler'sZ. PhysioL Chem., 1979,360,1217.

25. K. J. Chang, E. T. Wei, A. Killian, and J. K. Chang, J. PhannacoL Exp. Ther.,1983,227,403.

26. T. Yamazaki, S. Ro, M. Goodman, N. N. Chung, and P. W. Schiller, J. Med.Chem., 1993, 36, 708.

27. These have been reviewed by P. W. Schiller in The peptides, analysis,5Ynthesis,biology, S. Udenfriend and J. Meienhofer eds, Academie Press, 1984, pp 220­268.

28. J. F. Griffin, D. A. Langs, G. D. Smith, T. L. Blundell, J. J. Tiekle, and S.

Hedarkar, Proc. NalL Acad. Sei. USA., 1986, 83, 3272 and referô:nees therein.

29. J. DiMaio, and P. W. Schiller, Proc. NalL Acad. Sei. USA, 1980, 77, 7162.

30. P. W. Schiller, and J. DiMaio, Nature, 1982,297,74.

31. J. M. Herman, M. Goodman, T.M.-D. Nguyen, and P. W. Schiller, Biochem.Biophys. Res. Comm., 1983, 115, 864.

12

• 32. D. B. Sherman, A F. Spatola, W. S. Wire, T. F. Burles, T. M. -D. Nguyen, and

P. W. Schiller, Biochem. Biophys. Res. Comm., 1989, 162, 1126.33. P. W. Schiller, T. M. -D. Nguyen, 1.. Maziak, and C. Lemieux, Biochem. Biophys.

Res. Comm. , 1985, U7, 558.

34. P. W. Schiller, T. M. -D. Nguyen, 1.. Maziak, B. C. Wikes, and C. Lemieux, J.Med. Chem., 1987,30,2094.

35. H. J. Mosberg, R. Hurst, V. J. Hruby, J. J. Galligan, T. F. Burles, K. Gee, and

H. J, Yamamura, Biochem. Biophys. Res. Commun., 1982, 106,506.

36. H. J. Mosberg, R. Hurst, V. J. Hruby, J. J. Galligan, T. F. Burks, K. Gee, and

H. J, Yamamura, Life SeL, 1983,32,2565.

37. P. W. Schiller and J. DiMaio in Proceeding of the Eighth American PeptideSymposium., V. J. Hrubyand D. H. Rich Eds, 1983, Pierce Chemical company,

pp 269-278.38. N. S. Chandralromar, A Stapelfeld, P. M. Beardsley, O. T. Lopez, B. Drury, E.

Anthony, M. A Savage, 1.. N. Williamson, and M. Reichman, J. Med. Chem.,1992, 35, 2928.

39. H. J. Mosberg, J. R. Omnas, F. Medzihradsky, and G. B. Smith, Life. SeL, 1988,43,1013.

40. R. Schwyzer,Ann. N. y. Acad. SeL, 1977,247,3.

41. R. Schwyzer, Biochemistry, 1986, 25, 6335; R. Schwyzer, K. Shunsaku, N.

Kiichiro, D. Erne, R. Krzysztof, J. W. Bean, and D. F. Sargent, in PeptideChemistry, T. Shiba and S. Sakahibara Eds, 1987, pp 693-698;

42. P. W. Schiller, T. N. D. Nguyen, N. N. Chung, and C. Lemieux, l Med. Chem.,1989,32, 698.

43. 1.. H. Lazarus, S. Salvadori, M. Attila, P. Grieco, D. M. Bundy, W. E. Wilson,

and R. Tomatis, Peptides, 1993, 14, 21.44. A W. Lipkowski, S. W. Tarn, and P. S. Portoghese, l Med. Chem., 1986, 29,

1222.

45. P. S. Portoghese, M. Sultana, A E. Talcemori, l Med. Chem., 1990,33, 1714.

46. P. S. Portoghese, l Med. Chem., 1992,35, 1927 and reference therein.

47. P.S. Portoghese, D. 1.. Larson, 1.. M. Sayre, D. S. Fries, and A E. Takemori, J.Med. Chem., 1980,23,233.

48. 1.. M. Sayle, D. 1.. Larson, D. S. Fries, and A E. Takemori, P.S Portoghese, J.

Med. Chem., 1983,26, 1229.49. C. J. Evans, D. E. Keith Jr, H. Morrison, K. Magendzo, and R. H. Edwards,

Science, 1992, 258, 1952.

13

50. B. L Kieffer, K. Befort, C. Gaveriaux-Ruff, and C. G. Hirt, Proc. NalL Acad.Sei. USA, 1992, 89, 12048.

51. COS cells are semian cells that have been infeeted with SV40 (semian) virus in

arder to produce T antigen (cancerous). Foreing DNA is combined with anappropriate veetor and can he cleanly expressed into these cells. See J. D.

Watson, J. Tooze, and D. T. Kurtz in Recombinant DNA: A Short Course

Scientific American Books, W. H. Freeman and Co., New York, 1983; and Y.

Gluzman, Cel~ 1981,23, 175.52. G. J. IDte inPrinciples ofMedicinal Chemistry , W. 0, Foye and L Febiger eds.,

1981, Chap. 6

53. R. Maurer, B. H. Gaehwiler, H. H. Buescher, R. C. IDll, and D. Roemer, ProcNalL Acad. Sei USA , 1982, 79, 4815.

54. J. Bockaert, CU". Op. NeurobioL, 1991, 1,32.55. J. Deisenhofer, O. Epp, K. Miki, R. Huber, and H. Michel, Nature, 1985, 318,

618.

56. See for example on the muscarinic Ml receptor: G. Nordvall, and U. Hacksell,

J. Med. Chem., 1993,36, 867.

14

Chapter 2

A proposaI for the molecular basis of Il and cS opiate receptor differentiation

based on modeling of two types of cyclic enkephaIins and a narcotic alkaloid.

Authors contributions and notes

The results presented in this publication were from a molecular modeling

study accomplished by myself under the supervision of Pr. André Michel. The

draft of the experimental section was written by myself while most of theintroduction and the discussion was written by Dr. John DiMaio.

Color figures of the original publication were not suitable for the thesis

format and were removed. Instead black and white figures are provided but color

reprints could be request to myself.

Journal ofComputer-Aided Molec:uJar Design. oS (1991) SS3-S69ESCOM

J·CAMD 136

553

16

A proposa! for the molecular basis of Il and cS opiate receptordifferentiation based on modeling of two types ofcyclic

enkephalins and a narcotic alkaloid

André Michela••• Gérald Villeneuve" and John DiMaiob

"Loboraloirr dt' Chimie Strul"lufalt>. Dipar"mtnl ck Chimi,.. Facu/lédr.f Sdrncts. Un;"rs;lid~Shtrbrookt.Sh.,b,ook•• Qu.b<,. CanadaJ/K ZR/

"Th,. 8ioltC'hn%RJ' Rrsrarc!l Instilu,ro/TM National Rrgarch COlmei/ a/Canada. 6/00 RoyalMounl A"f'..

Manfrial. Qui~('.Canada H4P 2R2

RcœiYed 21 Scplember 1989Aceepled 14 March 1991

Kr." words: Cyclic opioid peptide: Receplor selec:livity; Conformational search: Enkephalin

SUMMARY

The moleeul.r basis underlying Ihe divergenl receplor seleeliYily of two cyelic opioid peplides Tyr.qN'·D'Orn'.G1y.Phe.Leu.) (c·ORN) .nd (o-Pen'. L-Cys']-enkeph.lin.mide (c-PEN) w.s inyestig.lcd using. mo­leeul.r modeling .ppro.ch. Ring e10sure .nd conform.lion.1 se.rching procedures were used 10 delerminelow·energy cyelic backbone conformers. Following reinsenion of .mino .cid side ch.ins. the n.rcolic .Ik.­loid 70-[(1 Rl-I-methyl.l.hydroxy.3·phenylpropyl]-6.I4-endoclhenote".hydro orip.yine (PEO) w.s used.s• nexiblelempl.le for bimoleeul.r superpositions with e.ch oflhe determincd peplide ring conformers usinglhe copl.n.rity .nd cocenlricity oflhe phenolic rings.s the minimum consll.int. A yeetor sp." ofPEO••c­counling for .n possible orienl.tions for the C"••rom.lic ring of PEO sel'\'ed as. geomelricallocus for lhe.rom.tic ring of lhe Phe' residue in lhe opioid peplides. Allhough • v.sl numher of polypeplide conform.·lions satisfied lhe crileri. of lhe opi.le ph.rm.cophore. lhey could he groupe<! inlo th... cl....s differing inm.gnilude .nd sign oflhetorsion.l.ngle v.lues oflhelyrosyl side ch.in. Only cl... mconformers for bolhc·ORN .nd c·PEN. h.vinglyr.mine dihcdr.l.ng'es XI =- 150' ± 30' .nd x,= _155' ±20'. h.d significanlslructural .nd conform.lion.' propenies lh.t Were mutu.lly comp.tible while respeeting the PEO veelorSpace. Comp.rison of Ih... prorerties in Ihe conlexl of Ihe divergent re"plor seleetivilY of Ihe studicdopioid peptides suggesls lh.tlhe incre.sed dislorlion of the peptide b.ckbone in the closure region ofc·PENlogelher with Ihe pend.nl p.ll-dimelhyl group. combine to genera... sterie yolume which is .bsenl in C'

ORN .nd Ih.1 m.y he incompatible with • restrictive topogr.phy of lhe ~ receplor. The n.lure .nd stereo·chemislry of substiluents .dj...nlto lhe closure region of Ihe peplides could .Iso modul.le receplor seJec·lion by inler.cting wilh. ch.rgcd (6) or neulral (~) subsite.

• Ta whom c:orrespondenc:e should be addressed.

092006S4XISS.OO © 1991 ESCOM Science Publishen B.V.

554

INTRODûCTION

The central and peripheral nervous systems contain a heterogeneous populalion of opiate re·ceptors whose interaction with narcotie opiates or opioid peptides manifests the characteristic an·algesic response [1-4]. Among the major opioid receptor families. the Il and 1\ subtypes have beensludied extensively. Pharmacologieal and biochemical evidence suggestlhattll:y are anatomicallydifferent and could be distinguished on the basis ofbrain region distribution [5.6J. differential af·finity for alkaloid and opioid peptide ligands [5.7]. divergent behavior toward alloslerie modula·lors such as Na + and GTP [8.9J and variable reactivity toward irreversible site·directed alkylatingagents [10].

Other important advances have becn made in the pharmacologic characterization of opiate re­ceptors through the rational design ofsynthetic opiate alkaloids [II Jand highly potent and recep­lOr·selective opioid peptide analogs [12.13J. Some of these. such as the conformationally con·strained analogs of [LeusJ-enkephalin prototyped by 1 (Fig. 1) have helped to define the Iimits ofstructural and conformationaltolerance of the receptors with which they interact [14-17]. In bind·ing studies morphine and related narcotic alkaloids typically have higher affinity for IIthan 1\ re­ceptors and the converse is true for Met and Leu enkephalin [18J; however. in spite oftheir peptid­ic nature. the cyclic compounds prototyped by 1 have been shown 10 display moderate to highselectivity for morphine receptors [19J.

ln another study Mosberg et al. [20.,11] have described a related series of enkerhalin analogscyclized through a disulfide bridge between a D-penicillamine residue in position two (Pen~) anda cysteine or another penicillamine residue atthe C-terminal position (2. Fig. 1). NotwithSlandingthe structural analogies between cyclic peptides of type 1 and 2. the former interacts selectivelywith lhe Il receptors while the laller with 1\ sites in functional isolated tissues preparations. As anexplanation. Mosberg [22] has proposed a model whereby the p.p-gem dimethyl group of c-PENconfers steric properties that are deleterious to binding to Il receptors. Alternatively. the possibili­ty that differenl bioactive conformations may be required by Il and 1\ receplors has becn investi·gated by Loew et al. [23.24] using computational melhods.

ln the Iight of opposing conclusions drawn from the above investigations. we reportlhe resultsof our own studies dealing with the conformational properties of the disulfide 21 and the cyclicamide le in order to derive a probable molecular basis for the divergent receptor seleclivity. Ourapproach utilized the 'template forcing' method to investigate key geometrical congruencies be­lween low-energy backbone conformers of the cyclic enkephalin analog le (Fig. 1) and the modelsynthetic alkaloid 7a-[( 1R)-I-methyl-l-hydroxy-3-phenylpropyIJ-6.14-endoethenotetrahydro ori·pavine (PEO) (3. Fig. 1) that could serve to explain the mutual affinity for a common receptor[25J. That PEO and its congener etorphine do not show any significant receptor discrimination[26.17J could be exploited sirce other than the 7a·aralkyl substituent. the remainder of the opiatepharmacophore is Iocked in an unambiguous atomic arrangement. Accordingly these alkaloids

Abhrf'IJ;alions. Symbols and abbrevialionsare in aœordanœ wilh recommendalion of the IUPAC·IUB Joinl Commission onBiochemical Nomenclalure. Biochemistry. 9 (1970) 3471. c·ORN. Tyr.qN"D·Om'·G1y.Phe·l.eu·l; c·PEN.ID·Pen'. L·Cys'l·enkephalinamide: Pen. penicillamine ~ p.p-dimelhyl cysleine: PEO. 7a·(( 1Rl-1.melhyl.l.hydroly.J.phenylpropyl)-6.14­endoethenotclrahydro oripavine: THT. 70-(( 1R)-'·mclhyl.l.hydrolly.J.melhylbulyJJ-6.14-endoelhenotelrahydr0 thebaine:CSO. comman space occupalion: NMR. nuclear magnelîc resonancc specuoscopy.

17

18

555

HO

+NH,

1-8n=11-bn=21 - C n = 3 H-Tyr-e[N" -D-Om2-G1Y-Phe-r.eu-j1-dn=4

+NH,

• 0

'NH:(NHyNHo 0

Me ,HO ~.S, NH 0S,:.. ....l.. NH,R""~ lr

1 R, 0

2 - a RI = R2 = H ( [D-Pen2, L-Cys'j enkephalinamide )2 - b RI = R2 = CH] ( [D-Pen2, L-Pen') enkephalinamide)

J (PEO)

Bond deranin,lOnkmal Mlles:%0' c,.c,.C... c.%0' c,. Co. c". c"x.: C,. c". c". c"zr c". c". c". c"

Fig. 1. Molecular slruc:ture ofc:yclic enkephalin analogs and PEO wilh sorne conronnalional variables under siudy.

556

have recognitive elements that define both the Il and ô receptor binding siles and. as such may beregarded as good semi-rigid models thal bridge the otherwise extreme behavior of lhe IWO relatedseries of cyclic enkephalin 1 and 2. Therefore we also investigated ail relevant conformers of thecyclic disulfide 2a in relation to PEO. Comparison of the resulting low·energy superpositionsserved to explain the possible molecular basis for the divergent receptor selectivily of the twopeptides.

MATERIALS AND METHODS

Mo/ecu/or mode/ andforcefie/dEnergy calculalions and conformational searches were performed using the MAXIMIN2 mini·

mizalion and the SEARCH function of the SYBYL 5.1 software package on a microVAX 2000computer [28].

Conformational energies were computed by the molecular mechanics approach. Standard pa­rameters supplied by the software were used in the energy function [29] but special paramelers had10 be added 10 take into account the torsional barrier of lhe disulfide bond which has a minimumat + or _90' [30]. The electrostatic component was computed assuming a distance-variable di·eleclric constant [31] and assigning partial alomic charges calculaled by the Pullman melhod.

Conformational searches were executed using the SEARCH program by the grid search proce·dure. to explore the conformational space and calculate the conformational energy ofgiven struc·tures while varying specified torsional angles. A general cutotl' of 0.85 times the van der Waals ra·dius served to reject conformations with bad atomic contact (bump check). The SEARCHprogram also allowed us to invoke specific constraints such as distance and orientation of vectors.

The function MULTIFIT was used to achieve the multimolecular flexible fits; this option al·lows the pharmacophore of many molecules to superimpose while minimizing the total strain en·ergy of ail the molecules. Standard strength constants were used to achieve the fit between thepharmacophoric elemems.

Molecular models for the peptide precursors were assembled from a slandard fragment Iibrarysupplied by lhe SYBYL system and ail the amide bonds were assigned the trallsplanar arrange·ment. The fused ring structure of PEO was constructed based on the crystal struclure of its con·gener 7a·[( 1R)·I·methyl·l·hydroxy-3-methylbutyl]·6.14-endoethenotetrahydro thebaine (THT)[32] as described previousJy [25].

Ring searching and ring e/osurefor the cye/opep/idesSince our primary concern resided in the low·energy conformation of the possible cyclic back­

bone structures of the respective cyclic amide le (c·ORN) and disulfide 2a (c·PEN), the a-carbonside chains were temporarily omined and replaced by hydrogen atoms while the tyrosine residuewas altogether removed for both molecules. A ring search and ring closure strategy based on dis·tance constraints was used in order to investigate the largest conformational space. Accordingly,the SEARCH program was applied to the models represented in Fig. 2. The bond distance con·straints were such that for c·ORN d, = 1.54 ± 0.15 A; d2 = d) = 2.51 ± 0.30 A; d. = 1.90 ±0.40 A; and for c·PEN dl = 1.80 ± 0.20 A; d2 = 2.85 ± 0.45 A; d) = 2.64 ± 0.40 A; d. = 1.90± 0.40 A. In both cases lhe dislance d, represents the bond forme<! by ring closure. and in orderto avoid nonbonded interactions in the closure region. three atoms were dummied (33) as shawn

19

557

20

Fig. 1. Distance constraÎnts and rolalable bonds as used in the ring c10surc and conformalional search procedure: (a) mod.01 for ORN.lblmodel for PEN.

in Fig. 2. The purpose of the distance parameters d~ and dJ was to invoke valid valence values forthe ncwly formed angles. The distance d. was imposed to simulate intramolecular hydrogen bondsas prcviously reporled for c-ORN [34J. A sereening of ail possible cyclic conformers generated forc-PEN demonstrated thatthe potentia) candidates for future superpositions with the c-ORN mole­cule were also those lhat featured the inlramolecular hydrogen bond Gly-C =0 L.;c NH. This as­sumption was also reinforced by experimenlal evidences obtained for similar peptides [35] andsupports our d. conslrainl.

1n the subsequent SEARCH procedure to affect ring closure. degrees of freedom were assignedto the rota table bonds for the abbreviated models shown in Fig. 2 and the corresponding torsionalangles were allowed to vary from 0' to 360' by 30' incremenls. Following the systematic searchail dummy atoms were replaced by appropriate groups and theconformers resulling from lhe ringc10sure were energy minimized using the MAXIMIN2 program in order to release local deforma·tions. No special component was used to preserve the pUlative intramolecular hydrogen bonds.For subse!luent studies the side chains and the exocyclic tyrosy) residue were reinserted inlo therespective ring conformers shown in Table 1.

Vectormapjor PEOThe high potency ofPEO and its C, a-alkyl congeners has been allributed to a well-defined site

on the opiate receptor which can accommodate a lipophilic alkyl or aralkyl group whose geome­try is deterrnined by the stereochemistry at C, and CIO (Fig. 1) [36]. In PEO the ftexibility of the70-[( 1R)-I-methyl-l-hydroxy.3.phenylpropyl] side chain precludes an accurate designation of thebioactive spatial geomelry of the C21 phenyl ring. Consequently a systematic conformationalsearch was conducted varying the torsional angles Xh X2. XJ and X. (Fig. 1) from 0' to 360'by 30'increments as previously deseribed [25]. Unlike the previous study np hydrogen bond distanceconmainl was invoked between lhe C.-OCHJ and Cio-OH groups. This variation permilled the.valuation of ail possible veClor loci corresponding to the C,,-aromatic ring. For each sterically

• 558

TABLE 1CONFORMERS RESUlTING FROM THE RING ClOSURE AND CONFORMATIONAl SEARCH APPLIEDTO THE MODEl OF ORN AND PEN (SHOWN IN FIG.!1

Ring Stcreochemical code of Energy' Ik\'lallllll from the nlldd!(conformer the ring ikcallmoll plane lrms \'aluesl

ORNI C' A' C' A a +a-s a 0.1 0.691ORN! C' P C G a +3-S a 1.4 0.420ORN3 C' F C' D -5-a+5+a 3.1 0.292ORN4 C' A C A' -s -3 +5 a 1.1 0.695ORN5 C C C' D a +5 -s-s 2.1 0.6S5ORN6 C A C A' il +5 -S-3 9.S 0.690ORN7 C F C' D il -s +a-a 0.0 0.5:!2ORNS C A C A' a -s +a +a 2.5 0.749

PENI E' A C G a +S +5 a+ 6.7 0.710PEN! A' G C E -s -s-s a- 10.5 0.611PEN3 D'A C D' -5 -s 3+a+ 4.4 0.637PEN4 C' C C' C' -5-a-a+5+ 4.7 0.452PEN5 A'C C· C· -3 +5 -s a- S.I 0.600PEN6 C' F C' A a +5 +S a+ 6.9 0.405PEN7 F' C' C A a a -s +s - 6.2 0.790PENS E' C· C G a +5 +5 a+ 0.0 0.S47PEN9 fi C· C E -s -8 a a+ 10.3 0.517PENIO F' A' C' A' a +5-S a+ 11.1 0.643PENil F' A' C' A' -s -s-s a- 4.5 0.675

"Energy is relative 10 the global minimum sella zero. Absolutc energies werc -19.3 and -26.7 kcal/mol for ORN7 andPEN8. respeclively.

allowed conformer the position of the vector normul to the centroid of the C,,·aromatic ring wasrecorded affording a vector map refiecting the preferential conformational space of the C, aralkylchain.

21

Fig. 3. Vector map c:orrcsponding 10 the confonnational spaœ of the C~l·aromalic ring of PEO (fol.lable bonds are Xl­X~. l.I and X. as indicalcd in Fig. 1).

559

Superposilion ofpharmacophoreThe coplanarity and cocentricity of the phenolic nuclei ofc·ORN. c-PEN and PEO were strictly

imposed while a distance deviation of up to 1.5 A was permined for the corresponding basic nitro­gens. FUrlhermore. the vector map previously generated for PEO served as an additional orienta­tion constraint for the proper alignment of the aromatic ring of the Phe' residue in the respectivepeptides. Using the computed backbone conformers ("ide supra: Table 1) of the peptides. a syste­matic conformational search was performed for each PEN-PEO and ORN-PEO pair. Degrees offreedom were assigned to the rotatable bonds of the peptide side chains as indicated in Fig. 1. Sub­sequently each bimolecular conformer emerging from the conformational search was resubminedto a bimolecular energy minimization using the funetion MULTI FIT. This operation perminedthe mutual convergenee towards the optimum bimolecular fit The fined structures were then al­lowed to relax freely ofany imposed constraints to the nearestlocal Ir.inimum with a convergencecriterion of 0.001 kcalimol. The energy differences for conformers before and after multifiningcan be used as an estimate for the conformational contributions to the binding energy.

22

Fi,. 4. Rtpresenlalive fit bctween PEO (reci Iines) and sorne ORN conformations (blue Iines). (a) fit No. 6. (b) fil No. 3.(cl fil No. Slsee Table 2).

• 560

Examinalion of the common and divergent conformational and sleric properlies exemplified bythe bimolecular filled pairs allowed Ihe identification of Ihose paramelers thal could account forthe dilferenl pallern of receptor selcclivily.

RESULTS AND DISCUSSION

The potenl narcotic alkaloid 7a-[( 1R)-I-methyl-l-hydroxy-)-phenylpropyl]-6.14-endoetheno­letrahydro oripavine (PEO) is a good semi-rigid templale to model more flexible cyclic opioidpeptides [37]. Despite its typical phenanthrene-like nucleus. this alkaloid displays lillie recoptorselectivity since it is equally elfective al both Il and ~ receptors. By inference. the flexibility of the7a-aralkyl substituent could account for the observed indiscriminant behavior by interacting withsimilar complementary binding siles on two dilferent receptors but in dilferenl orientations. Fig­ure 3 shows a stereoscopic veclor map corresponding to the geometrical space accessible to theC~I aromatic ring of PEO. Examination of the vector map indicates thatthe search generates aplethora of stable conformers scallered in almosttwo symmetrical vector regions: one concentra­ted in the pro-S and the other in the pro-R enantiotopic edge of the piperidine ring [38]. The pro-Rregion (blue) corresponds to that set of conformations with XI = 300· ± 30· whereas the pro-S

TABLE 2CONFORMATIONAL AND ENERGY PARAMETERS CHARACTERIZATION OF ORN FITTED ON TO PEO

23

Fit Cycli..: Class Sirain energ~ Characlerislie distancesb HydroseR bondsc F ansle" CSQ<no. peptide

ORN PEO Tyr·Ph. N·Tyr N·Ph. N·N No.1 No.2 No.3 No.4

ORNI li 12.5 4.7 9.22 5.17 11.09 1.11 1.48 1.50 33 ..2 ORNI III 6.7 4.6 10.90 5.13 11.68 1.10 1.55 1.58 42 •••3 ORN2 li 13.0 5.8 9.20 5.14 11.70 1.32 1.49 1.77 57 ..4 ORN3 1 13.9 1.3 tO.35 3.91 7.11 0.75 2.01 1.44 1.60 1015 ORN3 III 6.2 0.7 10.31 5.15 S.18 1.17 1.91 1.41 1.57 31 •••••6 ORN4 1 17.4 1.2 10.99 4.48 7.72 0.51 1.90 1.48 1.80 627 ORN4 III 4.1 0.4 7.73 5.IS 4.33 0.70 LVI 1.40 1.99 39 •••••8 ORN5 Il 13.2 2.3 9.81 5.14 11.68 1.53 2.50 1.62 70 ..9 ORN6 III 17.4 2.1 10.98 5.19 8.74 1.01 2.01 1.49 1.59 2.31 110 •

10 ORN7 III 14.6 3.0 9.94 5.16 10.15 0.72 1.58 1.89 1.61 109 ..Il ORN8 III 12.1 2.4 10.13 5.19 9.84 0.90 I.SI 1.54 1.57 2.28 95 •

•Conformalional cnerg)' in kal/mol fclalive to the global minimum sel 10 zero.~Tyr·Phe. distance between ccntroid ofaromalic nucleus orTyr and Phe (A).N·Tyr. distance between njlrogeR ofTyr and cenlroid ofaromalic nucleus ofTyr (A).N·Phe. distance bctween nitragcn ofTyr and œnlroid ofaromatie nucleus ofPhe (A).N·N. distance between nilrogen ofTyr in ORN and teniary nilrolen in PEO(A).

'H·bond No. 1C,..OH - O-C6: H·bond No. 2G1y·NH _ O=C·Tyr, H·bond No. 3(D)Om'NH _ O=C(D)Orn;H-bond No. 4 Leu·NH _ O=C-G1y.

~ Angular deviation betwecn the vcclor joining the Cw<:arbon and the ccntroid of phenyl rinS and Ibe vector joimnllbeCil of phenylalanine and Ihe cenlroid ofaromalie nuclei.

'Common spac:e occupalion. oblained by visual eSlimalion of the global overJap belween nonfilled atoms as observedafler the bimolecular flexible filling.

a

561

locus (yellowl coincides with X, = 180' ± 30'. Interestingly if the putative hydrogen bond [39J be·tween the C6-OCH3 and CI9-OH group is imposed, the resulting conformalional sampling of lheCl1·aromatic ring affords a locus concenlrated primarily in lhe pro·S c1uster. However. bolh iIIus·trated vector spaces were considered in subsequent flexible fit procedures since the putative intra·molecular hydrogen bond has been shown not to be an absolute requirement for high biologicalaClivily [40]. According to one hYPolhesis [41]. the C21·aromatic ring in PEO May have a functio·nal equivalent in opioid peptides of the enkephalin type; serving both as the 'message' and ·ad·dress' component of the opiate pharmacophore [42]. Although this analogy has nol been provenullequivocally. we have shown that using a single ring conformalion [34] of the cyclic opioid pep·tide Tyr.c[NO.o·Orn2, Leus] enkephalin (c·ORN), the Phe' residue satisfies this correspondence.Further, the alkaloid and peplide share other lopological similarities that could serve to explaintheir affinily for a mutually exclusive site [25].

ln lhe present study we have investigated other possible polypeptide backbone conformers us·ing a thorough conformational ring search of c·ORN and have included lhe cyclic disulfide (o·Pen2.L.CysS].enkephalinamide: a ô opioid receptor selective ligand [20]. The choice ofc·PEN (2a)was based on lhe observation lhat il is an established prototype upon which other more selectiveô·ligands have been modeled [43] and it displays a similar level albeit opposite selectivity to the

TABLE 3CONFORMAT10NAL AND ENERGY PARAMETERS CHARACTERIZATION OF PEN FITTED ON TO PEO

Fit Cyclic Class Strain cnergr- Characteristic distancesb Hydrogon bonds' Fangle' CSO'no. peptide

PEN PEO T~r·Ph. N·Tyr N·Ph. N·N No. 1 No.2

12 PENI III 9.6 1.0 10.12 5.18 8.71 1.67 1.86 1.51 56 ...13 PEN2 III 11.3 0.9 10.43 5.17 8.60 1.47 1.87 1.60 30 ••••14 PEN3 III 13.8 0.6 10.84 4.88 6.52 0.83 1.97 1.71 16 •••••15 PEN3 III 5.2 0.0 7.22 5.15 3.68 0.79 1.90 1.78 29 •••••16 PEN4 1 12.8 1.7 11.62 4.40 8.44 0.38 1.98 1.61 6217 PENS III 13.6 8.3 11.12 S.oI 11.20 1.20 1.64 53 ..18 PEN7 III 12.4 0.7 10.92 5.18 8.05 0.% 1.90 37 ••••19 PEN8 III 8.1 0.6 10.27 5.16 6.78 0.91 1.93 1.74 49 ...20 PEN9 1 14.8 5.8 9.85 4.38 8.29 0.41 1.99 1.54 16421 PEN9 Il 19.5 9.6 9.90 5.14 9.90 1.27 1.66 66 ..:!1 PENIO Il 18.4 3.2 10.83 5.13 12.44 1.39 1.54 57 ..23 PENil Il 14.0 5.8 10.60 5.16 11.47 1.64 1.56 114 ••

•Conformational cnersy in kcaltmol rclative to ahe glc.bal minimum set to zero.hTyr.Phe. distance between cenlraid ofaromatie nucleus ofTyr and Phe (A).N·Tyr. distance belween nitroseo ofTyr and centroid ofaramatic nucleus ofTyr (A).N·Phe. distance between nitraaen ofTyr and c:entroîd ofaromalic nucleus ofPhc (A).N·N. distance belween nitrolcD ofTyr in PEN and tertiary nitrogen in PEO(A).

'H·bond No. 1C,rOH - O·C6: H·bond No. 2Cy,·NH - 0 =C·G1y.... Ans,ular deviation between the vector joining the Cu-carbon and lhe c:entroid of phenyl ring and the vector joining theC· uf phenylalanine and the cenuoid of .romalie: nuc:lei.

'Common space occupation. obtained by visual estimation of the global overlap between nonfiued atoms as observedaher the bimolecular fte~ible filling.

24

562

prototypic cyclic amide 1< on isolated tissue preparations [16]. In addition the C-terminal residueis amidated and has the same relative stereochemistry as in c-ORN. The results of the ring·closureby cyclization and conformational search of the peptide backbone component of c-ORN and c­PEN are shown in Table 1. The conformational search generated eightlow-energy ring backbonesfor the cyclic amide and eleven correspondingly low-energy conformations for the cyclic disulfide.The resulting conformers are represented in Table 1. along with corresponding relative energies.using lhe stereochemical code adopted by Zimmerman et al. [44]. The small lener codes cor­respond to lhe values for x,. X~, x, and X4 of lhe side chain lorsional angles of the D-Orn~ residueof the cyclic amide analog and lhe Xl and X~ values of the torsional angle of the respective sidechains of the D-Pen~ and L-CyS' residue in the cyclic disulfide analog. The lener codes are definedas s = synperiplanar and a = antiperiplanar where s = 0' ± 30', + s = 60' ± 30'. - s = - 60'± 30', a = 180' ± 30', +a = 10' ± 30', -a = -120' ± 30'. For the disulfide torsional anglethe + sign stands for +90' ± 10' and the -sign for _90' ± 30'.

25

Fig. 5. Representative fil between PEO fred lines) and sorne PEN confonnalions (Ifeen linesl. CI) fil No. 16. (b) fil No. 21.(c) fil No. 14(... Table 3).

563

The last column in Table 1 Iists the rms deviation [45] from the plane constructed by filling thebest plane through the atoms comprising the cyclic backbone (i.e. N. C'. C' and atoms of the clo­sure region). The values provide an estimate ofplanarity of the peptide backbone. Il is intereslingto note that the most planar structures are found in the c-ORN series (ORN3 and ORN2 with rmsvalues of 0.292 and 0.420. respectively) while the most distorted is found for the global minimumin the c-PEN series (ORN8. rms = 0.847). The laller is stabilized by an extra H-bond betweenPen-C=Oand Phe-NH.

The final step in this study involved the mutual superposition of the essential pharmacophoricelements of the cyclic peptides with those of PEO. Using each ring conformer in Table 1 and thecoplanarity and cocentricity of the phenolic ring on one hand and a PEO vector map on the otheras minimum constraints. we obtained a large number of relaxed peptide conformees which couldbe grouped into three classes. These could be ditferentiated by the values of the torsional anglesX, and X~ of the tyramine moiety. Class 1 had X, = _90' ± 15' and X~ = -150' ± 15'; classIl had X, = 155' ± 15' and X~ = 115' ± 15'; class III had X, = -150' ± 30' and X~ = _155'± 20'.

The results in Tables 2 and 3 demonstrate that both [D-Pen~. L-Cys']-enkephalinamide and Tyr­c(N6-D-Orn~. Leu']-enkephalin could be modeled upon the alkaloid with only minor deviationfrom their lowest-energy conformation despite obvious ditferences in their backbone structure.however. the ORN and PEN conformations having the greatest common occupational space withPEO are concentraled in class III. This result is also iIlustrated in Figs. 4 and 5.

26

Fil· 6. Triple fil orPEO (rec!). ORN (blue) and PEN (smn) depiclins Ihe extlu.ion volume (yellowl orlhe di.ulfide bondand adjacent p.p~imelhyI8roup of PEN. (a) lyrosine-phenylalanine in the extcnded conformation (filS No. Sand 14).nd (b) tyrosmc...phcnylalanine in the falded conformation. (fils No. 7and 15).

564

Characlerislics ofthe bimolecular superposilionsTables 2 and 3 list key geometrical and energetic characteristics ofsorne bimolecular superposi.

tions corresponding to ORN-PEO and PEN-PEO. respectively. Interestingly. there was no majorreorientation of the polypeptide backbone during the bimoleeular fitting. consequently the va.rious designated intramoleeular hydrogen bonds remained intact. The C.-OCH). CIO-OH hydro­gen bond of PEO was also conserved in many superpositions; however. the greater global overlapwith PEO coincided with the accessory aromatic ring in lhe pro-S veelor space and having a con·served C.-OCH) < Cio-OH (see fits No. 5.7 and 14.15). The data also show thal the Phe' aromaticring of both peptides can assume either an exlended (exo) or folded (endo) orientalion with dis­tances inter-ring of 7.0 - Il A as already observed in related analogs [46]: lhe energy being slightlylower in the folded conformalion. This last phenomenon is presumably due to the better intramo·lecular van der Waals contacts in a folded structure while it is compensated in the ..tended con­formation by intermolecular contacts in lhe receptor microenvironment. The basic nitrogens ofthe fitted peptide-alkaloid pairs do not coincide resulting in generally more extended tyramineconformations in lhe cyclic peptides compared to the alkaloid.

The last column in Tables 2 and 3 denotes the common space occupation factor (CSO) whichwas estimaled by visual inspection and was assigned a value proportionalto the degree of globaloverlap within the fitted pairs ofmolecules. Figures 4 and 5 iIIustrale this poinl and depict repre·sentative bimoleeular superpositions wilhin the calculated classes of conformers. For bolh seriesof peplides those conformers comprising c1ass III. exemplified by Figs. 4c (ORN) and Sc (PEN)have lhe greatest common space occupation. In the conformation shown in Figs. 4c and Sc for fit5 and 14. respeclively, both aromalic rings of the peptides are localed on the Pface of lhe poly­peptide backbone in an exo configuration separated by approximately 10 A. The phenolic ringand Phe' side chain create a highly lipophilic surface in agreement with lhe arrangement proposedby Hruby et al. [47] for the solution conformation of the related peplide [D-Pen2, D-Pen'J·enke.phalin. The pUlative hydrogen bond between the G1y3-NH and the Tyr-C=O is not conserved inthis orientation for ORN3 but appears in PEN3 (Figs. 4c and Sc). The exocyclic tyrosyl residue isredirected over the cyclic backbone. For both cyclic peptides the polypeptide backbone sweepsundemeath the a face of the alkaloid such that the closure region of lhe respective peptides isproximalto the 6,I4-endoetheno bridge. However, because the PEN backbone is more dislorled(0.637 PEN3 vs 0.292 ORN3), the disulfide moiety descends further below the plane of PEO. Incontrast to the conformers resulting from class III superpositions, those emerging from c1ass 1(Figs. 4a and Sa; XI = -90'± 15', X2= - ISO' ±15') and class \1 (Figs. 4b and Sb; XI = 155' ±15'.X2 = 115° ±15°) have lheir respective cyclic backbone redirected in a region bearing little cor·respondence with PEO. Based on the reasonable assumplion that, as in other neurotransmitter re­ceplor superfamilies [48], the opioid receptor binding site is finite with conserved structural andstereochemical requirements in the active site of the Il and li receptor subtypes, the departure ofthe ring backbone as shown by Figs. 4a,b (ORN) and 5a,b (PEN) would not be expected to beconductive to favorable recoptor interaction.

Il has becn shown previously that substitution of the Phe' residue, in either cyclic or acyclic en·kephalin analogs. with eleetron·withdrawing or eleetron-donaling groups causes parallel qualira.tive and quantitative potency shifts at both reeeptors (in vitro) [49]. This result iIIustrales lhat lhePhe' aromatic ring is subjcct to lhe same elcctronic etrccls at both rcceptors suggestinillhat lhecrilical binding Sile associated with the side chain of the Phe' residue in PEN and ORN subserves

27

565

a similar role and may be a functional residue that inextricably links l'and 0 receptor subtypes.This argument is supported by the present model and is iIIustrated bya triple 'extended' fit in Fig.6a involving ORN3. PEN3 and PEO. Indeed the triple superposition shows thatthe aromatic ringoflhe Phe' residue in the peptides and the C21 ·aromatÎc ring of the alkaloid are coplanar while thefitting angle. defined as the angulardeviation of the bond bearing the CP·aromatic ring of the Phe'residue on one hand and the C21-aromalic ring in the alkaloid. is suflicienlly small rendering thering substiluent posilions congruent. and therefore subjeelto an analogous effeet. However. smallperturbations in the polypeptide backbones can confer substantially large huing angles (Tables 2and 3) such thatthe ring positions are not equivalent; however. the validity of the correspondencediscussed above should be extended to examine the effeets of biological activily of similar ringsubstilutions on the C21-aromatic ring ofPEO.

Comparison lI"ith o/her receplOr modelsEarly studies based on active analog design have generated several models regarding the deter­

minant features of the l'and 0 opioid reeeptors pharmacophore: among these. the greater com­pactness of the l' binding site and the nature of the side chain oflhe fourth residue in opioid pep­tides have formed the basis of distinction [12). One current model of the l' receptor has beenproposed by Keys et al. [24] using thp.oretical energy calculalions to study morphiceptin and otherl'.seleclive morphiceptin analogs. The conclusion from thal study and a subsequent one was lhatthe interaction with the l'and 0 reeeplor necessitates different polypeptide conformations. The re­sults from the present study which suggest no dramatic conformational difference between PENand ORN using PEO as a modeltemplate. are at variance with the conclusions derived by Loewet al. In the latter study the l' pharmacophore is defined by the conformational properties ofmor·phiceptin (Tyr·Pro·Phe·Pro·NH1) whose primary structure having two proline residues and a Pheresidue in posilion three. is a significant departure from c·PEN and c·ORN which have an acces·sory aromalic ring in position four and whose positional relation ofside chains is secured by intra·moleeular cyclization. Furthermore the present investigation allowed full freedom to the torsionalangles X1and X2 of the tyramine residue in the cyclic peptides while in the Loew modelthese valueswere confined to 267' and 193'. respectively. corresponding 10 those found in fused morphine andother alkaloids. Indeed we found thallhe rigid tyramine constraint described above redirects thepolypeptide backbones away from lhe PEO contour analogous to Class 1 superposition with theadded effeel that the Phe' aromatic ring fell outside of the veetor map for the C21 ring of PEO. Atyramine conformation different from rigid alkaloids is also justified on other grounds: for exam·pie: (1) the synthelic hybrids of enkephalinamide and metazocine. sharing in common the tyr·amine moiety. have been shown to be inactive [50]. and (2) the nilrogen locus. which is variable inanalgesics [51-53J. could also be different in these opioid peptides and. by extension. would notmanifest parallel struclure-activity relationships as rigid alkaloids [54]. Indeed the model pro·posed by Porloghese et al. [53] reeonciles sorne of these apparent anomalies.

Il is now generally aceepted thal the conformational heterogeneity of structurally diverse ana·logs of opioid peptides preclude a reliable assignment of the steric and geometric requirements ofthe various receptor subclasses owing to the propensity of such Iinear peptides to 'adapt to the to·pography of lhe aClive sile (17]. On lhe other hand rigidificalion of the enkephalin backbone bycyclization through an amide or disulfide bridge can be expected to reduce the number of intrinsicinterconverting conformalions. Cyclic peplides of the types 1 and 2 as weil as variants having the

28

• 566

phenylulanine residue in position three have becn the subjeet of considerable investigation byNMR und theoreticul energy calculations [47. 55-58]. NOlwithstanding that rigidifieation of theenkephalin backbone by cyclization via disulfide or amide bridge would be expecled to reduce thenumber of interconverting conformations. the degree of receptor selectivity is highly dependenton the mode ofcyclization. Accordingly. ahhough NMR data suggests that the overall conforma·tions corresponding to [D-Pen'. L-CysS]-enkephalinamide and [D-CyS'. L-Cysl]-enkephalinamideare similar. the laller is equally effective at both li and Il receptor sites [59]. Within the cyclic amideopioid peptides prototyped by Tyr-c[N"-D-Xxx-Gly-Phe-Leu] where Xxx is A,pr. A,Bu or Orn.similar conformations exist for ail three [56]. The receptor model derived from the present studyprovides a rationale for the above anomalies. Figure 6 shows two triple superpositions involving(a) PEO. ORN3 (fit No. 5). PEN3 (fit No. 14) and (b) PEO. ORN4 (fit No. 7) and PEN3 (fit No.15) adopting a class III tyramine conformation and with characteristic torsional angles Iisted in

TABLE4TORSIONAL ANGLES' OF TWO SELECTED CONFORMATIONS OF ORN AND PEN CORRESPONDING TOTHE PROPOSED ACTtVE CONFORMATIONS

Residue Torsional ORNfil No. S' ORNfil No. 7' PEN fit No. 14" PEN fil No. Il'angle IXxx=Orn. (Xxx=Orn. IXu=Pen. (X:\'l.=Pen.

Yyy= Leu) Yyy ~ Leu) Yyy=Cys) Yyy=Cysl

Tyr l W 96 -98 Ib2 lb:!

X' -162 -178 -127 180

X' -136 -159 -146 -131

U.>IXu: ~ 159 -14 76 108

w -56 -64 -76 -84

X' -48 -51 -39 -39

X' -113 -123 -73 -73

X' 94 74 88' 89'

X· 98 ISO

Gly·l ~ -(,6 -45 -63 -62w 162 -31 -64 -50

Ph'" ~ 74 -86 -66 -77

w -64 60 78 74

X' -44 -44 -73 32

X' 110 145 92 116

Yyy' ~ -161 52 167 167

W 62 46 -56 -61

X' -173 -176 137 134

X' 60 61 -172 -169

,... Angles are expressed in degrees.to Corresponding lorsional angles for PEO arc Xl = -177"', ~ = 58-. XJ = l77a

• x. =81~.

'Corresponding lorsional angles for PEOare Xl = -177'. X:= 58"', l.I= -91". x.= 123Q•

o.l C.S-S.C dihrdral angle in the c·PEN series.

29

567

Table 4. The stereoscopic view c1early demonstrales that the large dislortion of lhe backbone inthe arca of the disulfide bond of PEN forces this region and the adjacent gem dimethyl groupoUlsidc of the common occupational volume of the lhree molecules and suggests that lhis struc­turai clement. barring any major reorientation of the polypeptide backbone al the receptor micro­cnvironment. may be incompatible with the ~ site. Therefore lhe resulls of the approach used inthis study are more in agreement with a steric elfect which is deleterious 10 receplor binding assuggested by Mosberg [~~) based on NMR sludies. Expectedly the observed unfavorable stericbulk is exacerbaled by substitution of an additional Pen residue in position five. consequently theresulting analog (D-Pen'. L-Penl)-enkephalinamide would be ex;:.:cted to lose further affinily forthe ~ receptor as has been previously reported [~Ol. Interestingly the exclusion volume associatedwith the /l-gem dimethyl groups in the present model resides below the plane of the phenolic ringwhich etTectively deshieIds one methyl group in agreement wilh the anisotropic elfect previouslyobserved [1~]. The above observations may also serve to explain the fin ding by Portoghese el al.[4~1lhat alkaloids derived by fusion of an indole or benzofuran to the 6.7-position of naltrexoneor oxymorphone confers antagonist and agonist 1> receplor selectivity. respectively. According 10

the present modelthe additional aromatic ring of the above alkaloids coincides with the exclusionvolume shown in Fig. 6 and rnay account for the observed 1> selectivity. Thus it is conceivable thatthe greater distortion of the polypeptide backbone and the bulky steric volume of the pendant/l.Jl-dimethyl groups of Pen' residue in [D-Pen'. L-Cyslj-enkephalinamide or the bis-gem dimelhylgroup of the [D-Pen'. L-Penlj-enkephalinamide eongener are not compatible with a restrictive to­pography oflhe ~ site.

Finally. it should be noted that in the depicted triple superposition shown in Fig. 6. the pendantLee' -ide chain of c-ORN and the carboxamide function of c-PEN also maintain a similar orien­talion relative to their respective polypeptide backbone but. while the former is predisposed 10

confer additional lipophilic character to the /l surface. the laner is nol. The coincidence of theseside chains slems from the fact that the absolute configurations of L-CYS and L-Leu are Rand S.respectively. but while c-ORN is cyclized through the terminal carboxy groups. c-PEN is cyclizedthrough a disulfide bond involving side chains. Therefore it is tempting ta speculate that receptorinteraction may be related ta lhe hydrophobicity or hydrophilicity of this side chain. Indeed thecarboxylate derivative of c-PEN and congener display further reduced polency at the ~ receptor[~I]. bUI relaled data for c-ORN is unavailable.

CONCLUSION

The molecular basis of ""ioid receptor selectivity relies on the structural. conformational andslercoelectronic requirements of the receptor aClive site in a membrane microenvironmenl. Clear­Iy no singular approach can assimilate the above criteria. however. we have arrived at a tentalivemodel for the ~ and 1) receptor pharmacophore which does not necessitate drastically dilferentconformations for the related Tyr-c[N0-c Orn'. Leu']-enkephalin and [D-Pen'.L-Cys']-enkepha­linamide. For each opioid peptide two conformers emerge as good candidates representative ofthe biologically active conformation: for the c·ORN. fits No. 5 and No. 7 (Table ~). and for c­PEN. fits No. 14 and No. 15 (Table 3). The torsional angles of these conformers are provided inTable 4. In the present model. the interplay of steric and conformational elfects in the vicinity ofthe closure region of D-Pen generates an exclusion volume which may be incompatible with the ~

30

568

binding site: this result is in agreement with Mosberg's proposai who ascribed the reeeptor selee­tivity of PEN to a deleterious effect of the gem dimethyl group. The exclusion volume resides injuxtaposition to a site that could interact wilh a charged substituent on the li ligand but with aneutrallipophilic residue on the Il counterparl. This mode1 does not exclude the possibility thatthe respective binding sites may be buried within the cell surface whereby the membrane serves toscreen the effective net charge [60]: however. it is noteworthy that other sludies have alluded tosimilar models based on olher lines of evidence [61]. In the absence of sequence and structuraldata for individual reeeptors. the model derived above provides Ihe basis for a heuristic approachfor the design of receplor·seleclive opioid peptides.

ACKNOWLEOGEMENTS

We are indebted 10 the National Sciences and Engineering Research Council of Canada for fi·nancial assistance (operaling Granl A·1529) 10 J.O. and to A.M. (operaling grant A·0329).

REFERENCES

1 A preliminary report orthis work was presented al the American Crystallographic Association Annual Meeting. Phila-delphia. June 26-July 1. 1988.

1 Simon. E.J .. Hill... J.M. and Edelman.l .. Proc. Natl. Acad. SCi. U.S.A.. 70(1973) 1947.J Pert. C.B. and Snyder. S.H.. Science. 179 (1973) 1011.4 Goldstein. A.. Science. 193 (1976) 1081.5 Chang. K.J.. Halum. E. and Cuatrocasas. P.. Proc. Nall. Acad. SCi. U.S.A.. 78 (1981)4141.6 Duka. T.H .. Schubert. P.. Wüster. M.. Sloibet. R. and Herz. A.. Neurosci. Lell.. 21 (1981) 119.7 Chang. K.J .. Cooper. B.R.. Hazum. E. and Cuall'ccasas. P.. Mol. Pharmacol.. 16 (1979) 91.8 Chang. K.J.. Hazum. E.. Killian. A. and CU3trecasas. P.. Mol. Phannacol.. 20( 1981) 1.9 Childers. S.R.. Creesc.i .. Snowman. A.M.and Snyder. S.H.. Eur. J. Pharmacol.. SS (1979) Il.

iO Portoghese. P.S.. Larson. D.L.. Say... L.M .. Frics. O.S. and Takemori. A.E.. J. Mod. Chem.. 23 (1980) 233.Il Lipkowski. A.W.. Tam. S.W. and POrl08he... P.S.. J. Mod. Chem.. 29(1986) 1222.12 Fournië·Zaluski. M.C.. Gacel. G.. Maigrel. B.. Prëmilat. S. and Roques. B.P.. Mol. Pharmacol .. 20 (1981) 484.13 l\Dques. B.P.. Foumië-Zaluski. M.C.. Gacel. G., David. M.. Meunier. J.C.. Maigrel. B. and Morgal. lL..ln Cosla.

E. and Trabucchi. M. (Eds.) Regulatory Peptides: From Molecular Biology 10 Function. Raven Press. New York. NY.1981. pp. 321-331.

14 DiMaio.J. and Schiller. P.W.. Proc. Natl. Acad. SCi. U.S.A.. 77(19801 7162.IS Schiller. P.W. and DiMaio.J.. Nature. 297 (1982) 74.16 DiMaio.J .. Nguyen. T.M.D.. Lemieus.C. and Schiller. P.W .. J. Mod. Chem.. 25( 1982) 1432.17 Schiller. P.W. and DiMaio. Lin Hruby. V.J. and Rich. D.H. (Eds.) Peptides: Struclureand Function. IProcs.IUh Am.

Pep.. Symp.). Pierce Chemicai Co. Rockrord.IL. 1983. pp. 269-278.18 Lord. J.A.H.. Walerfield. A.A.. Hughes. J. and Koslerlitz. H.W.. Nalure. 267 (1977)49S.19 Sherman. 0.8.. Spalola. A.F.. Wire. W.S .. Burks. T.f.. Nguyen. T.M.D. and Schiller. P.W.. Biochem. Biophys. Res.

Comm.. 162(198911126.20 Mosberg. H.I .. Hurst. R.. Hruby. V.J .. Galligan. J,J .. Surks. T.F.. Gee. K. and Yamamura. H.I.. Biochem. Biophys.

Res. Commun.. 106(1982) S06.21 Mosberg. H.1.. Hurst. R.. Hruby, V,J .. Galligan. J,J .. Dur"s. T.F.. Gee. K. and Yamamura. H.I .. Life Sei .. 32 (1983)

:!S6S.22 Mosberg. H.I .. lnl. J. Pepl. Protein Res.. 29 (1987) 282.13 Loew. G.. Key•• C.. Luke. B.. Polgar. W. and ToU. L.. Mol. Phannacol .. 29 (1986) 546.24 Keys. C.. Payne. P.. AmSlerdam. P.. Toll. L. and Loew. G.. Mol. Pharmacol.. 33 (1988) S28.15 DiMaio. J.. Bayly. C.I.. Villeneuve. G. and Michel. A.. J. Med. Chem.. 29(19861 1658.

31

569

:!b Sn)'der. S.H .•and Goodman. R.R.. J. Neurochem.. 3S (1980) S.~7 DiMuio. J.. Unpublishcd observations ..28 SYBYl Molecular Modelling Software. Version 5.1. Tripos Associalc Ine.. April. 1988.19 Clark. M.• Cramer. j,O. and Van Opdenbosch. N.. J. Comput. Chem.. 10(1989) 982.30 Marshall. G. and Kataoka. X.• SYBYL Software Problcm Report. 2 (1990) 2.31 Gelin. 8. and Karplus. M.. Biochemislr)'. 18 (1979) 1256.31 Van den Hende.J.N. and Nelson. N.R.• J. Am. Chem. Soc.• 89(1967) 2901.33 A dummy alom is Jcomelrically defined in spate bui does nol contribute to lhe energy summations.34 Kessler. H.. Holumann. G. and Zechel. C.• lnt. J. Pept. Protein Res.• 2S (1985)267.3S Kesslcr. H. and Holzemann. G.. Angew. Chem.lnl. Ed. Engl.. 20(1981) 124.36 Bentley. K.W. and lewis.J.W.. In Kosterlitz. H.W.• Collier. H.O.S. and VilIarieaI.J.E. (Eds.) Agonist and Antagonist

Actions ofNarcotic Analgesie Druss. University Park Press. Baltimore. MD. 1972. pp. 7-16.37 loe"". G.H. and Pens. K.. Proc. Nat!. Acad. Sei. U.S.A.. 75 (1978) 7.38 Larson. D.L. and POr1oshese. P.. J. Med. Chem.• 16 (1973) 195.39 Michel. A.G.. Prou Il!.. M.• E\lrard. G.• Norberg. B. and Milchert. E.. Cano J. Chem.. 66 (1988) 2498.40 HUlchins. C.W. and Rapoport. H.. J. Mod. Chem.. 27 (1984)521.41 Bradbuf)·. A.F.. Smylh. O.G. and Snell. C.R.. Nalure. 260 (1976)165.42 Portoghesc:. P.S.. 5ultana. M.. Nagase. H. and Takemori. A.E.. J. Med. Chem.. JI (1988) 281.43 Mosberg. H.I .. Hurst. R.• Hruby. V.1 .. Gee. K.. Yamamura. H.t.. Galligan. J.1. and Burks. T.F.• Proc. Nad. Acad.

Sci. U.S.A.. 80 (1983) 5871.44 Zimmerman. S.S.• Poille. M.5.. Nemethy. G. and 5cheraga. H.A.. Macromolecules. 10 (1977) 1.45 RMS \lillues were calculated by SQRT (~:/n-I) where n is lhe number ofatoms within the macrocycle and â is the

minimum dislance between each atom and the middle plane.46 froimowilz. M. and Hruby. V.1 .• lnt. J. Pepl. Protein Res.• 34(1989) 88.47 Hruby. V.l.. Kao. L.F.. Pelli•• B.M. and Karplus. M.• J. Am. Chem. Soc.. 110(1988)3351.48 Kerla\'age. A.R.. fraser. C.M. and Venter. J.C.. Trends Pharmacol. Sei.. 8 (1987) 426.49 Schiller. P.W.. Nguyen. T.M.O.. OiMaio. J. and Lemieu•• C.. Life Sci.. l3 (Suppl. 1) (1983)319.50 Ramakrishnan. K. and Porto8hese. P.S.. J. Mod. Chem.. 25 (1982) 1423.51 Shiotani. S.. Kometani. T.. Mitsuhaski. K.• Nosawa. T.• Kurobe. A. and Futsukaishi. 0 .. J. Med. Chem.• 19 (1976)

803.52 Freed. M.E.. Poloski. J.R.. Frecd. E.H.• Conklin. G.L. and Bell. S.C.• J. Med. Chem.. 19 (1976) 476.53 Portoghese. P.S.. Alreja. B.D. and Larson. O.L.. J. Mod. Chem.• 24(198\) 782.S4 DiMaio. 1.. Schiller. P.W. and Belleau. B.• In Gross. E. and Mcienhofer. J. (Eds.) Peptides: Struclure and Function.

(Procs. 61h Am. Pepl. Symp.). PierceChemical Co.. Rockford.IL. 1979. pp. 889-892.55 Mammi. N.J .. Hassan. M. and Goodman. M.. J. Am. Chem. Soc.. 107(1985)4008.56 Hall. O. and Pavin. N.. Biopolymers. 24 (1985) 935.57 Wilkes. B.e. and Schiller. P.W.. Biopolymers. 26(1987)1431.58 Selleney. 1.. Roques. B.P. and Fournie·Zaluski. M.C.. lnt. J. Pept. ~rotein Res.. 3D (1987) 356.S9 Schiller. P.W.. Eggimann. B.. DiMaio. J.. Lemieu~. C. and Nguyen. T.M.D.. Biochem. Biophys. Res. Comm.. 101

(19811337.60 Schwyzer. R.. Biochemislry U.S.A.. 25 (1986) 6335.61 Schiller. P.W.. Npuyen. T.M.O.. ChunS. N.N. and Lemieu•• C.. J. Mod. Chein.. 32(1989)698.

32

33

Additional comments

One point conceming the discussion requires rectification. We stated inthe last part that the additional aromatic ring of naltrindole (Fig 1.1) couldcoincide with the exclusion volume generated by the gem-dimethyl group of Pen2 inH-Tyr-c[D-Pen-Gly-Phe-Cys]NH 2 (c-PEN). A careful fit performed later between

c-PEN and naltrindole showed that rather the additional aromatic ring innaltrindole coincided with the ~-methylene of CyS6. This fit is depicted in Fig. 7.

Portoghese ~ have demonstrated recendy the involvement of theadditional benzene ring in naltrindole (Fig. 1.1) with a specific subsite recognitionlocus. They have shown that the pyrrole ring in naltrindole aets as a spacer andconsequently can be substituted for a furan ring without causing significant loss ofactivity and seleetivity for the 1\ opiate receptor.82 They showed also that

modification of the geometry of the spacer such as the substitution of the pyrrolering by a six membered pyrazine caused loss of seleetivity presumably due to thereorientation of the benzene ring relative to the morphinane.82 Both results

argued in favor of a definite locus for the subsite that accommodates theadditional benzene ring in naltrindole.

Fig. 7 Superposition of c-PEN in the putative conformation bound at the 1\ opiate

receptor (Fig. 6a) with the narcotic alkaloid naltrindole.

• It is olwious that the fit presented in Fig. 7 does not assume the hypothesis

proposed originally by Portoghese~ regarding the fact that the newly inserted

aromatic ring in naltrindole mimics the phenyl group of Phe4• Although it may be

possible to achieve the putative superposition using Phe4 of cyclic enkephalinanalogs, we have sorne arguments in favor of the binding modalities presented in

Fig. 7. In our opinion, more pharmacological results can be taken into account

using this superposition and these are mentioned now.

1) The hypothesis of Portoghese~ implies that the Phe4 phenyl group is

an address and therefore the subsites that bind this pharmacophoric elementmust be located at a different spatial locus in \1 and ~ opiate receptors in order to

modulate the selectivity. In the previous paper we mentioned that para electron

withdrawing group substitution on Phe 4 induced parallel and quantitative potency

shift at both reeeptor (entry 1 and 3 of Table 5). This observation is best takeninto aecount if a common locus for the binding site of Phe4 for ligands with \1 or ~

increased selectivity assumed, as in our mode\.

2) It has been observed that cyclie disulfide enkephalins are more potent atmouse vas deferens (a tissue richer in ~ opiate receptors) than corresponding

cyclic disulfide enkephalinamides (entry 4,5,6,7 of Table 5).21 Moreover, this trend

is generally observed also for linear enkephalin analogstlS•84 Assuming that binding

modality is similar for both cyclic carboxamide and carboxylate compounds, it

could be proposed that the end groups interact with subsites that modulatespecificity towards \1 or ~ opiate receptors. Interestingly, this hypothesis receives

support from the fit presented in Fig. 7 since the terminal carboxamide is in the

vicinity of the newly inserted aromatie ring in naltrindole which also has beenshown to be a critical element to modulate ~ opiate preference.

3) The previous statement is not in contradiction with the fact that thesterie volume generated by the pendant P,P-dimethyl group on amino-acid 2 may

he incompatible with the topology of the \1 receptor. This factor could simply be a

supplementary steric requirement. Indeed this receives support from

pharmacological data since it is generally observed that increased selectivity of

cyclic disulfide enkephalin bearing a Pens residue is mainly the result of reducedpotency at \1 receptor (GPI) rather than increased potency at ~ receptor (MVD)

(see entry 6-11 of Table 5).

34

35

TableS Potencies (IC50) of enkephalin analogs to inhibit muscle contraction

in guinea pig ileum (GPI) and mouse vas deferens (MVD).

Analogs 1<;0 (nM) I<;o(GPI) Ref.GPI MVD II<;o(MVD)

1 [D-Cysz, D-Cys5]EAa 0.78±0.01 0.29±0.04 2.6 40

2 [D-Cysz, L-Cys5]EAa 1.51±0.03 0.76 ±0.09 2.0 40

3 [D-Cysz, p-(NOz)Phe4,D-Cys5]EAa 0.035±0.01 0.018 ±0.002 2.6 40

4 [D·Penz, L-Cys5]EAa 118±18 3.6±0.7 32.4 21

5 [D-PenZ, D-Cys5]EAa 11h21 16.8±3 6.9 21

6 [D-PenZ, L-Cys5]Eb 213±63 0.32 ±0.03 666 21

7 [D-Penz, D-CyS5]Eb 1350±340 6.27± 1.2 215 21

8 [D-Cysz, L-Pen5]Eb 39.9±1.5 0.75±0.05 53.2 2J

9 [D·Cysz, D-Pen5]Eb 66.7± 1.3 0.13 ±0.06 513 2J

10 [D-PenZ, L-Pen5]Eb 2720±50 2.50:!: 0.03 1088 2J

11 [D·Penz, D-Pen5]Eb 6930±124 2.19 ±0.03 3164 2J

a) Enkephalinamide. b) Enkephalin.

In brief, we propose that an accessory subsite, possibly of cationic nature,

may bind bath the supplementary aromatic ring of naltrindole and the terminalcarboxylate in cyclic disulfide enkephalins at the li receptor. This subsite is

presumably of different nature, or differs significantly in its location, at the II.

opiate receptor. Obviously, to arrive at this conclusion we must assume that this

subsite is different from the one which accommodates the aromatic ring of Phe4

for enkephalins.

fi our hypothesis hold, we reasoned that an aryl group added with the

appropriate stereochemistry on the methylene of CyS5 could reproduce the role

played by the supplementary aromatic ring in naltrindole, therefore causingsignificant increase in potency for li receptors for cyclic disulfide enkephalins of

type:la. To do so, we then have to prepare the amino acid p.phenyl-cysteine. p.

Phenyl·cysteine is an amino acid with two stereogenic centers and consequentlypossessing 4 possible stereoisomers. Since L or D a·amino acid configurations

are allowed at position 5 of enkephalins, we have envisioned a synthesis of the

racemates followed by enzymatic resolution. This part of the work is described in

chapters 4 and 5 of the present thesis.

Supplementary references.

62. P. S. Portoghese, H. Nagase, K. E. Maloney-Huss, C. -E. lin, and A E.

Takemori, J. Med. Chem., 1991,34, 1715.

63. J. Hughes, H. W. Kosterlitz, and F. M. Leslie, Br. J. PharmacoL, 1980,68,33.

64. K.-J. Chang, E. Hazum, and P. Cuatrecasas, Trend in Neurosci., 1980, 160.

36

Chapter 3

Structural study of the modeill. selective enkephalin analogN"'Cbz-c[(D)A2Bu-Gly-Phe-Leu]

In the preceding chapter we have described a molecular modeling studywhich aimed at determining the active conformation of cyclic enkephalins at theopiate receptor based on topological similarity with a narcotic alkaloid. Theconformations of the macrocycles were based on structures proposed frominformation available at this time from NMR data and molecular dynamic

simulations of 3.28 and 3.2b1,2 (Fig. 3.1). Notwithstanding the fact that thesepeptides were cyclic, no study in the literature had been focused on determiningthe protons spatial proximities by nuclear Overhauser effect (NOE) of 3.28. ln

this chapter, we describe the preparation of the related compound N"'Cbz­c[(D)A2Bu-Gly-Phe-Leu] 3.1 and the conformational study of this structuralmodel by IH NMR in [2He]DMSO with particular emphasis on 2D NOE data. The

compatibility of the revised macrocycle conformation with the previously proposedbound conformations at the II. opiate receptors is underlined. The putative

"bound" conformation is compared with recently proposed "active" conformationfor morphiceptin. Finally, we report the efforts thllt were made for obtaining

crystal structure of 3.1 by X-Ray diffraction.

3.1 3.28 n=23.2b n=3

Fig. 3.1 Chemical formula of N"'Cbz-c[D-A2bu-Gly-Phe-Leu] 3.1, H-Tyr-c[D­

~bu-Gly-Phe-Leu] 3.28, and H-Tyr-c[D-Orn-Gly-Phe-Leu] 3.2b.

3.1 Preparation of NUCbz-c[(D)A2Bu-Gly-Phe-Leu] 3.1.

The title compound was prepared by the solution phase synthesis. Scheme3.1 depieted the steps required. The C terminal Leu was protected as a methylenef1uorenylmethylene ester (OFm) which is resistant to acidolysis.3 The amino-acidt-Boc-Phe-OH was coupled to H-Leu-OFm using dicyclohexylcarbodiimide (DCC)as a coupling reagent. The t-Boc group was then removed from the elongatedpeptide by acidolysis with anhydrous trifluoroacetic acid (TFA) indicbloromethane (DCM). The procedure of coupling was repeated for t-Boc-Gly­OH with the elongated peptide and the terminal nitrogen of the tripeptide was

deprotected by acidolysis with the conditions mentioned earlier.

The next amino acid NIl,NY-(D)-diaminobutyric acid «D)A2Bu, 3.12) to be

coupled was not commercially available and had to be prepared. Since the Nilproteetive group should be resistant to the condition of removing the NYproteetive group, the carbobenzyloxy (Cbz) group was seleeted for the Nilposition. The t-Boc proteetive group was chosen for the NY position. Scheme 3.2depieted the steps required for the preparation of NIlCbz-NY-t-Boc-(D)A 2Bu from

(D)-Olo. This method is based on the preparation of NIlCbz-NP-t-Boc­diaminopropanoic acid from Asn described by Wald ~.4 Accordingly, the Nil

group of (D)-Gln was first protected with the Cbz group and the terminal amideconverted to an amine with bis(trifluoroacetoxy)iodobenzene as a Hofmann

rearrangement reagent. 6 Finally the N'i group was protected using 2-(t­butoxycarb:::nyl-oximino)-2-phenylacetonitrile (Boe-ON). NIlCbz-NY-t-Boc­(D)A2bu was coupled to the tripeptide 3.8 (Scheme 3.1) using conditions

described previously.

The f1uorenylmethylene ester group of the tetrapeptide 3.9 was cleaved

under mild basic condition (piperidine in dimethylformamide) and the side chainN't·t·Boc group of (D)~Bu freed by acidolysis with anhydrous TFA The

intramolecular peptide bond formation was performed under high dilutionconditions in DMF at O"C using diphenylphosphoryl azide (DPPA) as carboxyl

aetivating reagent.8

38

it-Boc-Leu-OH --... t-Boc-Leu-OFm (3.3)

lÜH-Leu-OFm· TFA (3.4)

t-Boc-Phe-OH------!üi

t-Boc-Phe-Leu-OFm (3.5)

liiH-Phe-Leu-OFm· TFA (3.6)

t-BOC-Gly-OH------!Hi

t-Boc-Gly-Phe-Leu-OFm (3.7)

liiH-Gly-Phe-Leu-OFm· TFA (3.8)

NCl

Cbz-NYt-Boc-(D)A2bu-OH~ Hi(3.12) ~

Cl YN Cbz-N t-Boc-(D)A 2bu-Gly-Phe-Leu-OFm (3.9)

1iv, iiCl

N Cbz-(D)A2bu-Gly-Phe-Leu-OH (3.10)

lVCl

N Cbz-c[(D)A2bu-Gly-Phe-Leu] (3.1)

Seheme 3.1 Reagents andconditions: i, FmOH, DMAP, DCC / THFO"C, 1 h, RT, 1 h; ii, TFA SO% in DCM, S% anisole, RT, O.S h; Hi, NEM orDIPEA, DCC, HOBt / THF, RT, 2-3 h; iv, Piperidine S% in DMF, RT, 3 h;v, 1 mM in DMF, 2 eq. TEA, 2 eq. DPPA, S·C, 12 h.

39

OH

OH

NH-tBoc

•

i.

iii.

Cbz-H-"J'

»

OH

3.11

40

SCheme 3.2. Reagents andconditions: i, 2 eq. NaOH /Water:MeOH1:1, Cbz-Cl; Ü. CeH61(CFaCOz)a / DMF:Water 1:1, pyridine cat.,RT, 1 h ; iii, 1.2 eq. Boc-ON, TEA / Dioxane: Water 1:1, RT, 2 h.

Although the solution phase synthesis is more time consuming than solid

phase peptide synthesis, it is cheaper for large scale preparation. Another

advantage is that the purity can be checked after each step. For instance, in our

case the peptides were purified by silica gel chromatography after each coupling

reaction and ail peptide trifluoroacetates were crystallized after NOt deproteetion.

The final peptide obtained was highly homogeneous as shown by lH NMR, lac

NMR and analytical HPLC. It is worth mentioning that the OFm protective group

facilitated thin layer chromatographic identification of the produets owing to the

very high UV absorption of the fluorenyl group.

3.2 NMR solution study ofNOtCbz-c[(D)AaBu-Gly-Phe-Leu] 3.1.

Fig. 3.2 shows the lH NMR speetrum of compound 3.1 in [aHe]DMSO. The

spectral assignment is given in Table 3.1. For diastereotopic protons the subscript

A designates the lowfield proton while the subscript B designates the upfield

proton. The same rule is applied for diastereotopic methyl groups. Owing to

41

! 1

1 i 1 (1 i:,

1

(I1

1 )~~!1 ,1 ~Iql

~ L~!W" \~i~~L1 ~I\l

~V VJ, , , ,.., ..• .., ... '" .~

Fig. 3.2 500 MHz lH NMR speetra of NaCbz-c[D-A2bu-G1y-Phe-Leu] in[2He]DMSO at 297 K.

Table 3.1. Comparative lH chemical shifts for NaCbz-c[(D)AzBu-Gly-Phe-Leu]

(this study, 500 MHz, [ZHe]DMSO, 297 K) and N'Tyr-c[(D)AzBu­

Gly-Phe-Leu] (ref l, 270 MHz, [zHe]DMSO, 296 K).

3.1 3.2aChemical shifts Chemical shifts

(±0.001 ppm ) (±0.05 ppm)a

(D)A2Bu

NaH 6.916 8.4 b

C"H 4.197 4.45 b

dlHA 1.862 1.7dlHB 1.830 1.6ClHA 3.593 3.4 c

ClHB 2.767 2.7NSH 6.613 6.65

GlyNaH 9.045 9.20C"HA 4.006 4.05C"HB 3.414 3.55

PheNaH 9.079 9.15C"H 4.129 4.10dlHA 3.045 3.10dlHB 2.883 2.80

LeuNaH 6.981 6.90C"H 4.263 4.30OlHA 1.767 1.75OlHB 1.372 1.35

OH 1.473 1.50MeS 0.877 0.90A

MeSB 0.823 0.85

a) Estimated rrom Fig l, rer. 1. b)Important difference is due to the ract that we havean urethane bond instead of an amide. c) This resonance was hidden in the water peakin their case.

42

43

the cleanliness of the speetrum at 500 MHz, all resonances could be analyzed byselective decoupling and spin simulation. Fig. 3.3 provides the protons couplingconstants within each amino aeid. The JNHCH coupling constants are given in

Table 3.2 along with the chemical shift variation of the NH resonances withtemperature (~S/~T).

3.2.1 Similitudes in NMR data between 3.1 and 3.28.

It isgenerally accepted that values below 3 ppb/K for ~/~T are indicative

of NH protons that are not exposed to the solvent 7 and presumably involved inintrarnolecular hydrogen bonds. Accordingly, Leu NH and (D)A,bu NBH should

be involved in intrarnolecular hydrogen bonds as it has been reported withcompound 3.2 (Table 3.2).8 The J;~HCH coupling constants (Table 3.2) and the

chemical shifts (Table 3.1) of 3.1 and 3.28 are also very similar. Therefore it is

reasonable to assume that the two 14 membered rings are of very similarconformations in order to give the very similar speetra in [2HalDMSO. Dy the

sarne token, the exocyclic Tyr in 3.28 or the Cbz protecting group in 3.1 should notcontribute significantly (such as hydrogen bond donor or acceptor) to the 14membered ring conformation. Since no resonance overlap occurs in the speetrumof 3.1, itis an ideal model for studying the macrocycle conformation by 2D NOE.

C"I1 9.12 d'HA 2.59 C!HA

8.37 NH NIl 6.35 C"HA

~14.6 Z·oy -%3.9 ~14.83.21 / 6.03

/9.88 3.85d'HB OHB C"HB3.57

Gly

C"~4'2d'rA-14.1

10.6

d'HB

Leu PheFig. 3.3 Coupling constants between protons of each amino acid of 3.1 obtained

after spin decoupling and spin simulation analysis.

Table 3.2 Comparative values of JNHCH (Hz) and NH 6.8/6.T (Ppb/K) for

NaCbz-c[(D)A%Bu-G1y-Phe-Leu] 3.1 (tbis study), and H-Tyr­

c[(D)A%Bu-G1y-Phe-Leu] 3.2a (ref. 1).

3.1 3.2a 3.2a([%Ha]DMSO) ([%Ha]DMSO) (H%O:[%Ha]DMSO)

(8:2)

J NHCH

A:Bu 8.08 7.93 6.84

G1ya 12.28 12.20 12.50

Phe 6.15 5.40 4.32

Leu 9.35 9.37 9.37

6.8/6.T

A:BuNH 7.7b 4.8 6.5

G1y 4.8 3.8 5.8

Phe 5.3 4.7 6.5

Leu 0.3 0.6 6.3A:BuN8H 2.2 1.5 2.8

a) For the sake of comparison the sum of JNHCHA and JNHCHB is given. b) Thechemical shift variation with tempearture in this case can be affected by the increase rateof exchange at higher temperature with the small peak at 6.40 ppm due to the minorcis-urethane NH.

3.2.2 NOE data, relative mobility and intemuclei distances.

Table 3.3 gives the intensities of observed cross-peaks (Ill) for interresidue

NOEs while Tables 3.4 and 3.5 provide the intensitics of observed cross-peaks for

intra-residue contacts for each amino aeid. Both negative and positive NOEs were

observed for 3.1 under the given experimental conditions.g Negative NOEs occurwhen the factor lalte >>1 (lal is the frequency and te is the correlation time) yielding

a maximum value of -1 (-100%) for 11!0 assuming sole relaxation by magnetic dipole

interaction. Il These conditions are usually encountered for macromolecules suchas proteins where the slow intemal motion increases te (te >10 -8 S for lal > 100

MHz).!2 Interestingly, in our case all the NOEs involving the protons attached to

44

•45

Table 3.3 Observed NOEs and corresponding interproton distances forinterresidue contacts for NUCbz-c[(D)A2Bu-G1y-Phe-Leu).

spin i

~~+l

PheNH~BuN8H

CH;NHi+ l

~BuCXH

G1yCXHA

G1yC"HB

PheCXHLeuCXH

LeuNH

spin j ~j'

Leu !\TH -5Leu NH -4

G1yNH -25

Phe NH -17

PheNH NS·

LeuNH NS·~BuN8H NS·

PheCIIHB -3

2.843.09

2.18

2.31

>3.3

>3.3>3.3

3.09

rij dyn

(±stdv)e

2.74±0.302.87±0.40

2.53 ±0.282.45±0.17

3.42±0.24

3.67±0.083.28±0.47

2.65±0.29

~ dyn(±stdV)d

7.8±5.06.9±5.3

1l.7±4.9

13.2±4.5

1.8± 1.0

1.1±0.23.5±3.4

9.5±5.4

a) Relative amplitude of the cross-peak. b) Calculated according to rlj-« 1.79) e XSO/~j)1/8 where SO was the average ~j for two geminal negative NOEs in the 2Dspectrum. c) Average distance calculated during the molecular dynamic simulation; theaverage is the mean of 400 values; stdv is the usual standard deviation. d) Expected ~~

obtained from the mean of 400 ~j calculated during the molecular dynamic simulation;each ~j was calculated according to ~j dyn~ (1.79)8 X SO/(rijdyn)8. e) Not significant.

the atorns forrning the 14 membered ring were negative, therefore indicating that

the macrocycle is quite rigid.

On the other hand, positive NOEs are observed in the extreme narrowingcondition (6)te < <1) where a maximum value of + 0.5 (+ 50%) for " is predicted

based on mathematical treatment assuming sole relaxation by magnetic dipole

interactions. la These conditions are usually encountered with small molecules innon-viscous solvents 14 where rapid motion gives rise to short correlation times (te

<10 -8 S for 6> > 100 MHz). Interestingiy, in the case of 3.1, positive NOEs were

observed within Leu and Phe side chain protons and in interactions involving theexocyclic (D)~bu NH. These protons are presumably more free to internai

rotation and consequently have shorter correlation times than the ones attached

to the atorns forrning the 14 membered ring.

Table 3.4 Observed NOEs and corresponding interproton distances for vicinal

contacts for NaCbz-c[(D)A2Bu·G1y-Phe-Leu].

46

spin i

~BuNH

~BuC"H

~BucPH2

~BucPH2

~BuOHA

~BuOHa

G1yNHG1yNH

PheNHPheC"HPheC"H

LeuNHLeuC"HLeuC"HLeu cPHALeuCllHaLeu OHLeu OH

spin j ~j'

~BuC"H -3

~BucPH2 -41~BuOHA ·141~BuOHa ·61~BuN8H ·12~BuN8H ·16

G1yC"HA NS·G1yC"Ha ·20

PheC"H ·10PhecPHA ·8PhecPHa NS·

LeuC"H ·2LeucPHA NSLeu CllHa .h

Leu OH .h

Leu OH .i

Leu Me8A +37

Leu Me8a +37

3.092.95

2.462.34

>3.32.25

2.532.63>3.3

3.31

2.17r

2.17r

rij dyn

(±stdv)c

2.88±0.16

2.78±0.24

2.73±0.252.81±0.172.41±0.19

2.89±0.112.50±0.133.07±0.07

2.98±0.072.49±0.133.05±0.072.97 ±0.07

2.63±0.24

2.27.2.50 1

2.27.2.50 1

~j dyn

(±stdv) d

5.0±2.920.5±6.0 j

18.7±6.0 j

19.5±6.7 j

6.9±4.5

7.8±4.95.9±3.014.8±4.8

4.6± 1.211.4±3.43.19±0.4

3.S±0.61l.8±3.63.3±0.44.0± 0.4 k

11.9±4.9 k

20-40 k,1

20-40 k,1

a) b) c) d) e) see Footnote Table 3.3 f) Calculated according to rijm«1.79) 6 X 120/~j)176where 120 was the amplitude of the cross-peak between the geminal protons of Leu inthe 2D spectrum g) The fact that it involved two strongly coupled nuclei (second order)may render unpredictable the observed NOE (see ref Bc p. 206) h) Could not bedetermined owing to the presence of an intense J cross-peak i) The putative signal waslocated too close to the diagonal cross-peak and was not resolved j) Evaluatedconsidering the sum of contribution involving the two protons k) Calculated accordingto 1... (1.79)8 X 120/(r\j)6 1) ln staggered methyl conformation there is 2 contacts at2.S0A white in the eclipsed methyl conformation there is one short contact at 2.27À andtwo contacts at 2.9À; the values for each conformations are given.

•47

Table 3.5 Observed NOEs and corresponding interproton distances forintraresidue contacts (not vicinal) for NaCbz-c[(D)A2Bu-G1y-Phe-Leu]

spin i

~BuNH

~BuNH

~BuNH

~BuOXH

~BuOXH

~BuOXH

~BuCllH2

PheNHPheNH

PheNHPheOXHPhedlHAPhedlHBLeuNHLeuNH

LeuNHLeuNH

LeuNH

LeuOXHLeuOXH

LeuOXHLeu dlHALeuCllHALeu dlHBLeu dlHB

spinj ~.

~BuCllH2 -5~BuOHA -17~BuOHB NS·~BuOHA -2~BuOHB -3~BuN6H NS·~BuN6H -91

PheCllHA NS·PheCllHB -15

PheAr +6PheAr +29Phe Ar +33

PheAr +35

Leu CIlHA NS·Leu CIlHB NS·

Leu OH NS·Leu Me6A NS·

Leu Me6B NS·

Leu OH +5Leu Me6A +9

Leu Me6B +20Leu Me6A +17Leu Me6B +17

Leu Me6A +14

Leu Me6B NS·

2.842.32>3.33.31

3.09>3.32.57

>3.32.37

2.94'2.27'2.22'

2.22'>3.5>3.5

>35>3.5

>3.5

3.04'2.75'

2.41'

2.48'2.48'

2.56'>3.5

r ij dyn

(±stdv)c

2.88±0.163.63±1.064.31±0.38

3.80±0.203.66±0.844.07 ±0.4

3.7±0.112.64 ±0.22

3.74,2.88 m

2.59,2.52 m

2.61,454 m

4.53,4.9 m

3.76,5.0 m

3.094.46

2.392.50

2.50

2.503.81

~j dJn

(±stdv)d

6.5±6.74.5±5.40.5±0.70.9±0.24.1±5.40.7±0.96.7±4.41.0±0.178.7±3.9

10.5±3.7 n

13.9±8.1 n

15.3±5.7 n

18.7±6.2 n

1.4,6.9 k

13.1,15.4 k

12.5,0.4 k

0.4,0.3 k

1.4,0.3 k

4.5 k

0.4 k

21.1 k

16.1 k

16.1 k

16.1 k

1.4 k

a) b) c) d) e) f) k) see footnote Table 3.4 m) For rigid conformations assuming g-' andIg+ side chain conformation respectively n) Evaluated considering the sum ofcontribution of 2.6-Aryl protons.

Within the linear dependency of cross-peak intensities (~) with the mixingtime (lmix)' the cross-peak intensities are proportional to 1'h/6 On the other hand,l'JIJ is proportional to rifG (1' is the intemuclei distance) assuming that mechanism

of relaxation other than magnetic dipole are constant within the molecule.lG It istherefo1'e possible to evaluate distance between nuclei given that ~J is measuredfor a known riJ' Obviously in our case, calibration had to be performed separatelyfor positive and negative NOEs. In order to check the relationship of ~J with r-Gwe

have estimated the values for sorne fairly weil known intemuclei distances for bothpositive and negative observed NOEs and these are mentioned now.

1) Geminal diastereotopic methylene protons experiencing fmt ordercoupling served usually as a calibration for estimation of intemuclei distancessince they are geometrically fixed at a distance of 1.79Â. For the positive NOEsthis happened for the methylene protons of G1y and for the geminals protons ono of (D)~Bu. Relative amplitudes of the cross-peaks were similar in both cases

within the limit of experimental error and the average amplitude of these signals

was used for calibration.

2) ln Table 3.3 it can be seen that the C~~+l NOEs were not observed

for G1y caH and Phe NH, Phe caH and Leu NH and also between Leu caH and(D)AJBu NaH. Since the maximum distance between these nuclei is ca. 3.6 A. this

flXed the lower limit that our relation ~J vs r-Gmust cover. This clearly states that

ail the NOE signals that were detected should involve intemuclei distance shorter

than 3.6 Â. In order to respect these limiting conditions, the relative amplitudes of

the cross-peaks were used instead of the relative peak volume.

3) The Leu coupling constant J NHen is quite large (9.35 Hz) which suggests

a preferred tram orientation of the respective protons. Indeed, a very week NOEwas observed (Table 3.4) and accordingly using the previous calibration we

calculated an intemuclei distance of 3.3 A, just at the border of the lower limit

mentioned eartier and in agreement with a relatively trans orientation.

4) For positive NOEs, the cross-peak between the geminal diastereotopic

protons of the methylene of Leu served as a calibration. In absolute value, this

cross-peak was significantly more intense than the cross-peaks observed for G1yand ~Bu geminal diastereotopic protons. Even though, usually more intense

48

49

NOE are expected in the domain <alte> > 1 compared to the extreme narrowingcondition (Calte < <1) (vide supra), it should be pointed out that NOEs are stronglyreduced in the zone where Calte is close to 19 which presumably is the case for the

protons within the macrocycle.

5) It is difficuit to verify the relation lij vs r-S for the positive NOEs since

they involved protons in very high motion such as in the constantly flipping phenyl

ring or on the freely rotating methyl groups. A relatively good agreement wasnevertheless obtained between the vaiues observed and the vaiues calculated for

the methyl groups of leucine using distances measured with flXed staggered methylgroup (vide infra).

3.2.3 Hydrogen-bond acceptors and consequences on the macrocycleconformation.

Kessler .tl.iù proposed that the intramolecular hydrogen bond acceptor wasGlYS CO for Leu6 NH (y tum) and (D)Om~ CO was the acceptor for (D)OrnZ

N~H (Ca) in the case of 3.2b.~ Similarly, Mammi â..iI. suggested that the

intramolecular hydrogen bond acceptor was GlYS CO for Leu NH (y turn) and(D)A~bu~ CO was the acceptor for (D)~bu~ WH (Cr) in the case of 3.28.1 While

the variation of NH chemicai shift with temperature is consistent with the

assignment of NH donors (vide supra), our 2D NOESY experiments suggest

different hydrogen-bond acceptors. Two significants NH NH NOEs wereobserved. One 17 is between (D)~bu~ N~H and Leu6 NH and could be compatible

with the previously proposed y tum centered on Phe4• However, a slightly more

intense NOE is observed between Phe4 NH and Leu6 NH which rule out theputative y tum centered on Phe4• This last observation is more compatible withthe presence of a P tum involving a hydrogen bond between Leu6 NH and

(D)A~bu~ CO while still keeping (D)~bu~ N~H and Leu6 NH in a close

environment.

Four possible types of p-tum are usuaily encountered in crystal structure of

peptides and proteins, Venkatachalam18 had classified them as type l, l', II and II',While type 1 is ailowed for any L-residues in positions 2 and 3 of the P-turn, 19 type

l' is the mirror image conformation and should he observed only with two D­residues or in combination with Gly. On the other hand, type II p·tum is mainly

• observed V/hen the residue in position 2 is a L-arnino-acid and the position 3 is

occupied by a D-residue or a Gly. Conversely, the mirror image type II' is mainly

observed when position 2 is occupied by a D-residue or a Gly and when position 3is taken by a L-residue. On this basis, Ptum type 1 and II' (since we have a Gly in

position 2 of the P tum) can be envisioned in order to respect the observed NH

NH contact. We finally favoured the Ptum type II' since it permits explanation of

other observed NOEs. 1) The Phe NH should be oriented in such a way as topermit short distance with one of the diastereotopic Gly caH's (Gly caHA)

according to the the strong CHi N1I;+1 NOE observed (Table 3.3). 2) Phe CPHB

(which is trans to Phe caH) should be in the proximity of Phe NH (Table 3.4). Thepresence of a Ptum centered on GlyS Phe4 is further supported by the fact !hat no

NOE is observed between Leu NH and Phe caH corresponding to a quite longdistance ( ca > 3.3Â). Table 3.6 summarized the reasons for the preferred P tum

type II' centered on GlysPhe4•

Table 3.6 Compatibility of the divers P tum type centered on Gly3 Phe4 toward

chirotopicity and observed NOEs.

Type chirotopicity GlycaHAPhe NH Phe dHB Phe NH •

1 yes no yes

II no yes no

l' no no no

II' yes yes yes

a) Phe C~HB has a large coupling constant with Phe C«H.

Assuming the P tum type II' centered on Gly3Phe4, several supplementary

constraints have to be respected in addition to the cyclic nature of the molecule.1) There must be an hydrogen bond acceptor for (D)~bu NBH. 2) There is a

very strong NOE between (D)~bu caH and Gly NH (Table 3.2). 3) (D)~bu2

NaH has a large and a small coupling constant with (D)~bu OH protons which

are very diastereotopic (Fig 3.2 and Table 3.1). 4) Large and small coupling

constants are observed in the endocyclic methylene side chain of (D)~bu (Fig

3.2) and this should reflect at most few interconverting side chain conformation at

this level.7 5) The dihedral angle Leu NHCH must be relatively near 180" owing

50

to the very large JNHCH observed (9.35 Hz). Let us now see how it is possible to

arrive at an acceptable model that takes into account ail these observations.

A1though there are five CO hydrogen bond acceptors in 3.1, only two canpotentially be hydrogen-bonded to (D)A ,bu NBH. Since we assume that Leu NH

and (D)Azbu NBH are not exposed to the solvent, this implies that CO Phe and

CO Leu are exposed to the solvent (owing to the trans planar geometry of the

peptide bonâ). Moreover, the earbonyl group of Cbz is a poor acceptor and

presumably is not involved in a permanent hydrogen bond with hydrogen-donorscoming from the macrocycle (vùiesupra). Therefore this leaves CO (D)~bu and

CO G1y as putative hydrogen-bond acceptor for (D)A,bu NBH. The first

possibility involves a three centers two hydrogen bonds while the second supposestwo intercalated ~-turns (centered respeetively on G1ySPhe4 and Phe4Leu6). Based

on ball-and-stick models, both con~truetions were geometrically feasible while stillrespeeting the proximity between {D)A,bu caH and G1y NH (Table 3.3). Table

3.7 shows the value of 41.' dihedral anglesZD that are compatible with twoembedded ~·turns consisting of type n' intercalated with type 1. Moreover in both

cases the dihedral angle Leu NHcaH could be kept close to 180" in agreement

with the large coupling constants observed (Table 3.2). While most of theconstraints were satisfied with these models, the behavior of the (D)Azbu side

chain in relation to the observed coupling constant was still unclear. We then

relied on computer molecular modeling and molecular dynamic at this point.

Table 3.7 41.' dihedral angles of idealized ~ turns types showing the possibility of

forming two intercalated ~ n', ~ 1 tum centered on G1y3 Phe4 and Phe4

LeuS respectively.

tum type

51

n'

1

60" -120" -80"

-60"

0"

-30" -90" 0"

3.2.4 Computer molecular modeling and molecular dynamic simulation ofcyclopeptide conformation.

A probable macrocycle conformation was built and energy minimized usingMAXINMIN2 of SYBYLZl as described in the experimental section. This

conformation (Conf!) is represented in Fig. 3.4 and consisted of the twointercalated P-turns mentioned earlier. The Cbz group was replaced by an acetyl

and the Phe and Leu side chains were oriented according to the coupling constant

values and intra-residue NOEs (vide infra). This structure was submitted to !OO psof molecular dynamic simulation (MOS) in order to evaluate the relative flexibilityand stability of this model and to obtain other representative conformations thatcould account for the observed coupling constants and NOEs. Fig. 3.4 shows also

three often occurring structures that were obse,ved during the MDS. Theseconformations have been energy minimized and their respective dihedral anglesare given in Table 3.8. Conf! and ConO are characterized both by the system oftwo embedded P-turns mentioned earlier but differ in their (D)AzBu side chain

conformation. Cont2 and Conf4 show both the envisioned three centers twohydrogen bonds and have the (D)AzBu side chain conformation corresponding to

Conf! and ConO respectively. Several other conformations that have brief

duration were also observed during the MDS but when these conformation wereenergy minimized none of them had conformational energy below that of Conf!and their energies was usually greater than that of Cont2. It is worth mentioningthat the conformations with two intercalated p·turns (Conf!, ConO) had lower

conformational energy than their counterpart with the system of three-centers two

hydrogen bonds (Cont2, Conf4). This could be partly due to the fact that an extra

hydrogen bond appeared in the formers upon energy minimization (Conf!:(D)AzBu NH -> OC Leu, ConO: Gly NH -> OC Ac).

Hydrogen bonds were constantly breaking and forming during the

simulation but as an estimation of their presence or absence we recorded thepercentage of time the distance between the acceptor atom and the donor atom

was below 3.SA. Table 3.9 gives';"1 result for the possible hydrogen bonds andthe related 66/6.T coefficient. The hydrogen bond involving Leu NH and CO

(D)AzBu was observed about 50 % of the time in agreement with the low thermal

coefficient of Leu NH and reinforced the hypothesis of the p-tum type II' relativeto the y tum structure. It seems however that the acceptor for (D)AzBu NH is

52

53

Fig. 3.4 Representative conformations of N"Ac-c[D-~bu·Gly-Phe-LeuJ

observed during the molecular dynamic simulation and that couldaccount for the lH NMR data observed. Labe!ing of protonscorrespond to speetrum assignment. Hydrogen bonds are representedwith dashed !ines. From top to bottom: ConfI, Cont2, ConO, andConf4.

54

Table 3.8 Dihedral angles e) of conformations of NaAc-c[(D)A2Bu-Gly-Phe-Leu]

observed during the molecular dynamic simulation that are compatible

with observed lH NMR data of 3.1.

Tossional angle Conf 1 Conf 2 Conf 3 Conf

III 166 163 74 82

.- -164 156 -87 -73(D)A2bu 2 Xl -42 -39 178 174

X2 c58 -60 78 75

X3 112 -96 76 -173

GIf III 66 65 53 71

lV -138 -108 -115 -127

III -58 -57 -67 -71

Phe4 lV -40 -50 -48 -51Xl -64 -62 -59 -58

X2 105 104 105 103

III -109 -118 ·123 -91Leu6 • 53 ·60 53 -52

Xl -52 -55 -56 -53

X2 179 175 172 -179

Eneru.. 5.22 7.75 6.42 7.08(Kca mol)

a). Evaluated using Tripos Force-Field including electrostatic contributions weighted bya dielectric constant of 35.

most of the time (D)~BuCO rather than Gly CO, favouring therefore the three

centers two hydrogen bonds rather than the two intercalated ~-turns on a dynamic

point of view. (N.B. the reverse would have been predieted based solely on static

energy calculation). Two other hydrogen bonds were observed to a minor extent

and interestingly their percentage of occurrence correlates with the magnitude ofthe detennined experimental coefficient l!J3/ t::.T.

55

Table 3.9 Presence of hydrogen bonds during the molecular dynamic simulationand related experimental data.

Donor Acceptor % of occurencea t:>.S/ t:>.T (ppb/K)

LeusNH CO (D)A2bu2 48 0.3

(D)A2bu2 N8H COGlyS 14 2.2

(D)A2bu 2 NlSH CO (D)A2bu2 85 2.2

(D)A2bu2 NH CO Leus 12 7.7Gly3NH CO Ac (Cbz) 20 4.8

a) Relative numbers of conformation where the distance acceptor donnor was below 3.6A during the molecular dynamic simulation; estimated from 400 conformations

The information obtained from the MDS were put in relation with ourexperimental data by cslculating at regular interval of 0.25 ps the observable data.The intemuclei rij distance and the corresponding expected Iij (according to the

experimental calibration) were calculated. Similarly at each increment, the Jcoupling values were calculated using a Karplus-Iike equation.22-24 The averagevalues of rij., Iij and J were computed along with their mean standard deviations.

These values are given in Table 3.3, 3.4, 3.5, and 3.10. These data are now

analyzed more in detail concerning the interresidue interactions and the specifie

conformation of each amino acid.

3.2.5 Interresidue interactions.

The interresidue contacts calculated from the MDS (Table 3.3) generally

reproduced weil the observed data within the standard deviation (which in thiscase should more properly be called the mean fluctuation). With the currentmodel we however predict a smaller Iij than the value observed for the contact

A:Bu C"H Gly NH. On the contrary, the contact Leu NH Phe cJIHB seems to he

overestimated with our mOI':1 but this may be due to unfavorable correlation time

in this region of the molecule that affected the experimental data (vide infra).

Table 3.10 ObseiVed coupling constants and corresponding calculated couplingconstant during molecular dynamic simulation for NCXAc-c[(D)A,Bu-

Gly-Phe-Leuj.

56

dihedral angle

A,BuNHCCXHA,Bu CCXHOlHAA,Bu CCXHOlHBA,BuOlHAOHAA,BuOlHAOHBA,Bu OlHBOHAA,Bu dlHBOHBA,Bu OHAN!HA,BuOHBN!HGlyNHCCXHAGlyNHCCXHBPheNHCCXHPheCCXHdlHAPheCCXHdlHBLeuNHCCXHLeuCCXHOlHALeuCCXHOlHBLeuCPHAOHLeuCPHBOH

8.089.123.212.596.079.883.578.373.856.106.10

6.154.0510.60

9.353.4511.09.993.81

6.79±3.03c

7.27±3.62d

2.98±1.72d

3.25±1.49d7.29±4.11d

8.84±3.61d

3.29± 1.47d

5.84±3.54c

5.32±3.41c

4.57±2.73c

7.36±1.94c

5.93±3.01c

3.33± 1.54d

11.76± 1.01d

9.22±1.84c

3.48±1.36d

11.85 ±0.75d

8.10± 1.5()e5.19± 1.4()e

-140±50-60±30, 180±3060±30, -60±30-60±30,60±3060±30, 180±30180±30,60±30-60±30,60±30180±100-60± 100103±6O2±50

-115±75-62± 15-172±15

180±35-60±15-175±15180± 15r

-54± 151

a) J values were calculated for 400 conformations during the simulation from themeasured dihedral angle according to the equation referred in the note after which theaverage was estimated bl Average dihedral angle values during the molecular dynamicsimulation cl J. 9.4 cos' 8 -1.1 cos8+ 0.4. dl J. 11.0 cos'8 -1.4 cos8 + 1.6 sin' 8 elJ • 4.5 cos' 8 -0.5 cos 8 + 4 fl The diheral angle was 600 during a short period. gl Thediheral angle was 1800 during a short period.

•57

3.2.6 Conformation of amino acids

~Bu

Since this side chain is involved in the formation of the 14 membered ring,

we took particular attention to extract ail the coupling constant by selective

decoupling and spin simulation (Fig 3.3). Assuming solely that large coupling

constants were associated with preferred trans orientation for the protons and

that small coupling constant were associated with gauche orientation, it was not

geometrically feasible to arrive at a single solution within the 14 membered ring.

Another ambiguity was also the presence of :l coupling constant value located inbetween (6.1 Hz for cPHA-OHB). The MOS solved in pan this problem in

showing the existence of two stable side chain conformations for ~Bu. Expressed

according to the principal dihedral angle these are designated gogo and tg+ Cg- =

-60", t =180", g+ = 60"). The former was found in Confl and Conf2 while the latter

in Conf3 and Conf4 (Fig 3.4). Each of the two side chain conformations were

observed about half of the time during the MOS which permitted relatively goodreproduction of the observed coupling constants (Table 3.10) except for OHA­

N6H and OHB-N6H. The NOEs calculated during the molecular dynamic

calculation were generally in agreement with the observed data except between~Bu NCXH and OHAwhere the observed value was significantly greater than the

calculated value. These last discordances may indicate that Confl (Fig. 3.4) may

have a greater contribution in the real dynamic process.

GifThe most important information on Gly conformation cornes from the fact

that the geminal protons are separated by 0.6 ppm and that the two JNHCH are

equal and estimated to be 6.10 Hz (Fig 3.3). The Newman projections shown in

Fig 3.5 represent the four possible rotamers that could give rise to equivalentvalues of JNHCH for two diastereotopic protons related by a valence angle of 120".

Ifwe consider however the absolute values of the calculated J values,22 rotamers

A and C should be ruled out. Moreover, the fact that a strong vicinal NOE wasobserved between Gly NH and Oly caHB and that no NOE was detected between

Gly NH and caHA (Table 3.4) confirmed the presence of rutamers B or D. The B

rotamer was mainly observed during the MOS as exhibited by the higher ~J

calculated between Gly NH and caHB compared to the one calculated between

Gly NH and caHA. The calculated values of J NHCH during the MDS were

dissimilar, although they tend to approach the value of 6.1 Hz. The involvementof GlyS in a ~-tum type II' is further supported by the faet that the value of the

geminal coupling constant (-14.1Hz) is in close agreement with the value derivedfrom semi-emperical calculations when the lll. IV torsional angles adopt the values60", -12()"26 as required in position two of a ~-tum type II' (Table 3.6). Finally it is

worth mentioning that Gly has been reported to adopt this conformation whenplaced in position 2 of a ~-tum type II' in the X-Ray structure of a cyclopeptide.26

58

9, 9,H H

COR

A

9, =60"9, =300"J9,= 2.2 HzJ 9,= 2.2 Hz

B

9 =145·,9 -25·,-19,= 6.76 HzJ 9,= 7.12 Hz

11

9'rrt)'H~H

COR

c

9, =240"9,= 120"J9,=3.3Hz

J 9,= 3.3 Hz

9,

9,

D

9 =335·,9,=215·

J9,=7.12HzJ9,= 7.61 Hz

Fig. 3.5 Newman projection along the N-CX bond for Glycine showing the fourrotamers that could give rise to two almost equivalent JNHCH '

Phe4

High diastereotopicity is also experienced by the methylene protons of Phein bath their chemical shifts, coupling constants and NOEs. The large couplingconstant caHdlHs corresponds to a preferred trans relationship between theprotons while the small coupling constant caHdlHA correspond to a preferred

gauche relationship (Fig. 3.3). Accordingly, this suggests g- or t side chainconformation. This is corroborated by NOE measurements since an intensecross-peak was deteeted between caH and dlHA but none was observed between

caH and CIIHs (Table 3.4). While the coupling constant values do not permit

discrimination between g- or t , the g- side chain conformation is more probabledue to the NOE observed between Phe NU and the aryl protons (Table 3.5)

59

whose effects are not expected with a t side chain conformation. Consideringthese observations, the Phe Xl dihedral angle was set initially at ·60" for the MDS.

No interconversion to the t rotamer was noticed during the simulation but thephenyl ring was constantly flipping. This led to a calculated coupling constantCXH.ctlHA slightly under the observed value and a larger calculated couplingconstant for CXH-ctlHB which may indicate that other rotamers exist in the real

dynarnic process.

The Phe JNHCH value warrants further discussion. In our model, the

dihedral angle in this case corresponds to about -140" and accordingly thecalculated coupling constant Phe NHCXH during the MDS is in good agreementwith the observed datum. An interesting observation cornes from Mammi~ 1

who noticed that this coupling constant was the most affected upon increasingwater content (Table 3.2). In parallel, they observed that the Leu NH thermalparameter .6./i/.6.T became large when the water ratio was increased while the oneassociatedwith (D)A2bu NH remained below 3 ppb/K. This observation can bebest taken into account with our model of two intercalated ~-turns where Gly COacts as a hydrogen-bond acceptor for (D)~bu NH. If the p·turn involving Leu

NH get disrupted, this gives the opportunity to Gly CO to turn more toward(D)A2bu NH, reinforcing the ~-turn type 1 around PhecLeu6• Since Phe NH is

antiperiplanar to Gly CO, this will reduce by the same token the dihedral anglePhe NHCH which may account for the observed smaller value of Phe J NHCH in

aqu~ous medium (Table 3.2).

Leu&

High diastereotopicity is also observed within the Leu side chain. Themethylene protons are separated by 0.4 ppm such that ctlHA is observed at the

position where methine protons usually occur (see chapter 6). On the other hand,OH shows up at quite highfield (1.5 vs 1.7 ppm). The analysis of the large andsmall coupling constants (Fig. 3.3) suggests either got or tg+ conformation for theside chain principal dihedral angles as shown in Fig. 3.6 (X1,X2.=-60",180" or 180",

60"). These two possible side chain conformations are encountered the most

often in crystal structure of peptides and proteins containing leucine.27 The two

possible conformations are further confirmed by the presence of NOEs betweenC"H and Me8B' CllHA with both methyls, and CllHB with Me8A (Fig. 3.6 and Table

3.5).

Owing to the very large and very small coupling constants obscrved forOXH-CIlHB and OXH-CIlHA, we expeeted that essentially ooly one rotamer must

exist in solution. Backbone to side-chain NOEs should permit decision between g'

or t. We expeeted NOE between NH and OH for g't conformation while weexpeeted NOE between NH and CIlHA for tg+conformation. A NOE between NH

and CIlHB is expeeted for both conformations. None of these NOE were observed

60

tg'"

Fig. 3.6 The two possible side chain conformation for Leucine that could give

rise to the observed coupling constants and NOEs. Atom labeling is in

relation with the NMR assignment (N.B atoms identity changes between

the two side chain conformations).

and this lack of information should be attributed to an unfavorable correlationtime at this level such that Calte = 1 and caused the vanishing of NOEs. In the

absence of this information, a tentative assignment was done based on theobservation that the highfield cllHA experienced small coupling constant with C"H

as it was observed for Phe, therefore favouring the g' rotamer. Molecular

mechanic energy calculation of both possible Leu side chain conformations for

different macrocycle conformation gave always the g't as the minimum energy

61

conformation and therefore the latter was u~,.;..i during the MDS. The g- rotamer

around ca-cP bond persisted all the time during the MDS which permittedreproduction of the observed coupling constants (Table 3.10). It is therefore not

required to invoke the presence of other rotamers since fluctuations around the

perfectly staggered conformation is sufficient to account for the observed coupling

constants. The case of rotation around cP-O is different and rotation to the g+rotamer at tbis level was observed during the MDS for a period of about 20 ps. It

occured however too often since the corresponding calculated values of coupling

constants were less dissimilar than the observed one (Table 3.10).

The coupling constant Leu NHCH is quite large and is almost insensitive toincreased water content (Table 3.2). Notably, this value was very weil reproduced

during the molecular dynamic simulation and the relatively small standard

deviation indicated that average fluctuations were small at this level of themolecule and presumably less dependant on exterior environment.

3.3. X-Ray crystallography

Although compound 3.1 is deprived of the essential Tyr residue for opiate

activity, the macrocycle is identical to the active analog 3.20. On the other hand,

the exocyclic carbobenzyloxy (Chz) protecting group could be a close isosteric

mimic of tyrosine (see Fig. 3.1). Compound 3.1 is neutral and accordingly we

expected to obtained crystals suitable for X-Ray diffraction more easily. This

statement was based on the fact that the crystal structure of several uncharged

cyclopentapeptides have been reported previously in the literature28-33 and that

crystallization methods are usually simpler. However, crystallization of 3.1 in pure

organic solvent such as methanol or acetonitrile by slow evaporation or by cooling

gave only very thin needles that were not suitable for X-Ray diffraction study.

Platelets of suitable dimensions were obtained by slow evaporation of a mixture of

acetonitrile and water at 5°C for more than one month. These crystals lose solvent

(presumably water) at ambient temperature and got broken if not preserved at

low temperature or soaked with the mother liquor. A crystal proved to he single

as estimated from the profile shape of the X-ray diffraction lines. Its lattice

parameters were a= 13.S686(6)Â, b=20.644S0Â, c=21.7216(23)Â,ex =105.1396(57), P=91.0701(34), V=97,82S2(69) for a volume of S809.3SÀ3 at

-60"C. A total of 11741 reflections were measured but only 5014 were considered

observed (1 >20(1». This suggested Z=8 in a Pl space group for a calculated

density of 1.261 without solvent and 1.301 with 8 molecules of water. Theexperimental crystal density as determined by the floatation technique (hexane­CC~) provided a value of 1.304 in close agreement with the monohydrate

compound. The structure could however not be solved by direct method owing tothe very large number of independent molecules.

These many independent conformations for a cyclic constraint peptidecould be caused mainly by the presence of the floppy Ncx-Cbz protective groupwhich moreover could undergo cis-trans isomerization around the urethane bond.The two almost energetically equivalent side chain conformations of (D)~Bu

should also contribute in part to the duplication of independent molecules.Efforts should be given to convert the NcxCbz protective group to a p­

bromobenzoyl which will eliminate the rotational freedom at this level and by thesame token introduce a heavier atom which should facilitate X-ray structuresolving in case crystals are suitable for X-rays diffraction study.

3.4 Consequence on opiate receptor binding conformation.

Interesting structural similarities between the NMR deduced conformations

(Fig. 3.4) were observed concerning the flatness of the macrocycle (r.m.s = 0.420)and the respective orientation of the Phe side chain with the macrocycle ORN3

used to propose "active" conformation in Chapter 2 (fit #5, Fig 6). Since it was

not possible to achieve sterically allowed fit between ORN3 and PEO in the foldedmodel (d. Tyr-Phe =7.7Â), the extended model was favoured (d. Tyr-Phe = 10.6

Â) for an eventual fit using the NMR deduced macrocycle conformations. The

missing tyrosines were then added to the structures shown in Fig. 3.4 and flexiblefits of the pharmacophoric elements performed with PEO in the respective

"extended" conformation using similar methodology. Confl and Conf2 did not

give rise to interesting superposition owing to the unfavorable relative orientation

of the exocyclic Tyr relative to the macrocyle. On the other hand, Conf3 andConf4 gave superpositions with striking similitudes to the previously proposed

model. The result of the multifit is presented in Fig. 3.7 when Conf3 was used as a

basic macrocycle structure. Corresponding dihedral angle values are given in

Table 3.11. The fit shows all the good attributes mentioned in Chapter 2 for a

probable opiate receptor bound conformation. 1) The whole of the molecules

62

•63

occupy the sarne volume of the space (high CSO). 2) A low fitting angle (F angle

= 35°) between the superimposed aromatic rings (defined by QI of Phe, the Arylcentroid, and C21 of PEO). 3) The alkaloid is in a minimum energy conformationwhere the intrarnolecular hydrogen bond C19-OH -> O-C6 is maintained. 4)

Moreover, this time, the fit is achieved using a low energy macrocycle

conformation for 3.28 which is supported by NOE measurement and withouthaving to invoke a reverse Vtum (ca) around Phe4 as previously.

Fig. 3.7 Superposition of 3.28 using the conformation Conf3 deduced from

NMR data with PEO leading to maximum common space occupation.

An "active" conformation was proposed recentiy for morphiceptin, H-Tyr­Pro-Phe-Pro-NH 2' a Il selective agonist.M ln our opinion, this gives further

support to the model elaborated so far by us. As mentioned in the Introduction,this derivative of ~-casomorphin is characterized by a L-proline in position 2

whereas D-proline derivatives are inactive.S6,S6 Based on IH NMR study

performed by Goodman and Mierke,S7 Mierke ~,S6 and and Yamazaki ~S9

on several analogs of morphiceptin, Yarnazaki âill.S4 concluded that the presence

of a cis amide bond between Tyrl_Pro2 is associated with bioactivity. This

reconciles, by the sarne token, the apparent discordance with the SAR of

enkephalins, deItorphins, and dermorphin. Secondly, they searched a common

spatial arrangement of the pharmacophoric elements of the active analogs Tyr­Pro-NMePhe-(D,L)Pro-NH 2' Tyr-Pro-Phe-(D or L)-l?ro-NH2, (which were

exclusive ta the common spatial arrangement of phar.macophoric groups of the. inactive analogs Tyr-Pro-NMe-D-Phe-(D or L)-Pro-NH 2 which was shown by

Table 3.11Dihedral angles (") of model structure ConO and correspondingdihedral angle for H-Tyr-c[(D)A2Bu-G1y-Phe-Leu] in the putative

opiate bound receptor conformation (depicted in Fig. 3.5).

Torsional angle ConO Bound Conf.

Ij)

Tyrl ljI -141

Xl -180

X2 -138

Ij) 74 95

ljI -87 -81

(D)A2bu2 Xl 178 -170

X2 78 80

Xa 76 74

G1y3 <il 53 35

ljI -115 -79

Ij) -67 -93

Phe4 ljI -48 -54

Xl -59 -55X2 105 90

Ij) -123 -124

Leu6 1V 53 55Xl -56 -57

X2 172 172

64

•65

Fig. 3.8 Rigid body fit of 3.28 in the putative II. bound conformation as

proposed by us with Tyr-Pro-NMe-Phe-D-Pro-NH in the conformation

proposed by Yarnazaki ili134

Yarnazaki~34 to be inactive. Relatively few conformation could achieve these

prerequisites and interestingly, the most probable conformation retained by

Yarnazakilli134 shows astonishing pharmacophoric spatial arrangement with our

current mode!. Fig 3.8 presents a rigid body fit realized between 3.28 in theputative 11.' conformation proposed by us and the conformation of H-Tyr-Pro­

NMePhe-D-Pro-NH 2 as deduced by Yamazaki lli134 using active analog approach

(built using Table VI entry 1 of ref 34). It should be mentioned that not only the

pharmacophoric elements present good congruency but the whole of the

molecules occupy a similar environment (high CSO).

The preceeding superposition between the morphiceptin analog H-Tyr­Pro-NMePhe-D-Pro-NH 2 and the enkephalin derivative 3.28 supposed that the

aromatic cycles of the two phenylalanines in different sequential positions (Phe3 or

Phe4) interact with the same subsite in the receptor. This assumption may receivesevere criticism f,'nce it is weil known that substitution of Phe4 with a P·N02 group

causes increase potency for enkephalin derivatives at both II. and Il receptors40

while the converse is true for morphiceptin analogs.41 This does not necessarily

argue in favor of the two subsites hypothesis since the modality of binding for the

two sequence positions for Phe can be different enough in each case that the

electronic effeets might be reversed. Indeed the fitting angle (F angle) between

the two phenylalanines aromatic ring in Fig. 3.8 is 58° which basically make the paraposition in Phe4becoming the meta position in Phes.

3.5 Conclusion and summary

Despite the not very favorable correlation times for accurate NOE

measurements with 3.1, it has been possible to analyze in detail the conformationof this compound in [2HulDMSO. The observed intemuclei distances, coupling

constants and assignment of non exposed NHs are compatible with a structurecharacterized by a ~ n' tum centered on GlyllPhe4 involving a hydrogen bond

between Leu6 NH -> OC ~BU2. In addition, a hydrogen bond involving HN

~BU2 which, depending on dynamic fluctuation, has for acceptor OC ~BU2 (three

centers, 2 hydrogen bonds) or OC GlyS (two intercalated ~-tums), is present. The

endocyclic side chain of ~BU2 adopts two conformations with approximately the

same residence time. The values possible for the side chain dihedral angles Xl,X%

are -60",-60" and 180",+60". The calculated data derived from an unconstrained

molecular dynamic simulation were shown to reproduce quite well the measureddata. Amongst the 4 representative macrocycle conformations found during the

dynamic simultion, two were found to be highly compatible with a previouslyproposed bound conformation at the Il. opiate receptor. Furthermore, good

pharmacophoric congruency as much as common steiic volume occupation were

shown between this model and a recently proposed active conformation for

morphiceptin. Unfortunately, crystallograpbic structure could not be obtained

regarding compound 3.1, but efforts should not be stopped since no X-ray

stmcture of cyclic enkepbalin bas been reported yet.42

3.6. Experimental section

3.6.1 Synthesis

Equipment.- Melting points were recorded on a Gallenkamp capillary apparatus

and are uncorrected. Infrared spectra were recorded on a Perkin-Elmer

instrument and were taken in KBr pellets. lH NMR spectra were recorded either

at 300 or 200 MHz on a Varian XL-300 or XL-200 respectively. lSe NMR were

recorded at 75 MHz on a Varian XL-300. AlI NMR spectra were referenced to theresidual solvent peak (CDCls 1H 7.24 ppm, lSe 77.0 ppll'. [2HulDMSO, 1 H 2.49

66

67

ppm, lSC39.45 ppm); J values are given in Hz. 1H NMR assignments were based

on 2D homoscalar correlated experiment (COSY). Thin layer chromatography

was carried out on aluminium backed Merck silica gel 60, 0.25 mm thickness.

Optical rotations were measured on a JASCO DIP-140 instrument at sodium \ine

wavelength at 20"C in a 1.0 dm cell and the values were integrated over a period of10 s. [a]D values are given in units of 10-1 deg cm2 g-l. Mass spectra were

determined with either a DuPont 21-492B (DP). Department of Chemistry. McGill

University, a HP 5980A (HP). a ZAB 2F HS (Z2F) Biomedical Mass Speetrometry

Unit, McGill University. a ZAB HF (ZH) Biotechnology Institute of Montreal. or

a ZAB 1F (ZlF). Departement de Chimie, Université de Sherbrooke as specified

in the text.

Reagents and Solvents. Ali reagents were commercially available and were used

without further purification. Tetrahydrofuran was dried by distillation from

sodium-benzophenone and dichloromethane by distillation from calcium hydride.

The TLC solvent butanol:acetic acid:water (4:1:5) upper phase (BAW) was used

for polar compounds.

Preparation ofCbz-(D)Gln 3.11- (D)Gln (10 mmol. 1.46 g) was suspended in

water (10 ml) and NaOH lN was added (10 mmol. 10 ml). Methanol (10 ml) was

added to the aqueous phase. Benzyl chloroformate (11 mmol, 1.87 g) was added

in small portion altematively with NaOH lN (11 mmol. 11 ml) in order to keep the

pH at around 10. More benzyl chloroformate and sodium hydroxide were added

until complete negative ninhydrin response on TLC. The aqueous solution was

evaporated under reduced pressure in order to remove the methanol. The

aqueous solution was extracted with diethyl ether (2 X 25 ml). This phase was

discarded. After acidification to pH 2 with conc. HCl, the aqueous phase wasextraeted with ethyl acetate (2 X 25 ml). The orga!lÎc phase was dried over MgSO.

and evaporated under reduced pressure to afford a thick oil. The thick oil saon

solidified to a white sa\id. The pure title compound was obtained almost in

quantitative yield by this procedure whereas the omission of the methanol gaveyield that never exceeded 70%. M.p.137-138; [a]D20 -7.3 (c 2, EtOH); TLC R" 0.74

(BAW); vmax /cm-1,3460 (NH). 3320. 3220, 1750 (CO), 1715 (CO). 16905 (CO).

1630, 1620, 1450, 1430. 1420, 1340, 1280, 1245, 1220. 1060, 760, 700; IIR (200 MHz;

[2He]DMSO) 12.0 (lH. br s. C02H). 7.52 (lH, d, J 7.9, N"'H), 7.34 (5H, br s, Ar),

7.28 (1H, s, NlH1.....).6.76 (1H, s, NYHw), 5.01 (2H, s, O-eH2), 3.93 (lH. m, CXH),

2.13 (2H, t, J 7.2, OH2), 2.0-1.89 (lU, m, C~HA)' 1.78-1.60 (1H, m, ClIHs ); ôe (75

MHz; [zHelDMSO) 173.66, 173.46, 156.02, 136.95, 128.27, 127.72, 127.64, 65.30,

5353, 31.29, 26.60.

Preparation ofNacbz-N1Boc-A jJu 3.12- .Bis-trifluoroacetoxyiodobenzene (15

mmol, 5.97 g) was dissolved in water:dimethylformamide 1:1 (50 ml). Cbz-(D)G1n

was added (10 mmol, 2.68 g) foliowed by pyridine (1 ml). The carbon dioxide

evolution was monitored with a bubbler conneeted to the reaetion flask. The

reaetion was usually tenninated after 1 h. The mixture was then evaporated to

dryness under reduced pressure which afforded a sticky oil. The residue was

dissolved in water and extraeted with diethyl ether (2 X 50 ml.). The aqueous

solution was then concentrated under reduced pressure to a reduced volume (5

ml) and dioxane (5 ml) added. The pH of the mixture was adjusted to 9 by

addition of triethylamine. 2-(t-Butyloxycarbonyl-oximino)-2-phenylacetonitrile

(Boe-ON) (10 mmol, 2.32 g) was added and the reaetion was allowed to proceed

for 3 h after which all the Boe-ON had gone into solution. The reaction mixture

was evaporated to dryness under reduced pressure and the residue dissolved in

water. The aqueous phase was extraeted with diethyl ether (2 X 25 ml) andacidified with NaHSO. (5%) to pH 2. The desired compound was extraeted with

ethyl acetate (2 X 25 ml). This phase was washed with water (2 X 25 ml), and driedwith MgSO•. The extraet was evaporated onder reduced pressure and the crude

produet was purified by silica gel chromatography using hexane:ethyl acetate (3:1)

as a first solvent followed by pure ethyl acetate. The pure produet was obtained as

a solid when triturated with diethyl ether:hexane. Yield 2.11g 60%. M.p. 102°C(dec.); [alD ZO +13.5 (c 2.2, MeOH); TLC R,. 0.08 (ethyl acetate); ôR (200 MHz;[ZHelDMSO) 12.05 (lU, br s, COOH), 7.57 (lU, d, J Il.!, NIlH), 7.34 (5H, br s, Ar),

6.80 (lU, t, J 5.1, N'tH), 5.02 (2H, s, O-CHz), 3.96 (lU, m, O'H), 2.95 (2H, q,OHz),

1.93·1.75 (lU, m, d'HA), 1.74-1.54 (lU, m, C~Hs), 1.35 (9H, s, t-butyl); m/z (ElZlF) 296 (M+ • C.He), 278 (M+ • C.HeO), 252 (M+ - C.He - COz), 234, 209.

General Method for Coupling with Dicyclohexylcarbodiimide (DCC)- C blocked

peptide or amine acid in the form of trifluoroacetate salt (5 mmol) was suspended

in anhydrous TIIF (10 ml) and a tertiary amine (düsopropyl-ethyl-amine or N­

ethylmorpholine) added (5 mmol). The mixture was cooled to O"C, and 1·

hydroxybenzotriazole (5 mmol, 0.67 g) followed by the t-Boc amino-acid (5 mmol)

were added. The mixture was stirred for 15 min after which DCC (5.5 mmol, 1.13

68

69

g) was added. The reaetion was allowed to proceed at O"C for 1 h and then 2 h at

room temperature. The reaetion mixture was evaporated to dryness, dissolved in

ethyl acetate, filtered to remove dicyclohexylurea (DCU) and reevaporated to

dryness. The oily residue was chromatographed on silica gel using the appropriate

hexane/ethyl acetate mixture as eluting solvent.

t-Boc-Leu-OFm 3.3 [a]o2D -19.1 (c 17, CH2C~); TI.C R,. 0.10 (hexane:ethyl

acetate 5:1); /)H (200 MHz; CDCls) 7.76 (2H, d, J 7.5, Ar), 7.60 (2H, t, J 6.8, Ar),

7.41 (2H, td, J 7.5, 2.6, Ar), 7.32 (2H, td, J 7.4, 3.5, Ar), 4.85 (lH, d, J 8.7, NH), 4.49

(2H, d, J 6.3, O-CH2), 4.33 (lH, q, J 5.0, Leu CXH), 4.22 (lH, t, J 6.6, O-CHsCH),

1.7-1.35 (3H, m, Leu Cl'H2-OH), 1.49 (9H, s, t-butyl), 0.92 (3H, d, J 6.5, Leu Me),

0.88 (3H, d, J 6.5, Leu Me); m/z (El, DP) 410 (MH+, 1%),409 (M+, 1.3%), 354(MH+ - C4Hs' 28%), 309 (M+ - C4Hs-COs, 54%).

t-Boc-Phe-Leu-OFm 3.5 M.p. 6D-61°C; [a]o2D -21.5 (c 2, CHsC~); TI.C R"

0.25 (hexane:ethyl acetate 2:1); /)H (200 MHz; CDCls) 7.76 (2H, d, J 7.5, Ar-Fm),

7.60 (2H, t, J 7.5, Ar·Fm), 7.45-7.27 (4H, m, Ar-Fm), 7.2 (5H, br d, Ar-Phe), 6.2

(lH, d, J 8, Leu NH), 4.95 (lH, br d, Phe NH), 4.53 (lH, m, Leu CXH), 4.47 (2H,

AB system, O-CH2), 4.31 (lH, q, J 8.7, Phe CXH), 4.19 (lH, t, J 6.5, O-CHsCH),

3.05 (2H, d, J 7.0, Phe dlH2), 1.4-1.2 (3H, m, Leu cllH2-OH), 1.40 (9H, s, t-butyl),

0.82 (3H, d, J 6.5, Leu Me), 0.78 (3H, d, J 6.5, Leu Me); m/z (CI NHs, HP) 557

(MH+, 17%),501 (MH+ - C4Hs, 43%), 457 (MH+ - C4Hs~C02' 100%).

t-Boc-Gly-Phe-Leu-OFm 3.7- [a]oSD -25.0 (c 2, MeOH); TI.C R,. 0.13

(hexane:ethyl acetate1:1); /)H (200 MHz; CDCls) 7.76 (2H, d, J 7.5, Ar-Fm), 7.60

(2H, t, J 7.5, Ar-Fm), 7.45-7.27 (4H, m, Ar-Fm), 7.2 (5H, m, Ar-Phe), 6.58 (lH, d, J8.5, Phe NH), 6.18 (lH, br d, Leu NH), 5.00 (lH, br s, Gly NH), 4.65 (lH, q, J 8.0,

Phe CXH), 4.50 (3H, m, Leu CXH, O-CH2), 4.22 (lH, t, J 6.5, O·CH2CR), 3.75 (2H,

d, J 6.5, Gly CXHs), 3.13 (lH, d, J 7.9, 14.0, J'he cPHA), 3.00 (lH, d, J 8.1, 14.0, Phe

dlHB), 1.5-1.3 (3H, m, Leu dlH2-OH), 1.43 (9H, s, t-butyl), 0.87 (3H, d, J 6.5, Leu

Me), 0.82 (3H, d, J 6.5, Leu Me).

Nacbz-Nlf-Boc-(D)AjJu-Gly-Phe-Leu-OFm 3.9 M.p. 155-157"C; [a]o20 -5.2

(c 2, THF); TI.C R,. 0.11 (hexane:ethyl acetate1:2), 0.50 (ethyl acetate); 8H (200

MHz;CDCla) 7.75 (2H, d, J 7.1, Ar.Fm), 7.70 (lH, br s, Gly NH), 7.60 (2R, t, ; 7.1,

Ar-Fm), 7.5-7.1 (14R, m, Ar-Fm, Ar-Phe, Ar-ebz), 7.0 (lH, d, J 8.5, Phe NH), 6.7

(lH, d, Leu NH), 5.72 (lH, d, A,bu NClH), 5.15 (lH, d J 14, Cbz CHAHB ), 5.07

(lH, d J 14, Cbz CHAHs ), 5.0 (lH, br s, A,bu NyH), 4.7 (lH, q, J 8.0, Phe Ü"H),

4.50 (lH, ID, Leu Ü"H), 4.45 (2H, dd, Fm-CH2), 4.22 (lH, 1, J 6.5, O-CH2CH), 4.15(lH, m, A"bu Ü"H), 3.88 (2H, d, J 6.5, Gly Ü"H2), 3.5-3.2 (lH, ID, A,bu OHA), 3.1

(2H, d, Phe cllH2), 3.00 (lH, ID, A,bu OHB), 2.0-1.7 (2H, ID, A,bu dlH2), 1.55-1.2

(3H, ID, Leu cllH2-OH), 1.46 (9H, s, t-butyl), 0.85 (6H, d, J 6.5, Leu Me's).

Generalprocedure for removal ilft-Boe group with anhydrous trifluoroacetic acid.The t-Boc peptide was dissolved in dichloromethane (5 ml) and anisole (0.5 ml)

added. Anhydrous trifluoroacetic acid (5 ml) was added and the mixture stirred

for 30 min. The reaetion mixture was then evaporated under reduced pressure.

The sticky solid was then treated with cold water which stimulated precipitation of

the salt. The mixture was kept at 5°C for 1 h after which it was filtered, washed

with diethyl ether and dried on high vacuum. The salts obtained in this manner

were white solid of high purity.

H-Leu-OFm TFA salt 3.4. M.p. 158-159"C; [a]D 2D -11.4 (c 10, MeOH); TI..CR,. 0.55 (BAW); «SR (200 MHz; [2HelDMSO) 8.1 (3H, br s, NHa)' 7.9 ( 2H, d, J 7.1,

Ar), 7.70 (2.H, dd, J 7.1, 11.9, Ar), 7.37 (4H, ID, Ar), 4.96 (lH, dd, J 4.6, 11.0, 0­

CHA)' 4.63 (lH, dd, J 4.6, 11.0, O-C~B)' 4.32 (lH, 1, J 4.6, O-CH2CH), 3.71 (lH, 1,

J 7.0, Leu Ü"H), 1.3-1.0 (3H, ID, Leu cllH2-OH), 0.61 (3H, d, J 5.6, Leu Me), 0.54

(3H, d, J 5.7, Leu Me).

H-Phe-Leu-OFm TFA salt 3.6 M.p. 175-176°C; [alD2D -9.9 (c 2, MeOH);

TI..C R,. 0.71 (BAW); «SR (200 MHz; [2HelDMSO) 8.77 (lH, d, J 6.3, Leu NH), 8.1

(3H, br s, NHa), 7.87 (2H, d, J 7.1, Ar-Fm), 7.65 (2H, dd, J 7.1, 11.9, Ar.Fm), 7.37

(4H, ID, Ar-Fm), 7.23 (5H, br 50 Ar·Phe), 4.66 (1H, dd, J 10.8, 5.5, O-CHARB)' 4.52(lH, dd, J 10.8,5.2, O-CHAHs), 4.27 (lH, 1, J 5.4, O-CH2CH), 4.15 (lH, ID, Leu

C"H), 3.97 (lH, br, Phe C"H), 3.00 (lH, dd, J 4.5, 14.0 Phe cPHA), 2.82 (lH, dd, J9.4,14.0, Phe cPHB), 1.5-1.0 (3H, ID, Leu OH, Leu cPH2), 0.75 (3H, d, J 6.5, Leu

Me), 0.68 (3H, d, J 6.4, Leu Me).

H-Gly-Phe-Leu-OFm TFA salt. 3.7 M.p. 173-174°C; [alD2D -11.5 (c 2,

MeOH); TI..C R,. 0.59 (BAW); «SR (200 MHz; [2HelDMSO) 8.58 (lH, d, J 8.3, Phe

NH), 8.50 (lH, d, J 7.6 Leu NH), 7.9 (3H, br s, NHa), 7.87 (2H, d, J 7.1, Ar·Fm),

7.70 (2H, dd, J 7.1, 11.9, Ar-Fm), 7.50-7.27 (4H, ID, Ar-Fm), 7.25-7.15 (5H, ID, Ar·

70

•71

Phe), 4.66 (lH, dd, J 10.8,5.5, O-CH.J:IB), 4.65 (lH, m, Phe C"H), 4.52 (lH, dd, J

10.8, 5.2, O-CHAHB), 4.30 (lH, 1, J 5.4, O-CH2CR), 4.15 (lH, m, Leu C"H), 3.52(lH, br d, Gly C"HA), 3.40 (lH, br d, Gly C"HB), 3.00 (lH, dd, J 3.9, 13.9 Phe

dlHA), 2.70 (lH, dd, J 10.0, 13.8, Phe dlHB), 1.42(lH, m, Leu OH), 1.35 (lH, nt,

Leu dlHA), 1.20 (lH, m, Leu dlHB ), 0.78 (3H, d, J 6.5, Leu Me), 0.74 (3H, d, J 6.4,Leu Me).

Preparation of Nacbz(D)AjJu-Gly-Phe-Leu-OH. 3.10 The C,N protected

peptide 3.9 (1.36 g, 1.60 mmol) was dissolved in 20 ml of dry DMF and cooled to

O"C. Piperidine (1 ml) was added and the reaction aIlowed to proceed for 3 h at

room temperature. DMF was evaporated at room temperature under reduced

pressure (using a rotary evaporator equipped with a dry ice condenser and

connected to a high vacuum mechanicaI pump). The crude product was left on

high vacuum overnight. Triturating with hexane:diethyl ether removed most ofthe f1uorene resulting from Cl • rm deprotection. The product was not further

purified and the Boc group was removed by acidolysis as described previously.The peptide obtained after this sequence of reaction was dissolved in a smaIl

volume of acetonitrile (0.2 ml) and water added (5 ml). The crude product wasapplied on a column filled with reverse phase ClI I1-Bondapack flash

chromatography gel and the elution started first with pure water. The elution was

pursued with slowly increasing proportion of acetonitrile until a ratio of 1:1 was

reached. The fractions containing the desired product (UV and ninhydrin

positive) were evaporated under reduced pressure to remov: the acetonitrile and

were freeze dried to remove the water and provided a white f1uffy soUd. Yield 182mg 20% M.p. 198~C (dec.); TLC Rr. 0.53 (EAW); liB (300 MHz; [2H.1DMSO) 8.27

(lH, d , Leu NH), 8.17 (lH, t , Gly NH), 8.12 (lH, d, Phe NH), 7.66 (lH, d , ~bu

NUH), 7.40-7.1 (10H, m, Ar-Phe, Ar-Cbz), 5.05 (2H, AB system, Cbz CHAHn ),4.58(lH, Dl, Phe C"H), 4.22 (lH, m, Leu C"H), 4.10 (lH, m, ~bu C"H), 3.8 (lH, àd,

GlyC"HA), 3.55 (lH, dd, Gly C"HB), 3.07 (lH, dd, Phe dlHA), 2.85 (2H, m, ~bu

OH2), 2.77 (lH, dd, Phe dlHB), 2.05-1.72 (2H, m, ~bu OH2), 1.70-1.47 (3H, m,

Leu dlH2-OH), 0.91 (3H, d, Leu Me), 0.87 (3H, d, Leu Me); m/z (FAB Glycerol,Z2F) 570 (MH+, 100%),436 (MH+ - C,H7-C02, 24%).

Preparation of NOCbz-cfN1(D)AjJu-Gly-Phe-LeuJ. 3.1. Peptide 3.10 (0.22 mmol,

126 mg) was dissolved in dry DMF (200 ml) and cooleè at O"C. Triethylamine

(0.45 mmol, 22 mg) was added followed by diphenylphosphorylazide (0.45 mmol,

60 mg). The mixture was left at 5°C for 12 h and monitored by the decrease ofninhydrin response on lLC. DMF was removed under reduced pressure with the

apparatus previously described and the residue obtained was triturated withdietbyl ether. The yellowish solid was washed witb ethyl acetate which removed

most of the color and recrystallized several times from water:acetonitrile. White

cristals well shaped were obtained after this process. Yield 60 mg 50%. M.p.241°C (corr.); lLC R,. 0.90 (BAW); /lB (500 MHz; [2HulDMSO) see table 3.1; Sc(75 MHz; [2HulDMSO) 173.74, 172.92, 171.04, 170.40, 155.29, 137.56, 136.91, 128.85,

128.28, 127.72, 127.55, 126.49, 65.38, 57.47, 51.79, 49.83, 42.68, 40.22, 35.79, 34.31,30.45,23.98,23.32,20.96. m/z (El DP) 443 (M+-CrHsO 0.2%), 387 (0.7%), (rAB

DTI/DTE, ZHF) 574 (M+Na+), 552 (MH+).

3.6.2 NMR solution study

NMR solution study of 3.1. -Solutions (0.010 mol dcrn-S ) of 3.1 in[2HulDMSO were degassed by two cycles of suc~essive freezing and pumping

under high vacuum. The NMR tubes were filled with argon and sealed. 10 and

2T) IH spectra were recorded both at 300.13 and 499.83 MHz with a Brüker AC·F

300 HC Spectrometer aild a Varian Unity 500. The temperature was controlled at

24 oC. ID normal and selective proton decoupled spectra were recorded with 32K

data points. The FIDs were zero filled to 64K data points and resolution

enhanced by multiplication with a gaussian funclion having its maximum at 0.568 s

for SOO MHz speetra and with a gaussian function having its maximum at 0.3 s for

300 MHz spectra.

Assignment of the ID spectra was done with the help of 2D COSY and 2D

relayed coherence transfer experiments at 300 MHz. The assignment of the

spectrum was quite straight forward since none of the signal overlapped

significantly. Selective proton decoupling confirmed the assignment obtained with2D spectra. Analysis of complex spin systems such as for Leu and (D)A2bu

residues was conducted with the help f?f the PANIC simulation program for the

selective decoupled spectra and the undecGupled spectrum. The geminal protons"HA and cPHB of (D)A,bu were only separated by ca 9.S Hz at 300 MHz which

excluded the unambiguous assignment of J coupling constants (whoseapproaching values can he extracted from the signals of (D)A2bu ClHA, ClHB and

C"H). lbe separation of geminal protons was 16 Hz at Soo MHz which rendered

72

• the assignment of J coupling constants possible by simulation of the spectra whenOHA, OHe or O"H were decoupled. Final refinement of the coupling constants

and cherrdcal shifts previously derived from the analysis of decoupled spectra were

obtained by simulation of the normal (undecoupled) spectrum. Simulated andexperimental spectrum are given in supplementary material.

2D NOE spectra43 were recorded at 297.0 K at 500 MHz in the phase

sensitive mode (allowing detection of J correlated cross-peaks in dispersion mode

and NOE cross-peaks in absorption mode) using the phase cycling method ofStates-Haberkom. 44 A relaxation delay of 3 s was used. The experiment was

repeated for two different mixing times (0.3 s and 0.5 s). A spectral widths of 5264Hz (10.53 ppm) was used. 2K data points were acquired in F2 dimension and 512t1 in FI. A total of 32 transients were accumulated for each t1• Zero filling and

multiplication with a half gaussian having its half decay at 0.022s was accomplish in

FI domain. Resolution enhancement by multiplication with a half-gaussian

function having its half decay at O.09s was accomplished in F2 domain before 2DFourier transformed. This resulted in a digital resolution of 2.60 Hz/point. The

NOE cross-peaks were significantly more intense with a mixing time of 0.5s which

indicated that we were presumably in the linear regime of cross-peaks intensitygrowth. Relative intensities were estimated from this spectrum. Cross-peak

amplitudes were obtained from cross sections taken at maximum diagonal peak

amplitude. Selected regions of 2D NOE spectrum are given in supplementary

material.

The variation of chemical shift of NH signais with temperature was

conducted at 300 MHz. The chemical shifts were measured at seven different

temperatures in the range 296 to 326 K. A minimum of 10 min of equilibration

was allowed between cach measurements. The chemical shifts were referenced tothe [1H12Hs]DMSO resonance at 2.490 ppm which remain constant at ail the

temperatures studied. Graphs of Il vs Tare given in supplementary material.

3.6.3 Molecular dynarnic simulation

AlI computations were performed using the SYBYL software running on a

mM RISC 6000 model 320H. Initial molecular structure was built from thestandard fragment library. The '.f dihedral angle of Gly,Phe,Leu were adjusted

73

at values that permitted the formation of a p-tum type n' centered on GlySPhe4

and a p-tum type 1 centered on Phe4Leu6 as given in table 3.7. (D)~bu side chain

Xl and X2 torsional angles were set both at -60" in such a way as to permit ring

ciosure while minimizing eclipsing of substituents. In order to avoidsupplementary torsional variables, the Cbz was replaced by an acetyl group. PheXl torsional angle was set at -60" and X2 at 90". Leu Xl torsional angle was set at-60" and X2,1 at 180". The structuro: was then energy minimized using MAXIMIN2

with the Tripos force-field21 until the convergence criterion of 0.001 Kcal/mol was

reached. At this point, partial atomic charge were derived using a Mulliken

analysis of the molecular orbital coefficient obtained from a semi-empericalcalculation using the AMI Harniltonian (MOPAC).46 The structure was then

minimized again with MAXIMIN2 including this time an electrostatic contribution

weighted by a dielectric constant of 35 (DMSO).

The molecular dynarnic was simulated by integration of Newton equation of

motion assuming ciassical mechanic treatment. The Verlet algorithm46 was used to

perform the numerical integration using a time step of 1.0 fs. Initial velocities were

calculated from Boltzmann distribution. The force acting on each atom was

calculated from the valence force-field of Tripos inciuding Van de Waal and

electrostatic contributions. Since we did not inciude explicitly molecules of solvent,

tho: simulation was run at 250K in order to simulate the inertia effect of the

solvent. The simulation was performed for a period of 100 ps. Conformations

were colleeted every 0.25 ps. The total simulation required about 2 h of CPU time.

The 400 resulting conformations were used to compute average couplingconstants, and expected Iij with the help of the funetion TABLE of SYBYL.

3.7 References and notes

1. N. J. Mammi, M. Hassan and M. Goodman, J. Am. Chem. Soc., 1985, 107,

4008.2. H. Kessler, G. Holzemann and C. Zechel, Int. J. Pept. Protein Res., 1985,25,

267.

3. For standard procedures related to peptide solution phase synthesis see M.

Bodansky, and A Bodansky in Principle ofPeptide Synthesis, Springer-Verlag,

1984; M. Bodansky, and A Bodansky inl'racticeofPeptideSynthesis, Springer­

Verlag, 1984.

74

•75

4. M. Wald, Y. Kutajima and N. Izumiya, Synthesis, 1981,266

5. A S. Radhakrishna, M. E. Parham, R. M. Riggs and G. M. Loudon, J. Org.Chem., 1979,44, 1746.

6. T. Shiori, K Ninomiya, and S. Yamada, J. Am Chem. Soc., 1972,94,6203.

7. H. Kessler, Angew. Chem. In!. Ed. EngL, 1982,21,512.

8. It is worth mentioning that the two NH presumably involved in

intramolecular hydrogen-bonding are found at higher field than the others

(excluding the carbamate NH) which is consistent with the fact that these

protons do not experience hydrogen bonding with the polar solvent.

9. Positive NOEs correspond to cross-peaks that are of opposed sign relative to

the diagonal peak in 2D phase sensitive NOESY. Negative NOEs

correspond to cross-peaks that are of same sign as the diagonal peaks. F.

Sauriol, personal communication.10. Tl is defined as the numerical value (expressed in %) for the difference

between the observed peak area of a particular NMR resonance when the

resonance of a neighboring nuclei is irradiated compared to the peak area of

the same resonance under normal condition (undecoupled).

11. D. Doddrell, V. Glushko, and A Allerhand, J. Chem Phys., 1972, 56, 3693; P.

Balararn, A A Bothner-By, and J. Dadok, J. Amer. Chem. Soc., 1972, 94,

4015. For a general treatment see E. B. Becker in High Resolution NMRTheory and ChemicalApplications , 2nd Ed. Academie Press, Orlando, 1980.

12. G. Wagner, and K Wüthrich,J. MoL BioL, 1982,155,347.

13. 1. Solomon, Phys. Rev., 1955, 99, 559; N. Bloemberger, J. Chem. Phys., 1957,27,

572.

14. D. W. Graden and D. G. Lynn,J.Am. Chem. Soc., 1984,106, 1119.

15. S. Macura, K Wüthrich, and R. R. Ernst, J. Magn. Reson., 1982,47,351.

16. J. H. Noggle, and R. E. Shirmer in The Nuclear Overhauser Effect, Academie

Press, New York, 1971. For the particular case of cyclopeptide see M. D.

Bruch, J. H. Noggle, and L. M. Gierasch, J. Am. Chem. Soc., 1985, 107, 1400.

17. For convention with the endogenous Leu6 enkephalin (Tyr! GlyZ Gif Phe4

Leu6) we keep the numbering (D)AzBuZ Gif Phe4 Leu6•

18. C. M. Venkatachalarn, Biopolymers, 1968,6, 1425.19. Four amino acids are involved in the formation of a ~-turn. The first one

furnishes the carbonyl acceptor while the last one furnishes the NH donor.

Residue at position 2 and 3 are conformationally constraint in order to allow

the formation of the hydrogen bond. By the same token, the direction of

propagation of the peptide chain is reversed by 180" (turn) over two

residues.20. Peptide backbone conformations are described according to Ill, 1jI torsional

angles while side chain conformations are described according to theprincipal dihedral angle X as defined by IUPAC-IUB Commission on

Biochemical Nomenclature, /. MoL BioL, 1970,245,6489.21. SYBYL Molecular Modeling, Version 5.5, Tripos Associate Inc. February

1992.22 V. F. Bystrov, V. T. Ivanov, S. L Portnova, T. A Balashova, T. A Balashova,

and Y. A Ovchinnikov, Tetrahedron, 1973,2',873.23. K. Kopple, G. R. Wiley, and R. Tauke, Biopolymers, 1973, 12, 627.

24. M. Karplus, /. Chem. Phys., 1959, 86, 5561; M. KarpIus, /. Am. Chem. Soc.1963, 85, 2870.

25. M. Barefield, V. J. Hruby, and J. P. Meraldi, /. Am. Chem. Soc., 1976, '8, 1308.

26. 1. L Karle, /. Am. Chem. Soc., 1979, lOI, 181.

27. E. Benedetti, G. Moreli, G. Némethy, and H. A Scheraga, Int. /. Pept. ProteinsRes., 1983, 22,1.

28. A B. Mauger, O. A Stuart, R. J. Highet, and J. V. Silverton, /. Am. Chem.Soc., 1982, 104, 174.

29. G. Kartha, K. K. Bhandary, K. D. Kopple, A Go, and P. P. Zhu, /. Am. Chem.Soc., 1984, 106, 3844.

30. I. L Karle,/. Am. Chem. Soc., 1979, 101, 181.

31. J. L Karie, Int. /. Pept Protein Res., 1986,28,420.

32. A N. Stoup, A. L Reingold, A. L Rockwell, and L M. Gierasch, /. Am.Chem. Soc., 1987, 109,7146.

33. L M. Gierasch, I. L KarIe, A. L Rockwell, and K. Yenal, /. Am. Chem. Soc.,1985, 107,3321.

34. T. Yamazaki, S. Ro, M. Goodman, N. N. Chung, and P. W. Schiller, /. Med.Chem., 1993,36,708.

35. H. Matthies, H. Stark, B. Hartrodt, H. L. Ruethrich, H. T. SpieIer, A. Barth,

and K. Neubert, Peptides, 1984, S, 463.

36. K. J. Chang, A Killian, E. Hazum, P. Catrecasas, and J. K. Chang, Science,1981, 121,75.

37. M. Goodman, and D. F. Mierke, /. Am. Chem. Soc., 1989, 111,3489.

38. D. F. Mierke, G. Nôssner, P. W. Schiller, and M. Goodman, Int. /. Pept ProteinRes., 1990, 35, 35.

76

• 39.

40.

41.

42.

43

44.45.

46.

T. Yamazaki, S. Ro, M. Goodman, Biochem. Biophys. Res. Commun., 1991,181,364.P. W. Schiller, T. M. -D. Nguyen, !.. DiMaio, and C. Lemieux, Life Sei., 1983,33,319.This is mentionned by G. R. Marshall, and R. D. Nelson in Peptide Chemistry,Y. Kiso Ed., Protein Research Foundation, 1986, Osaka, 239.J. L Fiippen-Anderson, C. George, K. B. Ward in Ameriean CrystallographieAssociationAnnualMeeting, 1993, Poster PA18.J. Jeener, B. H. Meier, P. Bachmann, and R. Ernst, J. Chem. Phys., 1979, 71,4546.D. J. States, R. A Haberkorn, and D. J. Ruben, J. Magn. Reson., 1982, 48, 286.M. J. S. Dewar, E. G. Zoebish, E. F. Healy, J. J. P. Stewart, J. Am. Chem. Soc.,1985, 107,3902.L Vedet, Phys. Rev., 1967, 159, 98.

77

Chapter 4

Preparing ~-phenylcysteine: First approaches

4.1 Introduction

Although ~-phenylcysteine is a simple amino acid, pure enantiomeric forms

were oniy reported during the course of this work. Both published methods rdiedon synthesis of the racemates followed by enantiomeric stparation. One relied onseparation on a chiral chromatographie support 1 while the other used (-)mentholto prepare diastereomeric esters which were partially separated bychromatography. 2 We based our approach also on synthesis of the racemates butwe envisionp.d the use of an enzyme to resolve the enantiomorphs. Several~­

substituted cJsteine have been prepared earlier by Carter ><i..il13 whose methodrelied on the facile addition of mercaptan to unsaturated azlactone under basiccatalytic condtion (Scheme 4.1). This chapter reports the limitations of Carteroriginal methodology when apply to the particular case of ~-phenylcysteine. The

following chapter report how this 'methodology can be adapted to provide theselective removal of the protecting groups, and the easy resolution of the

enantiomorphs.

4.2 Using N-Benzoyl and S-benzyl protecting groups.

4.2.1 Synthetic method

In a first attempt, we used the original method described by Carter~

Accordingly we used the azlactone derived from hippuric acid (N-benzoyl glycine)

and benzaidehyde.4 The azlactone, 4-benzylidene-2-phenyloxazol-5.one, was

reacted with benzylmercaptan using sodium methoxide as a catalyst in

methanol/THF at room temperature. This provided a 1:1 Erythro:Threo mixtureof N-Benzoyl-S-benzyl-~-phenylcysteinemethyl ester. (Scheme 4.1).

+Phyl

1 °P

HSii 1

.......Ar

79

DL Threo4.1. (mp 131·C) 4.2. (mp IIO·C)

CÛ2Me CÛ2Me

ACHN+H H+NHAC

ArCH:S+H H+SCH2Ar

Ph Ph

DL Erythro4.1b (mp 171·C) 4.2b (mp 142·C)

... x

Chymo.

C02Me

ACHN+H

H+SCH2Ar

Ph

CÛ2Me

H+NHAC

ArCH:S+H

Ph

Separate Ph S

l.Ar

4.1 Ar c Ph4.2 Ar = a-naphtyl

Scheme 4.1 Reagents and conditions: i, NaOAc-Ac,O, reflux, 2 h; ii, NaOMe (cat.)-THF, MeOH, room temp., 3h.

4.2.2 Separation.of diastereoisomers

The diastereoisomers of N-Benzoyl-S-benzyl-P-phenylcysteine methyl ester

were separated at this stage. The diastereoisomers had same R. f. on TLC which

forbade any chance of separation by this technique. The same behavior was

observed by HPLC. However, differential crystallization proved to be quite

convenient and moreover easy handling for large scale separation. The mixture

was dissolved in the minimum amount of hot ethanol which was allowed to cooled

slowly. After 3 h. of standing at room temperature, a crop of pure erythrodiastereoisomer (higher M. p.) was obtained. The mother liquor was evaporated

to dryness and retaken in a minimum amount of ethyl acetate which upon cooling

at SoC provided pure threo diastereoisomer (Jower mp) whose crystals were

suitable for X-Ray diffraction study.

80

4.2.3 Enzymatic resolution and comments on ex-chymotrypsin pocket.

Having performed the separation of diastereoisomers at the stage ofmethyl esters, the possibility of using an endopeptidase such as ex-chymotrypsin to

c1eave stereoselectively the esters with ex L-configuration could 00 explored. The

fust difficulty encountered was the very pOOl' solubility of the substrate (4.28 or

4.2b) in solvent containing water. Typically only 5 mg of substrate could be

dissolved in 2 ml of dioxane:water 1:1. Other cosolvents, addition of polyethylene

glycol or tris-(hydroxymethyl)aminomethane (mIS), did not improve the

solubility. No hydralysis was observed in this medium at pH varying from 6 to 9.6

Due to this difficulty, it was not c1ear if the medium was too denaturating for the

enzyme or the substrate was really unacceptable to the enzyme.

We then tried to improve the solubility of the substrate by chemicallymodifying it. It is known that phenylalanine ethyl ester is an acceptable substratefor ex-chymotrypsin providing that the optimum working pH is lowered to 6.5.8

We then estimated that S-benzyl-phenylcysteine methyl ester would be more

soluble in water than the N-benzoyl counterpal't, particularly in acidic medium.

Consequently, the benzoyl group and the methyl ester were removed by acid

hydrolysis (compound 4.5, scheme 4.2.) and the methyl ester (compound 4.7)formed using 2-2-dimethoxypropane in aqueous HCI.7 The amine hydrochloride

obtained was indeed very soluble in water at low pH but started to precipitate

soon when pH reached 4. The enzymatic reaction was nevertheless tried at pH

ranging from 5 to 7 and in presence of small amount of methanol but again no

hydrolysis of the substrate was deteeted. Control experiments with phenylalanine

methyl ester and leucine benzyl ester showed that rapid hydrolysis was observed in

pure water at pH ranging form 4.5 to 7 and also with increasing amount of

methanol (up to 40 %), although at a reduced rate. It was then quite obvious thatS·benzyl-~-phenylcysteine methyl ester was an unacceptable substrate for ex­

chymotrypsin.

a-Chymotrypsin is probably the enzyme which is the oost charaeterized

concerning the structure of the active site by X-ray crystallography and with

specifically designed substrates and suicide inhibitors.8 The poeket dimension

• 4.1 or 4.2

Hi

81

Chymo. ;/ 4.3 Ar = Ph4.4 Ar = a-naphtyl

4.5 Ar = Ph4.6 Ar • a-naphtyl

H~OM""'''__V__ H2JOH

Ph S Ph~SlAr lAr

4.7 Ar. Ph

CFyO 0

iv HX----l..~ OH

Ph S

lAr

4.8 Ar = Ph4.9 Ar = a-naphtyl

Sc:heme 4.2 Reagents and conditions: i, HCI 6N HOAc (1:1), reflux, 2 h; H. HCI 6Nformic acid (1:1), reflux, 13 h; Hi, HCI 6N formic acid (1:1), reflux, IS h;iv, (CF,CO),O, CF,COOH, O·C, 10 min; v, conc. HCI in 2,2-dimethoxypropane, roomtemp., 12 h

allotted to the binding of the side chain of the amino-acid being hydrolyzed has

been estimated to be 12 Aby sA, just enough to allow a snug fit for a Trp sidechain.V However, it has been reported that amino ~cid with p-branching such asVal and P,P-dimethyl phenylalanine are only hydrolyzed with difficulty by cx­

chymotrypsin. 10 The binding pocket of cx-chymotrypsin would probably bave been

sufficient toaccommodate the S-benzyl moiety of our unusual amino acid but theremaining P-branched phenyl group could not be adequately positioned such as to

permit the scission of the ester.

We then tumed to using the enzyme carboxypeptidase A.Carboxypeptidase A is an exopeptidase that cleaves off the last amino acid of a

peptide or protein with approximately the same preference for aromatic amino­acids as cx-chymotrypsin but is much less sensitive to p·branching. 11 Its optimum

activity pH is 7.S at which the terminal carboxylic group of the substrate is in theform of a salt. For these reasons, this enzyme was particularly attractive since we

• already knew that the corresponding sodium salt of 4.3 was water soluble. On theother hand, carboxypeptidase A usually accepts up to 5 residues in the bindingdeft which provide the tight binding required for catalysis and therefore re ndere~difficult and even unpractical selective amide bond c1eavage with a single acylamino-aeid as a substrate.12 Selective hydrolysis can however be achieved withsingl~ amino-aeid when a very good departing acyl group like trifluoroacetyl isused. 1S Consequently, the N,O deprotected diastereoisomers 4.5a,b weretrifluoroacetylated \".ith trifluoroacetic anhydride in trifluoroacetic aeid at O"C.14The resulting trifluoroacetamides 4.88 and 4.8b were dissolved carefully in water

taking care that the pH never exceeded 9.16 Carbo~)'peptidase A was added andslow amidolysis was this time observed as detected by a ninhydrine positive

response by TLC at R.f. =0.5 (BAW). The reaction was irreversibly inhibited ifthe substrate was not carefully recrystallized in carbon tetrachloride such that ailtrace of free thiol was elirninated16 which otherwise could bind irreversi"ly to theZn+2 in the active site of the enzyme. Even if the substrate was very pure,reversible inhibition occurred when the concentration of the substrate exceeded0.01 M and this phenomenon has been often reported with carboxypeptidase A,17

The resulting free amino aeids (presumably L) showed very high optical rotation

values (Scheme 4.3) as weil as the unreacted trifluoroacetamides (presumably D).The unreacted product was aeid hydrolyzed and the resulting free amino acid gavean optical rotation value of same magnitude albeit of opposite sign to the enzyme

hydrolyzed product (Scheme 4.3). We could therefore conclude that theenzymatic reaction had gone to completion when ail these precautions were taken.

4.2.4 Removing the benzyl protecting group on sulfur.

82

Although we had obtained the four pure enantiomers of ~.phenylcysteine

with the sulfur protected with a benzyl group, we raised the question: will it be

possible to deprotect cleanly the sulfur when the unusual amino-aeid will be

incorporated into a peptide? Indeed this was an important question since the S

atom is not only linIted with the benzyl proteeting group but similar relationshipexist with the ~-phenyl group which may induce cleavage leading to desulfurization

(Scheme 4.4). In the original method reported by Carter~ this was not aproblem since the ~'substituent was an alkyl group and c1ean debenzylation could

be achieved using sodium metal in liquid ammonia. Accordingly, we used

compound 4.38 as a model to determine the regioselectivity of cleavage.

D Threo (4.5ab)[a]o20 = +190'

tii

83

DL Threo

4.Ba

DL Erythro

4.Bb

C02H

__.......~ H2N+H

ArCH~+H~

L Erythro (4.5ba)

[alD 20 = +150'

CÛ2H

+ H+NHCOCFs

H+SCH2Ar

~

D Erythro (4.Bbb)[a)D20 = -129'

tHD Erythro (4.5bb)[a)D20 = -147'

Sc:heme 4.3 Reagents and conditions: i. substrate conc. 0.01 mol dm-3 in water, LiOH(1.0 equiv.), LiCI (0.1 mol dm"), pH 7.5, 37'C, CPA; H, HCI 6N formic acid (1:1)reflux 13 h

PhyO

HOH

Ar

".3. Ar. Ph"."a Ar • Ol-naphtyl

i, ii ..

4.10a , 4.10b 4.11

Sc:heme 4.4 Reagents and conditions: i, 2 to 6 equiv. Na, Iiq. NH" -33'C to -7S'C,2 to 20 min; ii, H'

84

Reductive fission using dissolved metal in liquid ammonia should be

performed with much care. Severa! authors have described experimentalconditions in detaiP8 but we would like to review sorne of the critical points

-Substrate must be thoroughly dried.

-AlI glassware is flaIne dried ur.der high vacuum after which dry argon is

introduced.

-Ammonia is first condensed using a dry ice condenser after which it is

distilled from sodium metal and ferric sulfate. Typically 100 ml of ammonia

are distilled for a reaetion using 0.5 mmol of substrate.

-Teflon coated magnetic stirrers should be avoided. Glass coated magnetic

stirred are more appropriate.

-Accurate and non-oxidized amount of sodium is obtained by cutting at

the appropriate length a low diameter glass tube filled with sodium.

-A small bit of sodium is added to the ammonia prior to the substrate in

order to check the dryness of ammonia. Persistence of a blue color

(associated with the presence of solvated e-) is indicative of a dry medium.

The sodium metal induced CoS bond fission can be regarded analogous to

the C-halogens (C-hal) bond fission by alkali or a!kali earth metals in various

solvents. It is generally accepted that the direction of these reactions is driven

according to the relative stability of the resulting carbanion or radical products.1G

The oxidation potentials of the metal in the particular solvent must obviously hesuperior to the reduetion potential of the substrate. Table 4.1 provides the

oxidation potential of sorne metals in water20 and in liquid ammonia21 while Table

4.2 gives the reduetion potentials for the bond fission of sorne organicsubstrates. 22,23 For instance, Mg can induce C-hal bond fission (Grignard

reagents) but not S-Bz! cleavage while Na cano In a manner similar to the C-hal

bond fission, the S-Bzl cleavage has been shown to require 2 e-.'. In the particular

case of compound 4.3, 3 e- would be required because 1 e- is consumed to convert

the acidic proton into molecular hydrogen (through a one e- process).

Several conditions were applied to realize the regioseleetive cleavage of

model 4.3a. The results are givçn in Table 4.3. Accurate amounts of sodium were

used since no turning point (persistent blue color) was observed. At most, the

lemon yellow solution tumed reddish when the theoretical amount of metal was

Table 4.1 Oxidation potential of sorne "active" rnetal

85

Equation Oxidation Potential (V vs SHE)in water- in liquid NH3b

Li =Li+ + le­Na = Na+ + le­K = K+ + le­Ca = Ca+ + le­Ca = Ca+2 + 2e­

Mg = Mg+2 + 2e-

+3.05+2.71+2.93

+3.02+2.76+2.37

+2.99+2.59+2.73

+2.39not soluble

a) from ref 20. b) from ref. 21

Table 4.2 Reduction potential for thecIeavage of sorne covalent bonds

Substrate

CH3-I­CH3-Br­

CH3-CI­

Et-Br­

A1lyl-BI"BzI-BI"Bzl-SRb

BzI-ORb

BzI-NHRb

Reduction potential(V, vs SCE)

-1.630

-2.01<

-2.23<

·2.l3d

-1.29d

-1.22d

-2.8d

-3.ld

-a) From ref 22. b) From rer. 23c) 30% water in dioxane d) in DMFe) not reduced before discharge of the sUPJlorteiectro(yte.

• •Table 4. 1 Different reaction conditions tried ta perfonned regioselective benzylic c1eavage of 4.3a.

Trial Metall Distilled tempo pr.:lton metal duration quenchingC Products ratio'

amounP NHJ' Cc) source first (min) 4.3a 4.10" 4.11 others

1 Na/2 0 no -33 none no 2 NH 40Ac S 4(4: 1) 1 atleastone

2 Na/3.0 no -33 none no 2 NH4CI 0 1(4: 1) 1 net significant

3 Na/3.0 yes -SO none no S NH 4Cl 0.7 2(1:1) 1 at least one

4 Na/3.0 no -60 none yes 10 NH 4CI 0.6 2(4:1) 1 ntleasttwo

S Na/3.0 yes -78 none no 20 NH 4Cl 1 1(1:1) 1 numerous

6 Na/3.S yes -33 none no 2 HOAc(1 ml) O.S 0 1 atleasttwo

7 Na/4.0 yes -78 iPrOH yes 10 NH4CI 0.5 0.S(4: 1) 1 at least one

8 Na/6.0 yes -78 iPrOH yes 10 NH 4Cl 0 0 1 atleastone

9 Ca/1.5 yes -33 none yes 5 NH4CI 1 0 0 not significant

10 Ca/2.0 yes -33 none yes 5 NH 4CI 1 0 0 not sigllificant

10 Cal3.0 yes -33 none yes 5 NH4CI 1 0 0 not significant

al -.. 01 mmoI io iDdic:alOd IIOftDI1izld ror a ...-tÎoa wilh 1 ...".1 or_. hl WboII tbo _a.... no\ diltilled..lIlllD chi,. ..... aUed lIIIIiI _ hl.. coIor ponioled _ wllich tbo nactioa .... otarlOd. cl The ..... auml>er or IIlIIIOl

u _ ..... addod .-__ ._d. dl dtllrmiDld hy 1 H NMR. el The nlio or liuw.lliaomor a IIId h io ai.... in porommaio.

00

'"

added. After appropriate work-up, N-benzoyl-IJ-phenylcysteine 4.10 and N­benzoyl-~-phenylalanine 4.11 were easily identified by IH NMR. Product 4.10

QCCUrs as a mixture of diastereoisomers (4.108, b) showing typical doublets for thefree SH resonance at 2.3 and 2.4 ppm and triplets at 4.6 a..t1.d 4.7 ppmcorresponding to CIlH's respectively. The presence of compounds possessing freethiol was alse assessed by positive response to nitroprusside test after TLCseparation. On the ather hand, the presence of N-benzoyl-phenylalanine (4.11)was quantified by the multiplet at 3.15 ppm corresponding to CIlH2 • The identities

of the major produets 4.10 and 4.11 were later confirmed by mass spectrometry offractions separated by high performance liquid chromatography. Interestingly,benzyl mercaptan was never detected and the desulfurization product washydrogen sulfide which could easily be detected by its characteristic smell after

acidification of the aqueous phase.

According to Table 4.3, it seems that the desired deprotection occurs to amajor extent relative to the unwanted desulfurization but cannot be avoided Ü

complete sulfur deprotection is achieved. Indeed the amount of 4.10 relative to

4.11 is greater when the amount of metal is lower than theoretically required butsignificant quantity of starting material is recovered (entry 1). Similarly, the ratioof the de3ired product is generally inereased when the temperature is lowered

and the reaetion time prolonged but unfortunately it is accompanied by significantamount of unidentified produets. These produets result probably from Wurtz

type coupling or biradical coupling (entries 3,4,5,7). Moreover, twice excess of

sodium metalled to complete desulfurization (entry 8).

A possible solution emerged by using a metal with a lower oxidationpotential such as calcium. Hwu~ have reported that calcium can he used

advantageously to promote selective benzyl ether cleavage in the presence ofother potentially reducible functions (such as alkyne, 2.furylmethylene ether).26

Larger amounts of calcium than the theoretical were required to affect o-Bzl

cleavage with substrates with more than one potentially reductive function. This

was explained by the authors in term of two pools of eleetron with each a different

oxidation potential. The first pool contains the "strong" and therefore less

c:hemoselective eleetrons coming from the process Ca .> Ca+l + le· while the

second encloses the "mild", more selective eleetrons, coming from the processCa+l .> Ca+1 + le-. AlI potentially reducible functions in the substrate accept one

87

88

e- from the fust peol. Then, a "mild" e· adds to the most reactive function and isresponsible for the chemoselectivity.

Control experiment was fust performed. We determined that 1.5 mmol ofCa was required to completely deprotect 1 mmol of S-benzyl-cysteine. Ail

ionizable e- were used when only one potentially reductive function was present inthis substrate which was consistent with their proposai. Sorne precautions hadhowever to be taken regarding the cleanliness of the calcium and its dissolution inammonia prior to the substrate.

The reductive fission results were quite different when the same experimentwas conducted with 4.3. Treatment with amount of metal varying from theminimum quantity to 2.5 fold excess (entry 9,10,11) led without difference to therecovery of the starting material as a single diastereoisomer. It should he pointedout that all the blue color disappeared when the substrate was added to the"metal" solution indicating therefore that the "solvated e·" were consumed butpresumably only in a reversible process.

According to the results shown in Table 4.3, a mechanism for the reductivefission of 4.38 can be proposed. It is depicted in Fig. 4.1. In the first step, the

carboxylic acid reaets with liquid ammonia to forro a carboxylate and anammonium salt. The ammonium ion is still a strong acid in this medium and

reaets quickly with the first equivalent of sodium added to liberate hydrogen.

Then, the reductive fission can begin. Since the thiolate 4.13 seems to be formedfavorably (and subsequently benzyl sodium) at low Metal ratio, it is envisioned

that a radical anion is generated after the addition of the first electron, whichirreversibly dissociates after addition of a second electron.Je N-Benzoyl-~­

phenylcysteine disodium salt 4.13 and benzylsodium are produced as main

produets (path A) with N-benzoyl-phenylalanine disodium salt 4.12 andbenzylmercaptide (path B) as minor produets. Since no benzylmercaptan was

detected in the produets, it presumably underwent further reaction. Metal couldpromote this process but this would not be compatible with the stoichiometry

obtained in entry 2. Alternatively, benzylsodium can he a source of eleetron to

achieve the desulfurization process. Indeed, Gerdil and Lucken have shawn thatbenzylpotassium promotes desulfurization of dibenzyl sulfide in dimethoxyethlille

by a mechanism that is not fully understood.:I'1 Similarly, Truce m..al observed that

89

Ph 0y 0 NH;

Iiq. NH, HXo­..

4.3.

IPh'fO 0 No' ••~

. Ph

s0l Na"Ph

Na"oCH2 +

o

Ph

~ 1 Na~A(majOr)

PhyO 0

H~ H"0- _4.10.4.10b

Ph S0Na"

4.13

.... 01""Ph~OHXO

Na"0- + S"

Ph 0 Na"

4.12

2 e-

Na"oÇH2Ph

Fig. 4.1 Proposed mechanism for the reaetion of 4.38 with sodium metal inliquid ammonia at ·3:'·C.

90

addition of sodium to a liquid ammonia solution of dibenzylsulfide gave onlytoluene and hydrogen sulfide as reaction produets after acidification.28 Benzylsodium se'::ms Lherefore responsible for desulfurization of benzylmercaptan (pathC). Of course similar events can happen to 4.13 J.ild contribute to the increasingamount of desulfurization with reaction progression. The formation of dianion ofsulfur may seem unlikely; however the stability of the departing S-2 group is asgood as an alkoxide as revealed by the pKa of the corresponding conjugate acid(16.7 for HS- and 16 for EtOH) and is likely to happen with a benzylic carbanion

counterpart.

Truce ~28.2g.30have investigated the direction of c1eavage of thioethers.

Diaikyl thioethers are c1eaved by lithium in methylamine by a process which hasbeen interpreted in term of gradation of mechanism depending on the nature ofthe substituents. t-Butyl groups were always c1eaved by a one e- process involvingthe formation of a t-butyl radical while radical, anionic or both mechanisms occur

with less branched alkyl substituents. The reductive fission of aryl-alkyl thioetherswith sodium in ammonia always produced the corresponding thiophenoxide and

alkane. This fact has been used advantageously in the preparation of aromatic diand tri mercaptans. 31 By the same token this demonstrates the impossibility ofusing an aryl group as a protecting group for the alkylthiol function. Finally thec1eavage of asymmetric diaryl sulfides was explored but the results were

complicated by side reactions involving the substituents on the aryl groups. Fromtheir experimentations, Truce~ proposed the following rule of "thumb" to

predict the preferred direction of c1eavage for thioethers: "cleavage occurs to

produce the least basic anions of the pair having the greatest difference in

basicities". This rule seems to apply to the cleavage of 4.3a since the difference in

basicity between 4.12 and benzyl sodium is probably only slightly greater than theone between 4.13 and benzyl mercaptide. The basis for the rule of "thumb"

proposed by Truce m..al is given in supplementary material.

Another problem which was encountered during the sulfur unmasking was

the fact that epimerization occurred as revealed by the presence of two

diastereoisomers bearing the SH group in the reaction produets. Interestingiy,

the starting materiai was recovered intact without trace of the otherdiastereoisomer indicating therefore that the epimerization occurred only on the

s-deblocked produets. The ratio of diastereoisomers 4.10a/4.10b varied

depending on reaetion conditions but we noticed that systematically the ratio waslarger when a proton donor source was voluntarily introduced or when theammonia was not distilled (entry 1,2,4,7).S2 Otherwise an equal amount of the twodiastereoisomers was observed by lH NMR when care was given to have ammoniawithout hydroxide or alkoxide (entry 3,5). The presence of alkoxide has been

reported to prevent the formation of sodamide, a very powerful base, whichpresumably may promote the epimerization process. 19 It is worth mentioning thatoptical integrity is preserved when S-benzyl cysteine is subrnitted to sirnilarcondition. ss This may point to the benzoyl as a possible group responsible for thisphenomenon in the case of 4.3. However, in the absence of results obtained fromquenching with deuterated acids it will be speculative to go further concerning the

mechanism of epimerization.

4.3 Using N-benzoyl and S-methylene-a-naphtyl protecting groups

As an alternative to the problem encountered in the sulfur deproteetion weenvisioned first to use a different sulfur proteetive group which rnight undergomore easily reductive cleavage than the benzyl group. Based on the previous

results obtained, it could be anticipated that a para eleetron withdrawing groupinserted ante the aromatic ring of the benzyl group should make thecorresponding benzylic anion more stable, leading therefore to regiospecific

cleavage.

The eleetron withdrawing group must however be stable itself to thereduetive conditions of sodium metal in ammonia. Accordingly, the p-N02, p-Hal,

and p-CFs group should be elirninated. On the other hand, a p-COOH group is

resistant to metallic sodium reduction owing to the formation of salt, but as an arylsubstituent it facilitates so much the Birch aromatic ring reduetionS4 that this may

compete significantly to the CoS fission reaetion. Considering the pKa value of

various substituted benzoic acid as an indication of benzylic carbanion stability(Table 4.5), we deduced that an a-naphthyl group may meet the desired criteria.

This protecting group will presumably be stable to strong aqueous acid hydrolysis

which will permit a sirnilar synthesis as described in Scheme 4.1 and 4.2.

91

•92

Table 4.5 The pICa of sorne substituted benzoic acid derivatives

Substituent pICa

p-NH2 4.92

p-OMe 4.47p-OH 4.48p-Me 4.46poEt 435

p-H 4.19~-naphthoic 4.17

p-Cl 3.98a-naphthoic 3.70

p-COOH 3.5;poN~, 3.41

p-F 2.90

4.3.1 Synthetic method

The same azlactone, 4-benzylidene-2-phenyloxazol-5-one, was reacted witha-naphthylmethylmercaptan under basic catalytic condition which lead similarly to

a 1:1 erythro:threo mixture of N-benzoyl-S-a-naphthylmethyl- ~-phenylcysteine

methyl ester (4.2a,b, Scheme 4.1).

4.3.2 Separation of diastereoisomers

The separation of the diastereoisomers at the stage of the methyl ester

proved to be very difficult. Similarly to the previous case, no separation was

possible based on chromatographic techniques. Furthermore, diastereoselective

crystallization was unsuccessful using absolute ethanol, 95% ethanol, methanol,

ethyl acetate, or acetonitrile. In a last resort, hot carbon tctrachloride was tried.

To our surprise the floating crust that formed upon cooling the solution consisted

in significantly enriched threo diastereoisomer.S6 Looking for a better solvent of

similar polarity we pointed out l-chlorobutane as a possible candidate. We were

this time pleased to observe that the erythro diastereoisomer crystallized out as the

• major one this time. Optimizing the process with the two solvents provided each

diastereoisomer in an analytically pure form.

It is noteworthy that diastereoisomers separation was unsuccessfuJ whenattempted on the corresponding acid 4.4 or by fraetional crystallization of the saltsformed with phenethylamine which were used advantageously by Carter~ for

related amino-acids.S

4.3.3 Removing the a-naphthylmethyl group on sulfur.

After having succeeded in separating the diastereoisomers, we preparedthe corresponding acid 4.4a and submitted it to metallic sodium in liquid ammoniain order to verify our hypothesis. This time, when 3 rnmols of sodium were addedto 1 rnmol of substrate a clear turning point was deteeted indicated by a quick

color change from lemon yellow to a persistent blue. After appropriate work-up,

no smell of hydrogen sulfide was deteeted and the 1H NMR showed that indeedthe deproteetion was regiospecific, giving essentially produets 4.10a,b. The factthat we obtained a clear turning point suggests that 4.13 is stable to excess metaland confirms therefore that benzyl sodium is responsible for excessivedesulfurization. The a-naphtylmethylene carbanion is probably stable enough to

survive until the work-up without causing damage to substrates or thiolates.

4.3.4 Enzymatic resolution and cornments on carboxypeptidase A pockets.

Encouraged by the cleaniiness of the sulfur deprotection reaetion on the

model compound 4.4a, the corresponding trifluoroacetamide 4.9a, b wereprepared and incubated with carboxypeptidase A. Unfortunately, no amidolysis

was observed this time with any of the diastereoisomers. Several batches of

carboXYPeptidase A were tried but with the sarne disappointing results. It was

then quite obvious that the newly inserted aromatic ring on the benzyl protectinggroup was incompatible with the dead-end poeket allotted to the side chain of the

amine acid undergoing N·acyl bond cleavage. This pocket has been described as

being relatively large. It can accornmodate easily a Tep side chain. Moreover,sorne unnatural amine acid such as phenylglycineS7, a-arninoisobutyrlc acidS8 and

D-alanineo are acceptable substrates for carboxypeptidase A Carb0XYPeptidaseA shows preference for aromatic and p·branched residues but can however slowly

93

•94

hydrolyzed terminal basic amino acid such as L-Lysine.S7 On the other hand, it hasbeen reported that Na-hippuryl-N <-benzyloxycarbony-L-Iysine and Na-hippuryl-L­

arginine38 can not be digested by carboxypeptidasc A. The resistance of the

former to hydrolysis can be ascribed easily to the steric effect created by the largeacylating substituent on the (-amino group but it is not clear whether steric or

electronic reasons are the cause for arginine. A solution to this problem Mayemerge from the fact that S-benzyl-p-phenylcysteine underwent hydrolysis while 50

a-naphthylmethyl- p-phenylcysteine did not. Alignment of Arg with S-benzyl-p­

phenylcysteine performed in order to maximize molecular volume overlap (Fig.4.2) brings out the fact that the imminium nitrogen of Arg cannot be assigned tothe volume occupied by the phenyl group of the benzyl moiety of S-benzyl-~­

phenylcysteine. Indeed the imminium nitrogen corresponds roughly to an ortho

substituent on the phenyl ring. Obviously this position is occupied in the case ofthe S-a-naphlylmethyl proteeting group which May explain why it is also resistant

to the action of carboxypeptidase A. Interestingly, a p-methyl group on the benzyl

group does not hinder the action of carboxypeptidase (vide infra).

Fig. 4.2 Alignment of arginine with S·benzyl-P·phenylcysteine done in order to

maximize common molecular volume.

• The possibility of realizing the resolution using an aminopeptidase such asleucine aminopeptidase (usually ca\led amidase) could have been explored. Thefaet that 3 steps of synthesis were required to convert 4.6 to the suitable subst;atealong with the high cost of the enzyme justify why this avenue was not explored.We decided rather to optimize the choice of our proteeting group during thesynthesis, keeping in mind that resolution was possible with a S-benzyl group using

carboxypeptidase A

4.4 Using N-benzoyl and S-p-N02-benzyl protective groups.

Although a p-N02 benzyl group on the sulfur is not suitable for removal

with sodium Metal in ammonia, catalytic reduction with hydrogen should providethe corresponding p-amino derivative which usually undergoes easy deprotectionwhen treated with mercuric acetate.41 Dy the same token, the p-N02 benzyl group

is presumably very resistant to acidic hydrolysis which could permit the use of the

same steps described in Scheme 4.1 and 4.2. This idea did not go very muchfurther since p-N02-benzylmercaptan did not add to the azlaetone under basic

catalytic condition. A deep blue color developed during the addition of theazlaetone to the mercaptide solution. This color was lost during the acidic work­

up, which provided only traces of the desired produets. Similar results wereobtained when sodium carbonate or triethylamine were used as catalytic bases.

4.5 Conclusion and summary

Owing to the presence of a P-phenyl group, the S-benzyl proteeting group isnot suitable for p-phenylcysteine. Replacement of the S-benzyl group by an Q­

naphthylmethyl group rendered the S unmasking regioselective using sodium

Metal in ammonia. Unfortunately no resolution can be achieved on thecorresponding trifluoroacetamide substrate using carboxypeptidase A with this

new protecting group. Application MaY perhaps be found for this proteetinggroup in organic synthesis where extremely high resistance to acidic condition and

ease of reductive fission is required. Dy the same token, we determined that thearder of ease of reductive cleavage for benzylic thioether is S-CH2-a-naphthyl >S-CHJ-phenyl > S-CHR-phenyl. This arder is in agreement with the relative arder

of carbanion stability and argue for a two eleetron process.

95

• 4.6 Experimental

-Melting points were recorded on a Gallenkamp capillary apparatus and are

uncorrected. Infrared spectra were recorded either on a Bornem Michelson (BM)series or a Perkin-Elmer (PE) 1600 FTIR as specified in the text and were taken inKBr pellets. IH and ISC n.m.r were recorded at 200 or 300 MHz for proton speetra

and at 50 or 75 MHz for carbon spectra either on a Varian XL-200, a Gemini 200

or a Varian XL-300. The speetra are referenced to the residual solvent peak(CDCIs tH 7.24 ppm, ISC 77.0 ppm, CDsOD, IH 3.3Oppm, lSC 49.0 ppm, C,DeSO,

IH 2.49 ppm,ISC 39.5 ppm); J values are given in Hz. Thin layer chromatography

were taken on glass backed Merck silica gel 60, 0.25 mm thickness. Optica1

rotations were measured on a JASCO DIP-140 instrument at sodium linewavelength at 20"C in a 1.0 dm cell and the values were integrated over a period of

60 s. Mass spectra and exact mass were determined either with a ZAB-IF (ZlF),

or a HP 5980 (HP) as specified in the text.

96

Reagents and Solvents- a-Naphthylmethylmercaptan was prepared from the

corresponding l-chioromethylnaphthalene using the Bunt's salt methodology; theproduct was disti11ed B.p. 104°C 0.02 mmHg, /lH 8.15 (d, lH), 7.9 (d, IH), 7.8 (d,

lH),7.7-7.4 (m, 4H), 4.25 (d, 2H, CH,), 1.95 (t, lH, SH) and was essentially

odorless. Benzylmercaptan from a commercial supplier was used, which had a

very pungent odor. Therefore, all reactions perforrned with it were done in a weil

ventilated fumehood (even invacuo evaporation). 2-Phenyl-4-benzal-5-oxlI?.olone

was obtained according to procedure already published in Organic Synthesis·.

Uquid ammonia was obtained by condensation from a commercial tank. Sodium

metal with low iron content was obtained from Aldrich. The metal was molten in

refluxing toluene after which it was aspirated into a low diameter glass tube.

Typica1ly, 1.6 mm internaI diameter tubes were used which account for 2.1 mg of

sodium by mm. A length of 17 mm of tube represented 1.5 mmol of sodium.

Calcium metal was obtained from Aldrich and was cleaned by washing with 2%

conc. Hel in ethyl alcohol. The metal was quickly rinsed with diethyl ether and

then kept in hexane and used in extenso. Ammonium chloride was purified bysublimation. Othe! reagents and solvents were purified as described in chapter 5.

Preparation of threo and erythro N-benzoyl-S-benzyl- fiphenylcysteine methylester (4.18 and 4.1b) and threo and erythro N-benzoyl-S- a-naphthylmethyl- fipheny/cysteine methylester 4.28 and 4.2b. Preparation is sunHar to that described inchapter S for threo and erythro N-acetyl-S-p-methylbenzyl-~-phenylcysteine methyl

ester.

Separation {yf Threo and Erythro N-benzoyl S-benzyl- fiphenylcysteine methyl

ester 4.18.and 4.1b. A quantity of Il g of the erythro:threo mixture was dissolved in

220 ml of hot absolute ethanoJ. The Erlenmeyer flask was kept on the hot plate to

ensure slow cooling. Thin needles of erythro diastereoisomer started to form

about O.S h after turning off the heat source. The optimal crop was collected by

suction filtration aiter 3 h (a longer standing caused the crop to he contaminated

with threo diastereoisomer); yield 4.5g, 82%. The remaining mother liquor was

evaporated to dryness and redissolved in hot ethyl acetate (ca 40 ml). The

solution was cooled slowly to room temperature and then kept at SoC for 12 h

after which pure threo diastereoisomer could be obtained as large prisms which

were suitable for X-ray diffraction study; yield 3.S8o 64%. Threo (4.18). -M.p.

131°C (from EtOH); ne R.f. 0.50 (EtOAc:hexane 1:1); vmax/cnr l (BM) 3361

(NH), 3027, 2947, 1726 (CO), 1666 (CO), 1531, 1491, 1359, 1220 1171; .sR (COCla.200MHz) 3.489 (d, IH, 2J 13.3, CH.Jls), 3.596 (s, 3H, C02CHa), 3.648 (d, lH,2J

13.3, CHAHa), 4.269 (d, lH, J 6.0, CH-S), 5.121 (dd, IH, J 6.0, 8.2, N-CH), 6.741 (d,

lH, J 8.2, NH), 7.2-7.8 (m, 15H, Ar); .sc (CDCla 50 MHz) 170.57, 166.94, 137.72,

137.21, 133.65, 131.84, 128.95, 128.58, 128.49, 128.10, 127.28. 127.10, 56.97, 52.45,51.16, 35.63; m/z (El, ZIF) 406 (MH+), 374 (M+ -OMe), 346 (M+ -C02Me), 314.

Exact Mass cale for ~4H24NOaS 406.1477 exp. 406.1471. Erythro (4.1b) -m.p.l71°C

(from EtOH) ne R.f. 0.50 (EtOAc:hexane 1:1); vmax/crrr 1 (BM) 3349 (NH) 3063,

2948, 1745 (CO), 1641 (CO), 1519, 1490, 1452, 1353, 1220, 1162; .sR (CaOeSO,

200MHz) 3.561 (d, lH, 2J 13.3, CH.Jls), 3.668 (s, 3H, COaCHa), 3.731 (d, lH,aJ

13.3, CHAHa), 4.294 (d, lH, J 5.1, CR-S), 5.322 (dd, lH, J 5.1, 8.9, N-CH), 6.519 (d,

lH, J 8.9, NH), 7.2-7.8 (m, 15H,Ar); .sc (COCla 50 MHz) 170.41, 166.95, 137.14,

136.88, 133.61, 131.77, 128.95, 128.73, 128.51, 128.40, 128.32, 128.23, 127.1, 127.01,

55.67, 52.33, 51.03, 35.72; m/z (El) identical to 4.18.

Separalion of threo and erytbro N-benzoyl s- a.naphthylmethyl-jJ

phenylcysteinemethylester 4.28 and 4.2b. A quantity of 25g of erythro:threo mixture

was dissolved in 600 ml of boiling carbon tetrachloride which was allowed to cool

97

• at room temperature overnight. The floating crust that formed consisted of 6g

(50% yield) of 95% pure threo diastereoisomer which can be brought to 100%

purity by further recrystallization with 200 ml of carbon tetrachloride. The

combined mother liquors were evaporated to dryness and retaken by 400 ml of

hot 1-chlorobutane. After 16 h of standing at room temperature, a crop of 12.6g

consisting of a 3:1 mixture of erythro:threo diastereoisomers was obtained which

when further recrystallized with 1-chlorobutane provided pure erythrodiastereoisomer. Yield 9.8 g (78%) Threo (4.28) -M.p. 1U)"C (from CCI.); TLÇ

R.f. 0.47 (EtOAc:hexane 1:1); vmax/crrr i (PE) 3319 (NH), 1739 (CO), 1642 (CO),

1530, 1345; liH (CDCls 3ooMHz) 3.41 (s, 3H, C02CHs), 3.96 (d, tH, 2J 13.0,

CHAHs), 4.13 (d, tH,2J 13.0, CHflB)' 4.33 (d, tH, J 6.1, CH-S ), 5.11 (dd, tH, J

6.1,8.0, N-CH), 6.67 (d, 1H, J 8.0, NH), 7.22 (m, tH, Ar), 7.29-7.51 (m, 11H, Ar),

~~~~~~~~~~~~~~~~~~1~

Ar); lie (CDCls 75 MHz) 170.37, 166.94, 137.68, 133.97, 133.49, 132.30, 131.69,

131.16, 128.68, 128.51, 128.49, 128.46, 128.40, 128.07, 127.47, 126.98, 125.99, 125.77,

124.97, 123.94, 57.02, 52.21, 51.82, 33.48; m/z (El, ZIF) 456 (MH+), 424 (M+ ­OMe), 396 (M+ -C02Me). Erythro (4.2b). -M.p. 142°C (from 1-chlorobutane);

TLC R.f. 0.47 (EtOAc:hexane 1:1); vmax/crrr i (PE) 3308 (NH), 1738 (CO), 1640

(CO), 1532, 1435, 1325, 1219; liB (CDCls 300 MHz) 3.620 (s, 3H, C02CHs), 4.038 (d,

tH,2J 13.0, CHAHs, 4.185 (d, tH,2J 13.0, CH.Pa), 4.374 (d, tH, J 5.1, CH-S ),

5.340 (dd, tH, J 5.1, 8.9, N-CH), 6.531 (d, tH, J 8.9, NB), 7.23 (m, tH, Ar), 7.24­7.51 (m, 11H,Ar), 7.58 (m, 2H,Ar), 7.60 (m, tH,Ar), 7.73 (m, tH,Ar), 7.81 (m, tH,Ar), 8.04 (m, tH, Ar); lie (CDCls 75 MHz) 170.35, 166.85, 136.92, 133.92, 133.49,

132.42, 131.68, 131.24, 128.69, 128.61, 128.45, 128.35, 128.26, 128.22, 127.38, 126.91,

125.99, 125.70, 124.92, 124.00, 55.71, 52.25, 51.86, 33.56; m/z (El, ZIF' identical to

4.28.

Preparation of threo or erythro N-benzoyl-S-benzyl- jJphenylcysteine 4.38 and

4.3b and threo or erythro N-benzoyl-S- anaphthylmethyl· jJphenylcysteine 4.48 and

4.4b. -The following procedure was applied to bath erythro and threodiastereomers. A quantity of 10.0 g of ester was dissolved in 100 ml of boiling

glacial acetic acid and while the solution was maintained at boiling, 50 ml of 6N HCl

was added by increments of 10 ml. The reaetion mixture was maintained at reflux

l "If 2.5 h after whicb it was coolerl to room temperature. In the case of 4.38 or b

the solid deposited upon cooling and was colleeted by filtration, washed with cold

98

ethanol, and dried in high vacuum. In the case of 4.48 and b the mixture was

evaporated to dryness, pumped under high vacuum and the residue was

recrystallized using absolute ethanol.

TItreo N-benzoyl S-benzyl-ftphenylcysteine 4.38. M.p. 177"C (from ethanol);

TLC Rf. 0.55 (EtOAc:MeOH 1:1); vmax/crrr i (PE) 3500-2500 (vbr, CO,H), 3424,

1725 (CO), 1624 (CO), 1535, 1239; ~ (CDCla 300 MHz) 3.565 (d, m, 'J 133,

CH.JIB)' 3.686 (d, m,'J 13.3, CHAHs), 4.410 (d, 1R, J 5.4, CH-S), 5.120 (dd, lH, J5.4,8.0, N-CH), 6.885 (d, lH, J 8.0, NH), 7.2-7.4 (m, 10H, Ar), 7.4-7.6 (m, 3H, Ar),7.75 (m, 2H,Ar); <Sc (COCla 75 MHz) 172.96, 167.56, 137.56, 136.83, 133.10, 132.04,

128.87, 128.60, 128.46, 128.23, 128.08, 127.27, 127.07, 56.83, 50.30, 35.80; m/z (El,ZlF) 391 (M+), 373 (M+ -H,O), 346 (M+ -CO,H).

Erythro N-benzoyl S-benzyl- ftphenylcysteine 4.3b. M.p. 184°C (from

ethanol); TLC Rf. 0.55 (EtOAc:MeOH 1:1); Vmax Icrrr1 (PE) 3500-2500 (vbr,

C02H), 3327 (NH), 1726 (CO), 1624 (CO), 1535, 1489, 1239,1183; <SH (COCla 300

MHz) 3.547 (d, lH, 'J 13.3, CH.J:IB)' 3.662 (d, 1H, 'J 13.3, CHAHe), 4.396 (d, 1H, J5.3, CH-S ), 5.147 (dd, lH, J 5.3, 8.2, N-CH), 6.918 (d, lH, J 8.2, NH), 7.2-7.4 (m,

10H,Ar), 7.4-7.6 (m, 3H, Ar), 7.75 (m, 2H, Ar); <Sc (COQa 75 MHz) 173.42, 167.58,

137.61, 136.84, 133.16, 132.02, 128.89, 128.58, 128.79, 128.59, 128.44, 128.25, 128.04,

127.23, 127.11,56.83,50.47,35.79; m/z (El, ZlF) identical to 4.38

TItreo N-benzoyl S- a-naphthylmethyl- ftphenylcysteine 4.48. M.p. 154°C (from

ethanol); TLC Rf. 0.59 (EtOAc:MeOH 1:1); vmax/cm-1 (PE) 3500-2500 (vbr,

C02H), 3340 (NH), 1709 (CO), 1635 (CO), 1542, 1334; <SH (COCla 300 MHz) 4.04

(d, m, 'J 12.8, CH.J:IB), 4.10 (d, 1H, 'J 12.8, CHAHs), 4.50 (d, lH, J 5.7, CH·S ),

5.15 (dd, lH, J 5.7, 8.0, N-CH), 6.2 (br s, lH, CO,H), 6.90 (d, lH, J 8.0, NH), 7.22­

7.50 (m, 12H,Ar), 7.64 (m, 2H,Ar), 7.65 (m, m,Ar), 7.80 (m, m,Ar), 7.81 (m, lH,

Ar), 7.94 (m, lH, Ar); <Sc (CDC!a 75 MHz) 172.61, 167.54, 137.80, 133.87, 133.14,

132.34, 131.90, 131.18, 128.63, 128.54, 128.52, 128.35, 128.02, 127.43, 127.06, 126.01,

125.70, 125.04, 123.81,56.86,51.55,33.74; m/z (El, ZlF) 441 (M+), 423 (M+ .H,O),

396 (M+ -CO,H)

Erythro N-benzoyl S- a-naphthylmethyl- pphenylcysteine 4.4b. M.p. 120"C

(from methanol); TLC Rf. 0.59 (EtOAc:MeOH 1:1); 'Jmaz/cm- I 3500-2500 (vbr,

99

•100

C02H), 3345 (NH), 1710 (CO), 1634 (CO), 1539, 1488, 1341, 1238; Sa (CDCls 300

MHz) 4.05 (d, lH, 2J 13.0, CH}:IB)' 4.19 (d, IH,2J 13.0, CHARD)' 4.45 (d, IH, J 4.7,

CH-S ), 5.35 (dd, IH, J 4.7, 9.0, N-CH), 5.4 (br s, lH, C02H), 6.60 (d, lH, J 9.0,

NH),7.20-7.49 (m, 12H, Ar), 7.57 (m, 2H, Ar): 7.60 (m, lH, Ar), 7.79 (m, IH, Ar),8.03 (m, IH,Ar); lie (COCls 75 MHz) 172.73, 167.57, 136.53, 133.89, 133.19, 132.42,

131.85, 131.24, 128.70, 128.58, 128.49, 128.21, 127.37, 127.00, 126.00, 125.67, 124.96,

123.97,55.74,51.35,33.63; m/z (El, ZIF) identical to 4.48.

Preparation ofthreo or erythro N-trijluoroacetyl-S-benzyl-pphenylcysteine 4.88and 4.8b and threo or erythro N-trijluoroacetyl-S- a.naphthylmethyl-pphenylcysteine

4.9a and 4.9b. -The following procedure was applied to both erythro and threodiastereomers. A quantity of 3.5 g of ester (or carboxylic aeid) was dissolved in

150 ml of boiling fonnic aeid and while the solution was maintained at boiling, 150

ml of 6N HCI was added by increments of 20 ml. The reaetion mixture was

maintained at reflux for 15 h (13 h when starting with carboxylic aeid) after which itwas cooled to room temperature. The reaction mixture was evaporated to dryness

under reduced pressure. Water was added and the evaporation was repeated.

The process was repeated until no smell of fonnic aeid could be detected. The

solid residue was then treated ,vith warm ethyl acetate in order to remove the

benzoic aeid produced by the hydrolysis and the insoluble amino-aeid filtered and

dried under high vacuum. The free amino aeid was trifluoroacetylated as

described in chapter 5.

Threo N·trijluoroacetyl-S-benzyl-pphenylcysteine 4.88. M.p. 130"C (from

CClt); TLC R.f. 0.67 (EtOAc:MeOH 1:1); vmax/crrr l (HM) 3500-2500 (vbr, C02H),

3330 (NH), 1713 (vs CO), 1543, 1452, 1252(s), 1185 (vs); IiH (COCls 200 MH:L)

3.522 (d, IH, 2J 13.3, CH.JIB)' 3.677 (d, IH,2J 13.3, CHARD), 4.295 (d, tH, J 4.8,

CH-S), 4.954 (dd, lH, J 5.3, 8.1, N-CH), 6.0 (br s, lH, C02H), 6.907 (d, lH, J 8.1,

NH), 7.1·7.4 (m, 10H, Ar); lie (COCls 50 MHz) 173.28, 157.14 (q, 2J ISC·IDF 38.1),

136.69, 136.57, 129.09, 128.77, 128.30, 127.71, 115.56 (q, IJ ISC_IDF 288.5), 56.74,

49.87,35.68; m/z (El, ZIF) 383 (MW), 365 (M+ '~O), 338 (M+ -C02H).

Erythro N-trijluoroacetyl-S-benzyl-pphenylcysteine 4.8b. M.p. 135'C (from

CC~); TLC R.f. 0.67 (EtOAc:MeOH 1:1); vmax/crrr l (BM) 3500-2500 (vbr, C02H),

3311 (NH), 1715 (vs CO), 1548, 1452, 1251 (s), 1187 (vs); IiH (CDCls 200 MHz)

101

3.568 (d, 1H, 2J 13.4, CHflB)' 3.731 (d, lH,2J 13.2, CHAHs), 4.250 (d, lH, J 4.4,

CH-S ), 5.103 (dd, 1H, J 4.4, 9.2, N-CH), 6.614 (d, lH, J 92, NH), 7.1-7.5 (m, 10H,Ar), 10.9 (s, lH, C02H); Ile (CDCI3 50 MHz) 173.86, 157.30 (q, 2J 13C_19F 38.4),

136.67, 135.20, 129.33, 129.10, 128.79, 128.32, 127.62, 115.61 (q, lJ 13C_19F 288.4),55.22,49.79,35.71; m/z (El, ZlF) identical to 4.88

Threo N-trijluoroacetyl-S- a-naphthylmethyl- jJphenylcysteine 4.98. M.p. 153°C(from TFA·water); TLC R.f. 0.67 (EtOAc:MeOH 1:1); vmax/crrr i (PE) 3500-2500(vbr, C02H), 3287 (NH), 1718 (COs), 1554 , 1174; IIR (CDCI3 300 MHz) 3.99 (d,lH, 2J 13.0, CHflB)' 4.10 (d, lH,2J 13.0, CHAHs), 4.38 (d, lH, J 4.8, CH-S ), 4.95

(dd, 1H, J 4.8, 8.7, N-CH), 6.87 (d, lH, J 8.7, NH), 7.17 (m, 1H, Ar), 7.26-7.37 (In,

6H,Ar), 7.43-7.51 (m, 2H,Ar), 7.74 (In, 1H,Ar), 7.81 (m, lH,Ar), 7.91 (m, lH,Ar,),8.2 (br s, lH, C02H); Ile (CDCI375 MHz) 172.38, 156.70 (q, 2J 13C_19F 38.3), 136.52,

133.87, 131.63, 131.06, 128.80, 128.69, 128.59, 128.50, 128.09, 127.47, 126.09, 125.79,124.92, 123.54, 115.31 (q, lJ 13C-19F 288.3), 56.76, 50.76, 33.76; m/z (El, ZlF) 433(W), 417 (M+ -OH), 314.

Erythro N-trijluoroacetyl-S- a-naphthylmethyl- jJphenylcysteine 4.9b. M.p. 70oC (from methanol); TLC R.f. 0.66 (EtOAc:MeOH 1:1); vmax/cm-1 (PE) 3500-2500(vbr, C02H), 1720 (COs), 1542, 1172; IIR (CDCI3300 MH) 4.065 (d, 1H, 2J 13.0,CHflB)' 4.169 (d, lH,2J 13.2, CHAHs), 4.383 (d, lH, J 4.7, CH-S ), 5.155 (dd, lH, J4.7,9.3, N-CH), 6.753 (d, lH, J 9.3, NH), 7.2-7.6 (m, 9H,C02H, Ar), 7.6-7.9 (m, 3H,Ar), 8.05 (m, lH,Ar); Ile (CDC1375 MHz) 172.69, 157.29 (q, 2J lSC_19F 38.1), 135.06,

133.99, 133.73, 131.86, 131.20, 129.96, 129.07, 128.82, 128.73, 128.55, 128.23, 127.42,126.18, 125.87, 124.97, 123.75, 115.47 (q, lJ lSC_19F 288.5), 55.52, 50.66, 35.67; m/z

(El, ZlF) identical to 4.98.

Resolution of Threo or Erythro N-trijluoroacetyl-S-benzyl- jJphenylcysteine

4.8a and 4.8b. The resolution was performed as described for Erythro or TheoN·trijluoroacetyl-S-p-methylbenzyl- jJphenylcysteine in chapter 5. Monitoring wasachieved by TLC. Threo: S-benzyl'~-phenylcysteine 4.588 M.p 170 oC [a]D2D -179 (c1,85% formic aeid) TLC R.f. 0.5 (BAW); vmax/crrr i (BM) 3500-2500 (vbr, C02H),3440 (NH), 1605 (vs CO), 1498 (vs), 1375 (vs), 1347, 1260, 1105; SR ([2He]DMSO+[2H]TFA 200 MHz) 3.52 (d, IH, 2J 13.2, CH,.HB)' 3.72 (d, IH, 2J 13.2, CHAHs),

4.28 (m, 2H, CH-S, N·CH), 7.1-7.5 (m, 10H, Ar); Sc ([2He]DMSO +[2H]TFA 50

•

•

102

MHz) 168.90, 137.42, 137.00, 129.23, 128.92, 128.80, 128.67, 128.37, 127.36, 56.26,49.44,35.37; m/z (CI NHs, HP) 288 (MH+), 244 ~MH+-C02H), Exact Mass calc forC16H16NOS 288.1058 exp. 288.1059. Erythro S-benzyl-~-phenylcysteine 4.5ba M.p165°C ne R.f. 0.5 (BAW); vmax/crrr l (PE) 3500-2500 (vbr, C02H), 3316 (NH),1770 (CO), 1721 (CO), 1570, 1491; /lH (COsOO 200 MHz) 3.660 (d, lH, 2J 13.2,CH.JIB)' 3.800 (d, 1H,2J 13.2, CHAHs), 4.30 ID, 2H, CH-S, N-CH), 7.2-7.5 (ID, 10H,Ar); /le (COsOO) 169.67, 138.48, 136.61, 130.43, 130.16, 130.10, 129.91, 128.74,58.08, 49.0, 36.78; m/z identical to 4.5aa. Threo: N-trifluoroacetyl-S-benzyl-~­

phenylcysteine 4.8ab M.p 136 [a]D2D +150 (c 1, methanol) spectroscopic dataidentical to 4.8a. Erythro N-trifluoroacetyl-8-benzyl- ~-phenylcysteine 4.8bb M.p

133°C [alD2D -129 (c 1, methanol) spectroscopic data identical to 4.8b. The

enantiomers that were not hydrolysed by the enzyme were hydrolyszed with strongacidic condition identical to that described for the conversion of 4.3 to 4.5. Threo:S-benzyl-~-phenylcysteine 4.5ab M.p 179 oC [alD

2D +190 (c 1, 85% formic acid);Erythro S-benzyl-~-phenylcysteine 4.5bb M.p 179°C [alD

2D -147 (c 1, 85% formic

acid).

Typical reductive fission with dissolved metal in liquid ammonia on modelcompounds 4.3a and 4.4a. • The setup used for the metal in liquid ammonia isdepicted in Fig. 4.2. Ali the equipment used was placed in a weil ventilatedfumehood. The apparatus was pumped under high vacuum (0.01 mm Hg) andflame dried after which it was slowly filled with a stream of dry argon. The

condenser#l was filled with a mixture of dry ice and acetone and gaseousarnmonia was allowed to flow slowly into the system. Condensation occurred

readily and the flow of ammonia was increased such as to ccllect about 100 dropsof liquid arnmonia/min. It was not necessary to cool flask#1 to -78°C. When 20 ml

of arnmonia had been collected, stirring was started and 2 or 3 chips of sodium

were introduced into flask#l along with few mg of anhydrous ferrous sulfate. 150ml of arnmonia were condensed and the dry ice condenser#2, filled with dry ice

and acetone. Condenser#l was removed and replaced by a glass stopper. A largedish filled with water at 20 oC was placed under flask#l and the distillation began.

When 100 ml of arnmonia was distilled, part#l of the setup was put apart and

part#2 connected to argon line. The stirring was started in flask#2 and a tiny

amount of sodium (about 1 mg) was added into f1ask#2 in order to check fordryness. More sodium was added if required. The substrate (0.5 mmol) wasquickly introduced into the liquid arnmonia and the reaction temperature

103

adjusted to the desired value with an appropriate cooling bath. Reactionperformed at boiling ammonia temperature (-33°C) were accomplished by

wa..,mng the flask with a cold water bath a regular intervaIs. Sodium metal in glass

tube was then cut at the appropriate length and added. When ail the metal wasconsumed, the reaction was quenched by addition of anhydrous ammonium

chloride, usually in the same ratio as the metal. Dry ice condenser#2 was removedand replaced by a glass stopper and the evaporation performed by placing a large

dish filled with water at 20"C under flask #2. Warmer water bath was added

towards the end of the evaporation in order to ensure complete removaI of the

ammonia. The residue was finalIy pumped under high vacuo after which water

was added to the residue and the aqueous phase acidified to pH 2 with 5%NaHSO•. The mixture was extraeted with diethyl ether which was dried (MgSO.),

and evaporated to dryness under reduced pressure.

A similar procedure was used with calcium but the metal was added first

and allowed to dissolve completely before the substrate was introduced. If the

preceeding precautions were not applied, an unreactive gray precipitate formed

upon mixing of the substrate with the metal.

Threo N-benzoyl-jJ.phenylcysteine 4.10a. From the crude reaetion mixture

after work·up. liB (CDCIs) 2.3 (d, lH,J 6.8, SH), 4.60 (t, lH, J 6.5, CH-S ), 5.1 (dd,

lH, J 6.0, 8.0, N-CH), 6.3 (d, lH, J 8.0, NH), 7.1-7.8 (m, 10H, Ar), 9.5 (s, 1H,CO,H).

Erythro N-benzoyl-jJ.phenylcysteine 4.10b. From the crude reaction mixture

after work·up. liB (CDCIs) 2.4 (d, lH,J 6.8, SH), 4.75 (t, lH, J 6.5, CH-S ), 5.35

(dd, lH, J 6.0, 8.0, N-CH), 6.85 (d, lH, J 8.0, NH), 7.1-7.8 (m, 10H, ù'), 9.5 (s, lH,CO,H).

Separation of N-benzoyl- fJ.phenylcysteine from N-benzoyl- fJ.phenylalanine.

The crude reaction mixture of a reductive fission reaetion was injeeted into anHPLC and a gradient starting with 20% CHsCN in water and going to 80% CHsCN

applied over a p:riod of 20 min. A reverse phase Cil II. Bondapack column

opcrating at 1 ml/min was used. The compound eluting at 16.1 min was shown tohave a mass of 270 (MH+) and the one at 17 min had a mass of 302 (MH+). The

104

mass measurement were performed with the electrospray technique on a SciexAPI ID mass spectrometer at Biotechnology Institute of Montréal.

Y..euumor _

....gon

Dry ieeeondenSl!r 1

--- Dry ie.eondens..r 2

Fluk 2

Stopper -----+ ~rgon

outl.t

.00­L4lb,::---..:t~==/tj ..cks

"stirr.rpl..tes

FI..sk 1

Glus eo..t.dr-~~~:::::~ magnetic

stirnrs

Fig 4.2 Setup for reduct-ive fission using dissolved metal in liquid ammonia

4.7 References and notes.

1. U. Nagai, and V. Pavane, Heterocycl~ 1989,28, 589.

2. O. Ploux, M. Caruso, G. Chassaing, and A Marquet, l Org. Chem., 1988,53,

3154.

3. H. E. Carter, C. M. Stevens, and L. F. Ney, l BioL Chem., 1941, 139,247. see

also for a more extensive review J. P. Greenstein and M. Winitz in Chemistry

ofthe AminoAcids, R. E. Krieger Publishing CO., Malabar, Florida, 1984,

chapter 50.

105

4. H. B. Gillespie, and H. R. Snyder in Organic SynJhesis Coll VoL 2, A H. Blatted., John Wiley and sons Inc., Ney York, 1959, p 489.

5. In one experiment we were drawn to wrong conclusions conceming thehydrolysis with a.chymotrypsin since a product having the expected R.f.

(according to the product which was prepared by chemical hydrolysis) wasdetected. We realized later that it was due to the presence of peroxide in

dioxane which presumably had oxidized the sulfur of the substrate to the

sulfoxide and by accident the resulting product has the same R.f. as the

expected acid. Moreover since the amount of peroxide was limited, sorne

unoxidized compound was still present which really make it look like a

resolution reaction.

6. H. Goldenberg, and V. Goldenberg, Arch. Biochem., 1950,22, 154.

7. J. R. Rachele, J. Org. Chem., 1963,28, 2898.

8. H. Dugas, and C. Penney in Bioorganic Chemistry: A Chemical Approach toEnzymeAction, C. R. Cantor Ed., Sringer-Verlag, New-York, 1981, p. 208-226.

9. D. Dressler, and H. Potter in Discovering Enzymes (Scientific American

Library series), W. H. Freeman, New York, 1991

10. G. E. Hein, and C. Niemann, 1. Am. Chem. Soc., 1962,84,4495.

11. M. A Stahmann, J. S. Fruton, and J. Bergmann, 1. Bio. Chem., 1946, 164,753.

12. N. Abramowitz, 1. Schechter, and A Berger, Biochem. Biophys. Res. Commun.,1967,29, 862.

13. J. P. Greenstein, and M. Winitz in ChemistryoftheAminoAcids, R. E. Krieger

Publishing co., Malabar, Florida, 1984, chapter 20.

14. F. Weygand, and R. Geiger, Chem. Ber., 1956, 84, 647.

15. It is worth mentioning that trifluoroacetamides were very sensitive to basic

conditions and cleavage was observed at pH 10. At this pH concomitant

epimerization occured.

16. Resulting from degradation products during the acid catalysed hydrolysis of

the benzoyl group.

17. S. Yanari, and M. A Mitz, 1. Am. Chem. Soc., 1957,79,1150; ibid 1154.18. J. M. Stewart, and J. D. Young in Solid Phase Peptide Synthesis, 2nd ed., Pierce

Chemical co., Rockford, Ill., 1984, P 95.

19. M. Smith in Reduction: Techniques and Application in Organic Synthesis, R. 1..Augustine Ed., Marcel Dekker, New York, 1968, 165 p.

20. Handbook of Chemistry and Physics 60th ed., R. <... Weast and M. J. Astle

eds., CRC Press, Boca Rato, Florida, 1980.

106

21. V. A. Pleskov,J. Phys. Chem. (USSR), 1937,9,12; H. L Dryden Jr. in Orga!licreactions in steroid chemistry, Vol 1, J. Fried and J. A. Edwards Eds., VanNostrand Reinhold co., New York, 1970, Chapter 1.

22. S. H. Pine, J. B. Hendrickson, D. J. Crarn and G. S. Hammond, in OrganicChemistry, 4 ed., McGraw Hill, 1980, P 990.

23. V. G. Mairanovsky, Angew. Chem. lnt. Ed. EngL, 1976,15,281.

24. M. Bodansky, and A. Bodansky in Practice of Peptide Synthesis, Springer

Verlag, New York, 1984, p 161; V. DuVigneaud, L F. Audrieth, and H. S.

Loring, J. Biol. Chem., 1930, 52, 4500.

25. J. R. Hwu, V. Chua, J. E. Schroeder, R. E. Barrans Jr., K. P. Khoudary, N.Wang, and J. M. Wetzel, 1. Org. Chem., 1986,51,4731.

26. A one electron process implies immediate rupture after the addition of the

first e- leading to the thiolate and a radical. The concommitant convertion ofthe radical to carbanion consume one more equivalent of metal (e-).

27. R. Gerdil, and EA C. Lucken,1. Chem. Soc., 1964,3916.

28. W. E. Truce, D. P. Tate, and D. N. Burdge, 1. Am. Chem. Soc., 1960, 82, 2872.

29. W. E. Truee, and J. Breiter,1. Am. Chem. Soc., 1962, 84, 1621.

30. W. E. Truee, and F. J. Frank, 1. Org. Chem., 1967,32,1918.

31. R. Adams, and A. Ferretti, 1. Am. Chem. Soc., 1959,81,4939.

32. The ratios were inverted when the other diastereoisomer was used as a

starting material in similar conditions indieating therefore that the presence

of a1koxide does not cause thermodynamie equilibration to the more stable

diastereoisomer.

33. J. L Wood and V. DuVigneaud,1. BioL C:lem., 1939, 130, 109.

34. J. M. Hook, and L. N. Mander, Nat. Prod. Rep., 1986,35.

35. The relative configuration was determined later by X-ray diffraction of the

eorresponding acid 4.4a (see end of chapter 5).

36. Hartsuk, J. A., and Upscomb, W. N in The Enzymes vol III, P. D Boyer ed.,

Academie Press, New York, 1971.

37. M. A Stahmann, J. S. Fruton, and M. Bergman, 1. BioL Chem., 1946, 164,753.

38. Carboxypeptidase B is more specifie to basie amino-acids and consequentiy is

complementary to carboxypeptidase A.

39. E. L Smith, Adv. EnzymoLogy, 1951, U, 191.

40. C. Berse, R. Boucher, and L Piché,1. Org. Chem., 1957,22,805.

Chapter 5S'p·Methylbenzyl·~-phenylcysteine: a potential tool for probing receptor

topologies

Introductory comments

The fact that the N·benzoyl group was used in all the synthesis descnbed inchapter 4 restricted the choice of S protective groups only to those that were verystable to strong hydrolysis condition (HCl 6P reflux 15 h.). The synthesis could

however be refonnulated with a S protecting group more labile to acidolysis suchas ap-methylbenzyl but the N-benzoyl group has to be exchanged for a more labilegroup. The method of preparation of unsaturated azlactones restricted howeverthe RI group to aryl or alkyl substituent.S9 Consequently, the synthesis was done

similarly to that described in chapter 4 but with a S-p-methylbenzyl protecting

group and a N-acetyl group. The N-acetyl group will be hopefully removed withhydrazine, a reagent that was shawn by U~ to be inefficient in removing thecorresponding N-benzoyl group. The hydrazinolysis conditious will have to be

considered with care also since decomposition of cystine is generally observedwhen this method is used to determine the C terminal amino-acid of peptides orproteins. 40

The following paper report the results of this sturly along with sorne of the

observations already elaborated in chapter 4. Ail chemical reactions and

enzymatic reactions described in this paper have been perfonned by myself and

the paper was written in bulk by myself. Pr. Michel provide considerablecontribution in the X-Ray diffraction study and in the molecuJar modeling section.

Pr. Chan and Dr. John DiMaio contributed significantly in advices and

proofreading on the parts concerning the synthesis and resolution.

J. CHEM. SOC. PERKlN TRANS. 1 1993

S-p-Methylbenzyl-~-phenylcysteine: A Potential Tooi for Probing ReceptorTopologies

Gérald Villeneuve,· John DiMaio,b Tak Hang Chan· and Andr. Miche"'c, Department of Chemistry. McGill University. Montréal. Québec. H3A 2K6. C~mld,

b Institut de Recherche en Biotechnologie. Montréal. Québec. H4P 2R2. Canad~C Département de Chimie. Université de Sherbrooke. Sherbrooke. Québec. J1K 2R1. Canad~

1897

108

erythro and threo N-AcetYl~S-p-methylbenzyl-p.phenyl-oL·cysteine methyl esters were obtained byaddition of p.methyltoluene-ot-thiol to 4~benzylidene-2-methyloxazol-5·one in methanol­tetrahydrofuran under basic conditions. The diastereoisomers were separated as their melhyl esters byfractional crystallization and the relative configuration assigned by X-ray crystallogrsphy. Bothdiast(:teoisomers were converted into their N·trifluoroacetyl derivstives and resolved usingCarboxypeptidase A. Hydrazinolysis of the unchanged N·trifluoroacetyl amine acids gave the otherenantiomers of the free amino-acids. Optical purity was determinod using Eu(hfc)~ chernieal shihreagent on N·trifluoroaeetyl-o-amino aeid methyl ester by both'H and "F NMR speetra. We discuss theconformation of this unique amino acid based on X·ray data and molecular mechanics calculations. Itsusefulness in probing the opiate receptor site is also demonstrated.

The biological response elicited by peptide honnones andneurotransmiuers is usually mcdiated by interactions withspecific recepton. 1 The magnitude and spec:ificity of suthinteractions are govemed by the primary and secondarystructure oflhe peptide. which combine ineltricably to generalea molecular arrangementlhat is recognized by the receptor.andby complementarily, should reIJectthe lopography oflhe activesite orthe receptor. 1

Struclural modifications of the peplide caused by C- invenedconfiguralions. i!>osieric replacemenl of Ihe funclional groups,altered side chain hydrophobicity, or inlramolecular cyc:liz·ation J may serve 10 slabiliu or penurb Ihe nalive conform·ation. Acc:ordinSly, sublle modulalions in Ihe peplide-reœplorcomplex may manifest inc:reaxd efficac:y. anlasonisl propeniesor enhanc:ed seleclivily in siluations of rec:cplor heteroseneily.

Cysteine serves a specialized func:tion in nalive peplides byengaging in disulfide bridge fonnalion. This propeny serves lajUltapose disc:onliguous functional elements. la slabilize activeconformalions or impan enhanced proteolytie resistalKlC.·Hruby and co--workers' have shawn Ihat penicillamine cansubstÎlule for cysleine bUI elens strons inIJuenœs on peplideconformalions owinS to lhe gtm-dimelhyl group.

On Ihe olher hand, phenylalanine possesses a paromalîc: rinlwhich is a c:rilical elemenl in lhe bindin. of severallipnds 10their reœptor (l'.g. enkephalin. substance Pl. The ftat aromaliering can interaci wilh hydrophobie microenvironmenll in thercceplor and may elhibil favourable intetaClions wilh disulfidebonds.·

p-Phenylc:ysteine combines lhe propenies of these twonalurally ocxurrins amine acids bUI po5SQSCS an addilionalasymmelric œntre al the p carbon which may he useful indccipherins slereochemic:al preferences Ihould the aryl groupenPSe in rcœplor inletldions. In Ihil rqard. MOIhefJ 1'1 al. 'have reponed the effoct on opioid nœplor inleractions exenedby an enkephalin analogue incorporalin, '"'1''' or try,lrro p.melhyl·D<ysteine.

Previous preparalions of P.phenyl·oLoC)'steine inc1ude thesynlhesis by Svoboda 1'1 4/.' whic:h Ied la Ihe t,y''''o dia~

slereoisomer and the one by HoUand and Mamalil' whichpve Ihe ''''1'0 îsomer. Thae two methodl were reinveslipledrc:œntly in a ICarth for new acelaldehyde Kquestratin,agents." Chiral rontlS or Il-phcnylcystein...... indcpenden.1yoblaincd by IWO leams. PlOUl " tIl. 11 bucd lheir approach GD

the regio-- and stereCKontrolled opening of 2-phenyl.)-menth·olycarbonylaziridine by 4-melbolytoluene·s·thiol. They how­ever experienced difficully in oblainins III four aziridines inenanliomerically pure (onn. Nalli and Pavane Il prepared thet,y''''o Ind r",to oL·amino acid by Ihe 1,4 addition t)( p.metholytoluene-o:-lhiolto N-ebz-dehydrophenylalanine. AOerN-S deprolection, they fonned the acelonide which WIS thebasis o( dilSlereoisomeric separalion by crystal1il.llion andresolulion by way of • preparalÏve chiral suppon. Thiscommunication repons the synlhesil and enzymatie rcsolulionof ail the four possible enantiomen of S·p-methylbenzyl·P.phenylcysleine. The X·ray slrudure delennination wasperfonncd (or ''''1'0 N·ac:etyl·S-p·lnelhylbenzyl·P.phenyl­oL.çysteine ntelhyl ester la in orderlo ISSiBR unambiluoull)' Ihecorra:l relative slereochemislry. Since Ibis amino lCid wouldhe evenlually incorporated in biolosically aClive peptides andlinked throulh a disulfide bridse la anolher Ihiol conlaininsamino acid. WC have also perfonned molecular mcc:haniCi cal·culalions on model strudures. The confonn'Iional preferem:esfor the side chain in relalion ta various blckbone eonfonn·alions were delennined and Ihelr compalibililY wilh previoullyproposed aClive confonnalionl for opioid peptides wasevaluated.

RosuU.SyntMSÎS cuul RtJolu,ÎOII.-The melhod we have adopted il •

variation of Ihe procedure dC\·~lolJ(d orisinally by Nicolel l )

and ellendcd Ialer by Caner t' al.'· This melhod relies on Ihefacile addilion ofthioll ta unlliuralcd azIaclones (ICC re(. 15 fora review ofazladone chemislry). AccordinslY, 4-benzylidene·2·melhylollZOl·5.one wu Irealed with sodium p melhylloluene·s·lhiol in methanol-tetrahydro(uran (MeOH-THf) .rrordin,• 1:1 trYI",o-,lr,to mixlure of N.acetyl·S·p-melhylbenzyl·P.phenyl,oLoC)'sleine melhyl esler al 1 consequence oflhe openinlof lhe IIluralcd IllaClone by lhe methanol in the medium(Sc:hcme 1).

separalion of diaslereoilOmen provee! la be very llrai&hl~

(orward alihis stase. DÎlIOlulion of Ihe crude reaClion millurein boilinl elhanol and coolinllO room temperalure pve onediulereoisomer in an analyticall)' pure fonn and whOte c:rystallwere suitable (or X·ra)' lll'UC'ture detmninalion. The ''''1'0relalive confiauralion wu .uil"cd 10 this diulereoilOmef.

t~N"*-:FH,N hC~FAc YXo 0__ HN SOMoChymo. DL lino (m.p.137-<:)01 StbtiI. 1.

CO.... CO,Mo l....-->L _N+H H+"'Ac~AfCH,s+H H+5ai,AI N·p·w.c,H.

PI> PI>

Dl ~1ro (m.p. 113-c),.~ 1 karrnlJ GNi C'ondiliOlU: i, NaOAc-Ac,O, rdu.. 2 h: ii.NaOMe (cat.)-THF. McOH. room temp., J h

Subsequent coaling or the ethanolie solution 104°C providedaRother crop or the Ih,~o isomer. uluaUy contaminated with aminor amOURI or the ~'YI"'o isomer. When the remainingmoiller liquor was evapora.cd 10 dryness and redissolved incarbon lcn.chloride-penlane and cooled al _20 Ge,lhc purerry,nro diaSlercoisomcr was oblained as shawn by NMRspectroscopy.

HaviRa in hand the IWO pure diaslcreoisomen. altempls 10rcsolve the racemic millures usinB ll-e:hymotrypsin andsublilisin were .boni"e for either diaslcreoisomer (Scheme 1).AUempts 10 increase lhe subsn.le binding by repl.cins the N·acelyl gloup wilh N·benzoyl also proved unsua:essful. EvenIhoulh Ihese trials had 10 be conducled in saluliolls rich inorlanic solvent (500/0 of melhanol or dionDe in watCf). theincrtness of Ihesc subslralcs may be allribuled 10 bulty pbranchinl.·· Il is ~ole"'orthy Ihal only the ~,yl"ro diulerea-­isomer of p·p.nitrophenylscrine melhyl esler is hydrolyscd bylI-chymotrypsin. l'

HOI renlilcylisc 1was allempled as Ihe nexl choic:e. Owinl10 the acid labilily of Ihe p-methylbcnzyl aroup." lhemelhyl cslers Iroup c.,uld nol be rtmoved by acid hydrolysis.Allernalively. saponificalion usinl NaOH (0.1 mol dm-") inTHF-MeOH-waler mixture (1:3:1) al room lempertllurecauKd complele epimeriulion which could be suppressed usinl2: 5 methanol-wlter binlry solventll reftux for 2 h. The pureIcid.ls revelled by NMR coukl he isolated in ,000 yield. Belhdilstereoisomen. however. were raillanl 10 the action ofacyllsc 1. Simillrly. N.acctyl.P.pheny1serine l' Ind N·aœlyl·S·benzylpenicillimine lO Ire notalllCted by Ic:ylase 1.

The N·lcclyl sroup wu Ihen elthanllUl for triftuoroac:clylwilh the hope Ihll Ihe more elCl:trophilic carbonyl would bemore susceplible 10 IlIlCt by enzyme. The N·accly1aroup wuremoved by trealment wilh hydrazine Il 60 -c for 15 hli andthe crude free amine .cid reacylated with Iri8uoroaccticanhydride in lriftuoro,cclte acid It 0 -Cu in 66" overall yieldfor lhe IWO step!. The .cids wcre carefully converted inlo Iheirwaler soluble lilhium salis and subjecled 10 .cylasc 1hydrolysis.HoWC\ltr. Ihe enzyme wu inca:-ble of deaYins lhe IClivaled.mide. This Plrt of Ihe work is summarimi in Sc:heme 2.

When lbese Iri8uoroacclamides were incubaled with Carb­olypqnidut A (CPA) al a œnc:entralion of 0.01 mol dm·J •uslow amidolysls wu observed for bolh diaslereoïsomm. Theresolulion was t'onveniently monitorcd by followinl lhe

1898

'po OHHN~O •

HyN 0

1PI>

J. CHEM. soc. PERKIN TRANS. 1 1993

yoo -,1.,1b ---!.-

HNrOH --r

PI> S

;/l.....

2.,2b

0 CF~yO 0

H~rOH XOH ......,----!L- --r

PI> S

l.... l.......... ......sma. 2 RqntU tWJ CONiilioru: i. NaOH (1.1 equiv.. 0.15 moldm-Il. HzG-McOH (5:2). rcftWi. 2 h: li. NzH•• 6O DC. IS h: iii.(Cf)COhO. CfICOzH. 0 -Co 10 min

auenualion of Ihe peak correspondinl 10 the starling malerialby hilh performance liquid chromalolraphy (HPLC) al 254nm. The peak area decreascd sleadily unlil halr of theconsumption was reached whereupon il remaincd esscntiallyconstanl ror a prolonsed period (1 wect). Typically. 10000unils ofCPA were required 10 hydrolyse 10 mmol ofsubslrate in48 h at 20 De. The HPLC measurcmenls also pcnnillcd us 10evaluale the relalive reaetion raIes: (i) The raie was doubledwhcn ehe amount of enzyme was increascd by a faclor of IWo,and Ihis was independent ofenzyme balches. (ii) No significanldifTerencc in the raIes of hydrolysis was obsef'\'ed belween Ihelwo diaslereoisomers. In here. we are meeling the reasonableassumption thal ail Ihe L·tlrr~o or L~rJ·tlrro isomers werehydrolysed. by lhe enzyme. and that the o-isomers were notac:ccptable subserates A·CPA.

The unchansed N.ui8uoroacetyl-D-Cnanliomers were iso·taled by acidificalion and diethyl elher eltraction while Ihe L·zwiuerions. which were very slightly soluble in waler. wereprecipilaled almesl quantilali...ely by t'onccntralion of theaqueous phase. The final rernoval of the triftuoroacclyl oflhe 0­isomcr could nol be affCl:ted using lhe conditions 200/0 acelicacid reflux 4 h. dcscribed by Fones for p-phenylserine l'andhydrazinolysis WIS apin used for Ihis reaction. The mislanccof lhe lrinuoroacclyl Jl'OUp to acidic hydrolysis can beexplaincd by Ihe absencc ora proximale nuc:leophilic OH Broupwhich can promole lhe acid hydrolysis in the case of p.phenylserine. The resolution sequenœ and the relalcd oplicalrOlalion values .re given in Sc:heme 3. The 0plic:al rolalionobtaincd for each enanliomorph wu of the same sign '5reponcd for P.phenylscrine [Lit. (- )L·,lrrm and (+ }Lof'rythro]in acidic ~diJlm." P.phenylc:ysleinr. melhyl ester.Il and N·I.Boc.S-3.nitro-2.pyridylsulfenyl.p.phenylcysleine. Il

Sincc optical rolilion is not a criterion for oplical purilY, theresolved compounds were analyscd by NMR. Farsdy. theracemtc Iriftuoro'CClamidcs .. and ... were eslerified wilhdiazomethane and Ihen milcd wÎlh chemicalshifl reasenl up 10a motar ralio or 1:0.3 amino ac:id-Eufhfch. Allhis ratio the IHNMR speçlrum showcd considerable chanp. The doublel ordoublel (dd) resonanc:e correspondinl 10~H reverted to 1 Silline multiplel. while lhe CtH doublel and the methyl eslersinglel 'Nere doubled. The S·benzylic CHzAB syslem alsoelpericnccd splillÎns. The l'F speclrum also showed two linesror the CFJ absorplion. When this elperiment wu performedw;th lhe resolved c:ompounds at Ihe same chemicalshiR ragenlconcenlralion. no .pliltinl œuld be detCl:lcd. indicalins anoptical purilY of Il luit 9S".

109

• J. CHEM. SOC. PERKtN TRANS. 1 1993 1899

110

DL ·tlno

••CO""

H"'+HH+SCH,Ar

""

+

D.r~ J,ab(a 10.,117

1·CO""

H+NHCOCF1

M:H,s+H

""

H H a

MlIY~~''''o I~ - ~37,

.s...,.:

• .. (11'no)

Cl..erythto -!­'b

L·lIno J ••la):-1~

HaN+"':ArCH,S+H

""+

o ·rlno "lblal~+ 1511

Co,H

H+NHCOCF,

H+SCH,Ar

""

Fia•• ORTEPplolorI.

Ob (.,.IVo)

FIa.1 Madel mokcules used in enerlY cakuillion. Variable lors;onalanlles Mre~: 1. 2. J. 4;.,: 2. J,". 5; Il: 2. J. 6. 1; X,: ;.6,1, B; 1)'6.7.8.9.

,") general1y found in C)'1teine or cystine derivutives. u Theorientalion of the Pphenyl ring is Irans (,) with respecllo thenilrogen atom. Dy comparison. ail slassered rolamers <'C-. 1.,0) have been observed by X.ray diffraclion studies ofphenylalanine derivillives.1• Several IlIrro p·phenykysteinederivatives have becn analyscd by X-ray diffraction in ourlaOOralory and ail have shawn this side chain arranlemenl.n

ln order 10 evaluale the confonnalional repercussionsinduced by the pphenyl ring in an evenlual disultlde bridge, wehave perfonned a systemalic conformational analysis bymolecular mechanic:s on models struclures S. 61 and 6b shownin Fig. 2. We have considered the fivc most stable bacltboneconformations A, C, D, E and F following lhe code ofZimmennan " al. n to idcntify the (\J, ,,) map local energyminima. Thesc fi' ! bac:kbone confonnations were combinedwith 18 side chain orienlation (corrcsponding la combinltionofg" ,1.'- for XI' g+ • 1,,- for 11 and +90, _90° for 1)' Thercsulting ninety conformers \lIere energy minimized. Fromenergy dirrerence observed for cach bacltbone conformations,the side chlin rolamer populalions were calculalcd Il T. 300K (sec ellperimental section). Table 1 lives the calc\!I:tledrelalive populations.

ln the case of 5. Ihe XI population is evenly distribulcd forbackbone A and D. The g - rolamer constitutes half of Ihepopulalion for backbone C and F while the, 0 rolamers is Ihemost popullted for an extended backbone (E). The' rotamc:r isncverthelcss observe<! wilh ail bac:kbone confonnations.

For compound 6a (,lrrto), lhe XI population is also evenlydistributed for il D backbone, but this lime the,· rotamer isstron&ly flvoured for a C and F backbone and shows animponanl contribulion for backbone E and D. The X-raystruclure corresponds la Ihe F backbone conformalionassoc:ialed wilh lhe same,- rotamcr of the side chain. 1be ,rolamer is hilhly preferred for an A backbone and hu animportanl contribution for Ihe Econformation. The, 0 rotlmeris only found la an apprec:iab~ eAlenl wilh a D bacltboneconformalion. Therefore, il appears for slrutlure 6e thallhe,­rolamer is highly populaled for four batltbones. This iscorroboralcd by the larae (8.S Hz) couplinl constanl meuuredbelwcen H' and HI in [1H.]dimethyl sulfoAide ([1H.]DMSO)for Il CH' and HI are , when N and S lire ,. (Fil. 3). Thisoverall tendençy of Ihe,· rolamer for lhe 11 lonional an.'emay be eAplained in tcnns of reduc:ed steric clTectl. In fae:t lhcreare IWo puche inlerlelions with the,' rotamer whi'e lbere areIhree wilh the , or ," rotamen (Fil. 3).

ln the case of compound 6It (~rYI"ro). the 11 populalionanalysis reveals tbat the , rotarner is (avoured Wilh 1 C or D

C119)

O'~tro 4bb101:-,7S1

C(7) 6IfI""'I1~~;....o;ll

CI91'l"";!J-.,~ ...

X-Ray Strudll'~ and COII!orfMlional Ana/ysiJ.-A perspec·tive view or the crystal structure of lhrto N·aœtyl·S·p­R1Cthylbenzyl-L.phcnyl'DLoC)'slcine melhyl ester la is shown inFig. 1 for Ihe L-enantiomcr. The ac:etylaled amino aad adopts 1

semi eJ.tended bac:kbone c:onfonnation where lonional anBles, and 1/1 U adopt values of - 61 and 1390 respectivel)' (Table4), This baclcbone confonnalion gjves rise 10 an infinitc H·bondchain along the crystallographic Z axis where the NH (x, y.:) ishydrogen bonded 10 lhe armde carboDyl 0(3) of Ihe mirrorimage mokcule (x. 0.5 - y,O.5 - :). Respec:live dislances are:N ···0(3) 2.951(2): H .. • 0(3), 2.15(2) A. The lhr« alom.No •• H .··0(3) fonn an angle of 165(2)°. The anBle belweenIhe planes ofhydrogeJ\ bonded amides is SOO.

One imponanl feature of Ihis unique amino acid is lhe sidechain conformalion. The sulfur atom adopts the, - orienlationrelalive to the nilrogen atom Idihedral ansle Xl - -6,.),correspondinslo one of the Iwo side chain conformations (, - •

l'o·~'bb

(aJ:- 175

Sc:bant 3 RlQgmu and cDNiitions: i. substrale cone. 0.01 mol dm') inwaler, LiOH (1.0 equiv.l. LiCl (0.1 mol dm-'), pH 7.5. 2O'C. CPA; ii.NJH•• 60'C.ISh

111

,- , ,1.

.,.. J Newmann projections Ilonl the c--el axis (xl) (or the Ihm:siauered rollmen ofcompounds" and"

X, with compound S. slight preference (60:40) for One of thetwo rotamers appears with compounds 6a and 6b and is relatedto the chirality of the ncighbouring C· stercogenic centre (+90for 6a and -9QO for 6b).

J. CHEM. soc. PERKIN TRANS. 1 1993

Fil. 4 ProPf'scd active confonnalion for the d selechve opiale Tyr.D-~en-Gly.Phe·CY5.NH:H allowing for the insenion of a rrytJ.rophenyl.LoCyslcine in place of L-CyS. Van der Waal surface com·sponding 10 0.5 times the aC'lual radii is shown for the n~'ly inxncdaromaliC' ring.

Exporimetlta'Equipmmt.-Melting poinu were recordcd on a Gallcnkamp

c:apillary apparatus and are uncorrectcd. In sorne cases.epimerization ofdiutcrcoisomcr oocurrcd upon slow heatina: aspecial note is indicatcd in the text in this case. This bchaviourwas ascertained by runnin.an NMR spectrum bcfore and aflerhcatin. the sample to complete fusion. Infrared speetra wereRCOrded on a Perkin-E1mer 1600 FT1R and were taken as KRrpellets. 'H. "F and "c NMR wm recorded al 300. 282 and 71MHz respectively on a Varian XL-300 and are rtferenced to the

DlscussiooThe presenl melhocl for obtaining ail four isomers of li-phcnyl­cysteine has the advantap: over previous methods of heingsuilable for large scale preparation since no chromatography isrequired. Ali purifications and separations are pcrfonned byextrdction and cl')'stallization. Enzymatic resolution is efficienland low cost espccially using immobilizcd enzyme preparation.

N-TriOuoroacetyl·S·p·methylhenzyl·p·phenylcysteine is oneof the largest amino acids to he hydrolyscd by CPA. The Iimitof structural tolerano: was investigated by preparing Ihecorresponding S-a.·naphthylmethyl derivative" (Ar = z·CH,­CIoH,) using similar mclhodology. Neither diastereoisomerwas hydrolysed byCPA. Thcse observations might bc helpful indelincaûng the sizc of the dead end poeket alloucd to the sidechain of the amino acid hydrolyscd by CPA.

Compared to cysteine or jJ.phenylalanine. p·phenylcysteinepossesses a rigidified side chain when engaged in a disulfidebridge (lIitk SIIpra). Thercfore, if the P.aromatic ring is involvcdin specific binding wilh a putative rc:c:eptor molcc:ulc, dramatie:efTcc:u should he expcc:tcd dcpendina on the diastereoisomerchosen. In order to demonstrate the potenlial intCl'CSt in usingthe P.phcnylcyslCine residue in the context of probing rcc:eptorsites. we have inçorporatcd Ihis panicular residue with itsprcdictcd most stable confonnation ioto a potential opiateligand. This simulation is to he relatcd to our prcc:eding paper"on the scarch for the active confonnation of the d selectiveopiale Tyr-D-~·Gly·Phe·Çys·NH,. The cysteine adopts a Dbackbone conformation and Xl and X, were bath transwhich is compalibk with the preferred conformation of an~rJ'lliro-phenyl-L<ysteine (sce Table 1). The peptide substitutedwith an ~r)·tllro-phcnyl·L<ysteine is represealed in the proposedcanfonnation in Fia. 4. Examining this structure, wc observcdthat the ncwly ioscned phcnyl ring creatcs an interaction withthe putative bindina subsite associatcd with the tyraminemoiety. Progras in preparing Ihis peptide is under way. andthe repcrc:ussion on binding propenics wiU he investigatcd.

::$::"H

..

1900

Ta" 1 Cakul.llcd relative lide chain rolamer populations u arunclion or b&ckbone confonnations for mode! molecua S." and QIlelpreued in %)

80lumann probability

1. l, l,Compoundlbackbone)" "

,"

, +90" -90"

• lA> JI l7 l2 JJ l4 II " 49ICI 9 II l6 l2 l6 l2 li 49,D, l6 20 44 29 II l6 47 II(E) 68 19 Il l8 26 l6 l2 48IFI 14 l4 l2 II li l4 l2 48

"(A) l 77 18 21 79 0 6l l7ICI 10 0 90 7 8l 10 61 19101 44 18 l8 14 8l l 6l l7'El Il J9 JO 20 80 0 68 l2IFI 2 10 88 25 74 1 64 l6

AbIAI Il 27 60 71 27 42 SRICI l 97 0 l 91 6 li ..101 4 66 JO 2 91 7 l7 6llEI 71 10 19 0 7J 27 l4 66IF) 56 l4 10 0 89 Il II 6l

• Conformai ion is described a"aldina 10 ,. " Zimmc:rman et al.code Ielltr: U "''''CfaF lonion angla(-) wele: AC -63 :!: B, -41 ::l: 6},CI -12 t J, 69 :!: 4), D( -145 t 10, S9 ± 10), lU -154 ± ID, 160 t81. FI -79 t 9, 154 ± 10); 11 and II .tdc chain tonion anaJes (.)wttt.·(-60 ± 10I,,'(+60:!: 10),1(180 t 10)

:* :* H:$:Ph..C NH Ph S.- .-

blckbone çonfonnltion while the , + rotamer is flvoured foran Eor F backbone. 'The: ~ rolamer is the mOit ravoureJ whenthe backbone adoplS an Aconfonnalion. Il is more dif1ku11 inIbis tise 10 aueu an overall preference for the XI dihcdralan,le. The Newmann projections (Fil. 3) show that there arctwo Bauche inttnClions in the, rowner while there are thrœ inthe ,. and ,. rotamen. Howevcr, up 10 no'll, wc have beenunable to oblain suitablc aystals orthe try,Aro diastereoisomerfor an X-ray diffraction st\ld)', but • larae couplinl canstanl(10.0 Hz) be.wcen H'IJld H' illlJo obsc..ce1 in ['H.JDMSOwhich scems to indiCile a predominance orthe' rolamer iD tbismedium (H· and H' are' when Nand S are 1).

The distribution over X, is much more inftumœd by theintrodPCtion of the Pphenyl substituenl. While aU X, rolamenwere cqually populatcd for model compound 5 indepcndent:yof the blckbone confonnation, the 1 rolamcT is much moref....oumi for bolh 6a and". A small population of,· and,­rolamer however pcrsÎSts for. and" respectively.

Finally, whilc both confonnations were cqually probable for

J. CHEM. SOC. l'ERKIN TRANS. 1 1993

residual solvent peak (CDCI) IH 7.24 ppm. De 77.0 ppm.CO)OD. IH 3.30 ppm, ue 49.0 ppm, (2H,]DMSO, IH 2.49l'pm. ue 39.5 ppm); J-values are given in Hz. Thin layerchromatography was carried out on glass backed Merck silicagcl60. 0.25 mm thickncss. OpticaJ rolations were mcasurcd on aJASCO DIP-I40 instrument at sodium line wavelength at 20 0 ein a 0.5 dm ecll and the values were inl~iraled OYer a p:riod of60 s; [:X]D values arc given in units of 10" Cleg cm2 g-l. Massspcttra and exact mass were determined with a ZAB-IF massspectromeler. Enzymatic relclions were monitored using a HPI090M analytical HPLC equippcd with an aUlomatie injcc:torand a 15 cm Il Bondapack reverscd phase eIl column.

Reogents and SolworJts.-p-Methyltoluene-lI.lhiol was pre­pared from the corresponding 4-methylbcnzyl chloride usingthe Bunte's salt methodology (reaction with sodium Ihiosulfaleand Ireatment WÎth hot dilule sulfuric acid).3D The malerial sooblained was dislilled (b.p. 97 oC 12 mmHg) and was esscnûallyodourlcss. N·Aeclylglycine and 4-benzylidene·2·melhyloxazol·5·one were obtaincd ac:c:ording 10 the procedures alreadypublished in Orgonic Synrhesis. 31 The azlaetone can be usedwithout further purification but rccrystallization from chlora.form al 4 oC faeililated purification al the nexl slep. Methanol'was dricd by dislillalion over Magnesium and telrahydrofuraoover sodium-benzophenone. Triftuoroacctic aeid (TFA) andtriftuoroaectie anhydride were from Aldrich and were wcdwithoui further purificalion. Carboxypeplidase A Type 1 wasoblained from Sigma, 4 cm' of suspension accounting for SOOOunits of enzymatic acûvily.

Preparation oflhreo and erythro N-Autyl.S-p-melhyllw,.:yl.p-pheny,,"ysleine Methyl EJler Il and Ib.-Ioto a dry 1 dm)three neck ftasil: equipped with a SOO cm' droppina f\lnnelunder positive argon pressure wu added dry methanol (150cm)). The solvent was cooled to 0 oC and freshly cul 50dium(0.5 g) was addcd with vigorous stirring. When aUlhe sodiumhad reacled. p-melhyltoluene..-thiol (24.9 8, 180 mmol) wuadded using a syringe. The mixture was Ihen allowcd to reachroom tempcrature and the addition funnel was fillcd wilh asolution of 4-benzylidenc-2-methyloxazol-5-one (33.4 10 17gmmol) in dry THF (400cm3). The solulion was addcd dropwiseover a pcriod of 3 h. The mixture was then treated wilhconecnlrated hydrochlorie acid unlil pH = 2 followed b)' ethylaectate (100 cm)). The layers were separated and the organiephase was washcd funher with water. The organie extl'llet wudried and evaporatcd under hÎgh vacuum affording a yellowail. The erude mixture c:onsisted of al: 1 mixlure of lheerYlhro/llIrto diaslereoisomcr U shown by NMR.

Separatio" oflhreo N.Arelyl-S-p-metllylbe":yl-p..plw,,ylrys.teille Melhyl Esle, Il.-The prccedina erudc mixlure WlS dis­solvcd in hoiling absolute ethanol (200 cm) and trealed withactivated eharcoal (1 a). The hOI mixlure wu fillered b)' lflvit)'and alJowcd 10 sland at room temperalure for 3 h. Larae prismsof the rllreo diulereoisomer precipitalcd from solulion. Thcscerystals were colleelcd by suc:tion filtralion and washed wilhcold ethanol (25 cm') providina pure la (II a). Funherc:oolinlof the fillrate 10 4 OC for 24 h alTordcd anolher erop of Ihuocrystals whieh were sliahdy contaminated wilh lhe: otherdiastereoisomcr. Recrystallizalion in the minimum amounl ofboiling elhanol (ca. 30 an') alTordcd 5 1 of additional pureIhrto diastcreoisomcr. M.p. 13~137 "C (from EIOH); nc Rt0.30 (EtOAc-hexa.. 1: 1); '.../an·' 3257 (NH), 3032, 2924,1742 (CO), 1643 (CO), 1537, 14l5, 1364, 1214, 1168;.l.(['H.)DMSO) 1.856 h, 3 H, CH,CO), 2.254 h, 3 H, ArMe),3.360 (d, 1 H, CH,H" 'J 12.9), 3.367 (s, l H, CO,CH,),l.531(d, 1H,CH,H" 'J 12.9),4.122(d, 1 H,CH-S,/8.4),4.n4(dd,1 H, N-eH, 1 8.5, 8.8), 7.00-7.10 (m, 4 H, SCH,A'), 7.26-7.35

1901

(m, 5 H, CH.~,), 8.491 (d, 1 H, NH, 1 8.9); .ldCDCI,) 170.57,169.64, 137.71, Il6.8l, 134.04, 129.09, 128.76, 128.42, 127.9l,56.60.52.21, 50.82, 35.22. 22.89. 21.00; ml: [elettron impact(El)) 357 (M'), l26 (M' - OM.), 298 (M' - CO,M'I, 141.217.210, 183. 168 (Found: M·, 357.1392. Cale. for CJDHU­NO,S. M, l57.ll98).

Separalioll of erythro N·Acelyl·S-p-ml'rJry/~n:)·I·p·plarn.\·I·

c.vste't1t Methyl Ester Ib.-The rcmaining mother liquo" werecvaporaled to drynes.s underreduccd pressure. The stlcky rcsiduewas then diuolvcd in hot carbon letrachloride (Ioo cm l ) andpenlane added unlillhc solution became cloud)'. The mixturewas kepl at - 20 oC for Iwo days whereupon a voluminouspowder prccipitaled. This precipitate was fihered quickly bysuction and washcd with the minimum amount o( cold diclhylether. The while solid was :lllowed to air dry afTording pure lb(10 g), m.p. 92-93 oC (from CCI.-pcnlane); TLC Rt O.JO(EIOAc-hexane 1:1); '...tan·' 3287 (NH) lO6O, 2946, 1717(CO), 1655 (COI, 15l6. 14l5, 1370, 1197, 1172; .l.(['H.]­DMSO) 1.621 (s, l H, CH,CO), 2.265 (s, l H. ArMel,l.l4l (d.1 H,CH,H" 'J Il.I), l.585 (d, 1H,CH,H" '113.1), l.627 (s,3 H, CO,CH,), l.942 (d, 1 H, CH-S, J 10.0), 4.812 (dd. 1 H.N-eH, 1 9.1, 10.0),7.02-7.12 (m, 4 H, SCH,A,). 7.22-7.34 (m,5 H, CHA'), 8.317 (d, 1 H, NH, J 9.01; .ldCDCI,) 170.5l,169.64, 137.02, 136.78, 134.06, 129.13, 128.88, 128.65. 128.34,128.13, 55.36, 52.2l, 50.86, l5.42. 2l.0l, 21.05; ml: (El) 357(M"), 326, 298, 242, 217, 210, 18l. 168.

Preparatio" 01 N-Acelyl-S·p·me'''yllwn:.l'I·p.phrn.yIfYSle'ne11 and 2b.-The followina procedure was applied to balherYlhro and threo diastereoisomen. Ester la or lb (14.31) ~'as

dissolvcd in boiling methanol (80 cm') (Ihe dissolulion heinlmoredifficult with Il) and while the solution was mllinlaincd ItreOux. water (160 an) WIS added by incremcnls of20 an). ToIhe resultina suspension (or solution for lb) was added NaOH(1 mol dm-); 45 cm') by 15 cm) incremenls. The reactionmixlure was maintaincd Il reftu~ for 2 h, afler whK:h themethanol wu removed under reduced pressure. The aqueousphase wu extracled wilh 2 ponions of 100 cm' ofethyl Ketale,placcd under reduc.cd pressure for a few minutcs 10 remove thesmall amounl of ethyl acctate sliJI present in the waJer phue.The aqucous phase WIS acidi6ed 10 pH 2 wilh concentraledhydrochloric acid and allowed to stand al 4 oC for 1 h. Thewhite solid obtaincd wu 6ltered by suction and allowcd to airdry al 50 OC for 12 h to alTord dialtereoisomerically pure 21 orlb (11.0 a, SOOI. yield). threo: M.p. 134-135'C (fast healin,);TLC R, 0.50 (EIOAc-MeOH 1:1); '_/an·' llOO-2lOOvbr(CO,H),ll57 (NH), 1707 (CO). 1659 (CO), 1lJ5, 1430, 1280,1237, 1138; .l'<CD,OD) 1.951 (s, l H, CH,CO), 2.276 Is, 3 H.ArM.), 3.387 (d,1 H, CH.H" '/13.1),3.529 (d, 1H, CH.H" '113.1),4.223(d, 1 H, CH-S, J7.2),4.866 (d, 1 H, N-eH, 17.11,4.91 (br s, CO,H + NH), 7.035 (s, 4 H, 5-CH,A'), 7.24-7.l7(m, 5 H, CHA,); 6dCD,OD) 173.15, 172.75, 140.10, Il7.80,135.81, 129.99, 129.89, 129.40, 128.75, 58.28, 52.4l, l6.O9,22.32,21.11; mlz (El) 344 (MW), 298 (M' - CO,H), 284,274,266,256,250,238,229,228,227,220,207,194,179,178(Found: M', 344.llIO. Cal<:. rorC,.H"NO,S, M,344.ll2O).erYlltro: M.p. 17~177'C «(rom EIOH-waler); TLC ~ 0.50(EtOAc-MeOH 1: 1); •.../an·' llOO-2lOOvbr (CO,HI, ll58(NH), 1718 (CO), 1630 ICOI, 1540, 1422, 1284, 1129:.l'<CD,OD) 1.796 (s, l H, CH,CO), 2.28l h,l H, ArM.), 3.446(d, 1 H, CH,H., '1 Il.II,l.62O(d, 1 H. CH,H" '1 Il.0),4.106(d, 1 H,5-CH,/8.1),4.92Ibrs,CO,H + NH),4.9l9(d,1 H,N-eH, J8.1), 7.07 (brs. 4 H, CH,A,), 7.28 Ibrs, 5 H, CHA,);.ldCD,OD) 173.00, 172.7l, Il9.4O, 137.81, 1ll.78, 130.06,IlO.02, 129.93, 129.l7, 128.74, 57.22,51.26,l6.27, 22.21, 21.1l;mlz (El) 344 (MH'), 298, 284, 274, 266, 256, 250, 238, 229, 228,227,220,207,194,179,178.

112

1902

Pr~pa'alio" of N·Trijluoroauly[.S.p-lTWlhyIMn:yl'P.phttf"'.C'YJtrinr~. and6.-18 or 2b (7.S BI was dissolved in hydrazine(100 cm)). The reaction mixlure was heated to 60 oC for IS h.afler which il was conccnlraled under reduced pressure. Coldwaler (200 cm) was added which resulled in the precipitationof the ami no acid. The suspension was kcpt al 4 oC rOt 1 hand liltercd under reduced pressure. The white precipitale waswashed succeuivcly wilh waler. ethanol and linally with diclhylether. The solid obtained was dried in a vacuum oven al sooeovernighl. The dry solid (4.5 g) was !aken up;:> :iiliuoroaceticacid (25 cm), cooled to 0 oC rollowcd 0)' the addition ofIriftuoroacelic anhydride (10 g). The: reaclion was esscnlial1yjnslantancous and thercrore could be checkcd on TLC as 500nas the addition was compleled and more trinuoroac:elicanhydr'.1e added if required. The reaction product was isolatedby pouring the mixture inlo crushed ice (200 cm'). The solidwhich fonned immediatcly was broken up and alJowed 10 standfor 30 min before being fillered under suction and washed wilha copious amount of waler. The while solid was dried in avacuum oyen al SO oC ovemighl afTording pure"a or"b (5.7 8,for Ihe two sleps). tlrrto: M.p. 119-121 oC (from dielhyl etMr­li@htpetroleuml:TLC R, 0.70 IEIOAc-MeOH 1:1); v_~cm·13316 (NH). 3500-2lOOvbr ICO,H). 1707s (COs). 1548. 1432.1406. 1254. 1183br: é.ICDCI,) 2.3271s. 3 H. CH,CO). 3.503 (d.1 H. CH,H•. '1 13.1l. 3.638(d.1 H.CH,H•• 'J13.1).4.319(d.1 H. S-CH. 14.7).4.957(dd. 1 H. N-CH./4.9. 8.8). 6.978 (d.1H. NH. '/8.6). 7.04-7.13 (m.4 H.SCH,Ar). 7.26-7.40 (m. 5 H.CH.•r) 11.0 lb". 1 H. CO,H): é.,{CDCI,) 172.55. 156.83 (q.'l''c.''' 37.9). 137.24. 136.76. 1ll.31. 129.25. 128.84. 128.80.128.45. 128.17. 115.50 (q. '/"c.'" 287.8). 56.77. 50.01. 35.61.21.07: ml: (Ell 397 (M·). 379 (M· - OH). 332. 331. 312. 292.284.274. 259(Found: M· . 397.09.50. Cale. forCI,H1,F,NO,S.M. 397.(959). trytlrro: M.p. 139-140 OC (from dielhyl ether­liaht petroleum); TLC Rf 0.70 (EIOAc-MeOH 1: 1); v....,·cm·1

3300 (NHI. 3500-2500vbr (CO,H). 1710vs (COS). 1550. 1405.1250.1212. 1180:é.(CDCI,). 2.343. (s. 3 H. ArM.). 3.558 (d.1H. CH,H•• '1 Il. Il. 3.713 (d. 1 H. CH,H•• '1 13.0). 4.273 (d. 1H. S-CH./4.4). 5.115 (dd.l H. NCH./4.4. 9.3). 6.635(d. 1 H.NH. 19.3). 7.11 (brs. 4 H. CH,Ar). 7.26-7.40 (m. 5 H. CHAr)10.8 (br s. 1 H. CO,H): é,(CDCI,) 173.43. 157.05 (q. 'J"c.'"38.1l. 137.20. 135.26. 133.40. 129.33. 129.14. 128.85. 128.24.115.54 (q. 'I"c.'" 287.7). 55.43. 49.91. 35.62. 21.0: ml: (Ell 397(M·). 379 (M· - OH). ))2. 331. 312. 292. 284. 274. 259.

RtsolutiOII oferythro or Ihreo NoTrif/uoroartt.v/·S-p-mttlJ.l·/·htn:.v/,p-l'lttnyl·DLoC.l·sttiM" and4b.~ or "b (3.98 a) wassuspended in water (900 an') and lithium chlotide (4.24 a) wasadded. The suspension was slirred viaorously and trealed wllhUOH (0.1 mol dm') dropwise being carefullhalthe pH neve!'ellct'Cded 7.5. This process is Icdious bUllhe use ofan lutomlticlilralor or a slow deliverinl s)'llem Isuc:h as a syrinae pump) isadvlntllt0us. When Ihe .ddilion w.scompleled (C'O. IOOcm'),the pH WIS adjuslcd 10 7.5 .nd a sman aliquoi sel apan rorrcference. A suspension ofCPA (4 cm'; 5000 unils) was addcdand the pH asain reldjustcd. Afler 3 h lhe pH had usuallydropped 10 7.I.nd wu brouahl bae:k 10 7.5. The reaetion wuallowcd 10 procecd Il room lemper'iure for 24 h. afler whie:hthe pH was readjuslcd Ind more cnzyme (4 cm') wu Idded.The reatlion wn monilorcd by injecling. al relul.r intervals. asample (10 mm') of the rcaclion millure and elutina il wilh agradienl of waler-aœlonilrile 0.1% TFA (100-1.. waler la WI..water 10 min!~" water 10 minllypical t, were:'" 10.6 min. lu8.0 min). The Cillent or resolulion was measurcd by compatinsIhe peak lru of lhe remaininl slanina malerial to lhe arearound Wllh the solution nollre.lcd wllh enzyme. Usually. lheruClion wu compltle within .q h Il 20 -Co The re.etionmillure wu Kidified 10 pH 2 wllh cone. hydrochloric "id andlhe Oounhydrolysed amino Kid ellraclcd wilh dinhyl elher

J. CHEM. soc. PERKIN TRANS. 1 1993

(2 x 250 cm'). dricd o"'cr MgSO. and e...aporatcd undcrreduced pressure to aive ,lIrto or tr}'thTo N.lrinuoroac:etyl.S-~

methylbenzyl-fl-phenyl·D<)'sleine (1.95 a. 98%). ,lrrto: M.p.11S-116OC (from diethyl ether-lighl petroleum); [2]6° + 159le 2. McOH); all spec1roscopic data were idcnlicallo racemiccompound. trYlltro: M.p. 140-141 oC (from diethyl ethcr-lightpelroleum): [a]6° -179 (c 2. MeOH): ail spectroscopie datawere identical 10 rac:emic compound. The L-enanliomer whichoflen precipilaled ur-on acidificalion \\'as reco\'cred from theseparalory funnel by ....·ashing ....ith ronnie: acid and .....Ols addcdto Ihe aqucous phase. The aqueous phase: .....as e\'aporated 10dryness under rcduccd pressure and wann .....aler added.Evaporation was repeated unlil no smcll of forroic acidpersisted. The residuc was finally laken up in boiling water 1100cm) and allowed to cool 10 room temperalurc before fiheringunder reduc:ed pressure and .....ashing suecessively with ethanoland dielhyl elher. The solid WBS dried ovemight under vacuumafTording the zwillerionics~es (1.42 g. 94%). Ilrrto: M.p. 169­170 oC (decomp.• from aœtic acid-waler) [2]bO -1% (c 2.HCO:H); TLC R, 0.59 (butanol-acctk acid-v:ater 4: 1: 5 upperphase); ".../em- ' 3500-2500vbr (CO:H) 1650. 16035. 14965.1374s. 1345: é,,(['H.lDMSO. CF,CO,D) 2.251 (s. 3 H. Ar­Me). 3.467 (d. 1 H. CH,H•• 'J 13.01.3.688 (d. 1 H. CH,H•• '113.1). 4.209(d. 1 H. S-CH. 17.0). 4.307 (d. 1 H. N-CH. 17.0).7.075 (br s. 4 H. SCH,A,). 7.35 (h, s. 5 H. CHAr):é,(['H.lDMSO. CF,CO,D) 168.84. 136.95. 136.65. 134.21.129.26.129.16.128.85.128.44.56.58.49.69.35.38.20.79: ml:[ehcmical ioniulion (CI) methane] 302 (MH O

). 285 lM· ­OH). 256 (M· - CO,H). 239. 227 (M· - NH,CHCO,H)(Found: M·. 256.1138. Calc. for CI"H,1NS. M. 256.1160.Found: M· •227.0892. Cale. forCI ,H l'S. M. 227.0894).ery,ltTo:M.p. 178-179°C (decomp.. from AcOH-H:O) [a]bO + 179 (c2. HCO:H): TLC R, 0.59 (bulanol-aoelic acid-water 4:1:5.upper phase): v_./cm·' 35~2.soovbr (CO:H). 1618vs. 1SIS.1495. 1396. 1355: é.(['H.lDMSO. CF,CO,D) 2.234 (s. 3 H.ArMe). 3.SlI (d. 1 H. CH,H•• '1 IJ.O). 3.687 (d. 1 H. CH,H••'1 13.0).4.248 (m. 2 H. N-CH and CH-S). 7.08 (b, s. 4 H.SCH,Ar). 7.34 (br s. 5 H. CHAr): é.,{['H.lDMSO.CF,CO,D) 169.03. 1)6.89. 136.37. 134.30. 129.42. 129.26.129.22. 129.14. 128.68. 56.69. 48.71. 35.32. 20.83: ml: (CIm..hane) 302 (MW). 285 (M· - OH). 256 (M· - CO,H).239.227 (M· - NH,CHCO,H).

Prtparation oferylhro and Ihreo S·poMttlrylbtn:.v/o~·phm.vl·

D-rYJ,tine 31b and3tIb.-The procedure uscd .....as esscntially thesame as the one described for removing Ihe acctyl group ofcompounds 2a and :lb (sec the preparation of'" and ..b). tlrrto:M.p. 17S-176'C (decomp.. from AcOH-H,O): [.l~o +197Ir 2. HCOzH); ail spectroscopie: data identicallo 31•. erythro:M.p. 179-180'C (decomp.. from AcOH-H,O): ['l~o -175(r 2. HCO:H); ail spec1roscopk data idenlicallo 3blI.

DtltrmÎna';Olf of Optica/ Purit.v.-Racemic ... or "b orresolved "ab or ..bit (100 mg) was dissolvcd in dielhyt nher(5 cmJ ) and a solulion of ctiazomelhane in dielhyl elher(prepared from 100 mg. of Nonilroso-N·melhylurea in 5 cm' ofdiethyl ether Ind 0.3 cm' of 40'1.. KOH) wa5 added slowly wilhswirlins al room temperalure until a fainl yellow colourpenisted. The solvenl was evaporllcd under reduc:ed prnsureand the residue wu recryttallizcd from diethyl ethcr-1ightpetroleum. A panion (50 mg) was dissolvcd in CDCI, (0.4 cm')and the NMR spec1rum recordcd. Tris[)'(heplaftuoropra.pylhydrollyme1hyleneH + kamphoralo]europiumllll) [( +)0Eu(hfch) was added by increments of 1S mg undl Ihe efTmappearcd al 0.3 cquivalents.

X.RD." Dota MtaSUrttMnrs pd Proussing.-lnlensiIY dalawerecollcc:lcd 11293 K on an Enraf-Nonius CA04 aUlomalic

113

J. CHEM. SOC. PERKIN TRANS. 1 1993

Table 2 Crystal data and details of structure deu:nnination

1903

TaWe" Se1ecled torsion anlles (") in compound la

114

Cryslal data5yslem and space groupQ.b.~(A)/11..)V(A!)ZD.(gem-)F(OOO) (e-)~(Mo-K::dem"

Crystalsizc lmm)

Monoclinie. P21/~8.3708(8). 24.5094( Il).9.4068(5)92.885(311927.5(2)

•1.2327601..0.20 li: v.25 )Il 0.35

ClIOI-N(IKI2Kll)0l2I-CtIK(2I-NII)N( 1)-C(2K(3}-CI4}CI2I-Cl)KI4I-C(5)Nil l-C12l-C'l>-S( 1)ct 121-$( 11-C()K'C2)C131-S< 1K( 12K'( 1IIS(I)-C( 12KI13,,-C(141

-61.01.21139.0(21Ifl7.9(21

-IIO.J(21-67.0('1111.9(2)167.Jt:\1I0.It::!1

Tablit J Selcc:ted bond distanc:cs" (A) and angles ID) in compound 1.

S(1l-CCl)SOl-C(l2)OOl-CCI)O(2)-C(I}0l2K(20)O(ll-CIIO)NI'l-C121Nill-COOIC(I"-C(2)C(2)-CmCCll-C(4)CIiOl-C1I1)C1I2l-CllllC(1'l-CCI9I

Cm-SOl-CII2)CIl Kl(2l-C120)C(2)-NIll-CIlOI0( .l-CC 1Kl(2)0(.l-C1ll-C1210(2f-CIll-C12)Nlll-C12l-C1I)NIll-C12l-CCl)CIll-CC2l-CCl)S(1l-Cllf-C(2)SOl-CClf-C(4)C12l-CCll-C(4)C'll-CC4l-C(5)CCll-C14l-C(6)O(lf-CC 10f-NIlIO(ll-CIlOl-C(1I1NIll-CIlOl-CC IIIS( Il-CII 2f-C( IIICCI2l-CIIll-C(l4)C( I2l-CUll-CC Il)C(16l-C(I'f-C(I9)C(l7l-Cl l8l-Cl 19)

Data collectionTemperalure (K)Radiation (A)omin-mal. (0)Scan typeScan (0)DatasctTot. uniq. dataObscrved rcfteclion: 1 :> 2.Sa(/)

RetinementN.or.N...R.R•• SMax. shifllenorMin. and mu.. fCSd. dens. le - A'!)

2930.709 301.5.24.9wl280.8 + 0.35 Lan (8)-9:9;0:28;0:1134522354

2364.2210.041.0.036.1.980.001-0.17.0.18

1.819(211.818(2)1.198(3)I.mm1.447(3)1.226(3)1.448(3)I.ll9(l)U24(3)1.542(l)I.5()8Cl)I.49SCll1.498(3)1.S08(4)

100.06(10)115.75(18)121.25(17)124.63(191124.61(19)110.69(11)110.95(17)109.92(16)108.S7CI7)108.1 I(14)'12.4lllS)I1l.00(I7)119.28(20)122.26(19)121.06(20)122.70(20)116.24(18)108.98(16)121.62(21)120.14(20)122.1I12l)120.65(23)

the range of 30 < 20 < 40°. Reftcetions were measured witha constant scan spced of 2.7"' min- I • During dala collection.the intc:nsitics of Ihree standard n:ftections were monilorcdevery 60 min. No decay 'Nas obscrved.

The structure was solved by the application ofdirect mclhodsand refincd by leasl squares usinSlhe NRCVAX program. H

Weight based on countinl statistics 'Nere used. A secondaryextinction coefficient was ulilizcd in the retinement.lit its finalvalue was 0.6S(4). Atomic scauerins raclors slaml in theNRCVAX prOSram were those of Cromer and Wabcr. H

Hydrogen atomic posilions were calculated; lhe)' were auisnedthe isotropic thennal paramcler of lheir respccli\'C auachedatoms and 'Nere refined. Ali non·H Itoms were rc6nedanisotropically. Bond lensths and angles arc Iistcd in Table 3,selceted torsional angh:s are given in Table 4. Tables of alomiccoordinatcs. bond lenll.hs and angles, and thennal parametersha\'C becn depositcd al the Cambridge Crystallographic DataCenlrc:.-

Enf.r;y Calrvla'iolU on Mod~1 MlJI~("III~J 5. 611 and".-ThemolceL:les were built rrom the standard SYBVL frllsmentlibrary. 90 starting conronnalions were construelcd rrom thecombinations orfive mosl probable backbone confonnations A.C. D. E and F (~. " - -60. -60: -'0.80: -80. 1.0: 180.180; 180, 60 correspondins rcspeclively 10 the Zimmerman~t al. letter code Ja with 18 side chain conformations ex· ,,- .,ror XI and Xl and ±9QCI ror Ihe X, disulfide bond). These wereminimized usin, lhe MAXIMIN2 rorce·field or SYBVL S.$ J.

includins elcetrastatic contributions. The partial charles wcrecalculatcd usinS the method or Berthocl and Pullmann.11 Thecomputations were pcrronned on an IBM RISC 6000 machine.The amide bonds were hcld ",ms while lhe dihedral anllesde6ncd by the C". C·, C'Ar. C'Ar were maintaincd al9QCI ± Joe for 61 and 611. Minimiations were pcrronned al Il

convergence 1e\'C1 or O.cXU kcal mol-·. (1 ~al CI 4.184 J). Theminimized conronnalions were analysed usina lhe TABLErunction or SYBVL. The Boltzmann probabilities were cal·culatcd as desc:ribed by Visquez r' al.JI

A.know.............G. V. thanks NSERC ror a doctoralscholarship. Our lhanklalso 80 to Marc Drouin ror X·ray data collcelion and 10 GauonBoulay ror man lpec:trometry measurements.

°For details or the œposilion IChtme. let 'Ins'ruclionl ror Authon'.J. CIa,,,,. Soc.• P"kiI, TrtllU. J, 1993. issue 1.

" Benune C-C min. 1.360(4) mu. 1.387(4). avenp 1.376 A.

dirrractometer. Table 2 provides crystallosraphic and datacollection details. The NRCCAD proSrams H were used rorcentrins. inde.ins and dala collmion. The unit œil dimension.were oblained by a least·squares fil or 24 œnlred refkctionl in

R.r.,......1 5. H. Snyder.Stlntc,. 1984. lU. 22i 1980. ZOt. 976;5. H. 5nydtrand

R. R. Goodman,J. Nftlroclv",.• 1910.35.5.2 M. C. FoumÎef.l.aIUlJr.i.G. Gliel. B. Mail"t. S. Premillaland B. P.

Roques, Mot. PIuI,trfIICOi.• 1911. ZI.4....l J. OiMaio. T. M. D. N.uycn.C. LcmICUJ and p. W. Schiller.J. Mrd.

C/vm.• 1912, 25, 1432; P. W. Schiller and J. DiMaio. ""IdI,:

1904

SlruCllUt lIIfd FtIN'wn. PrMut/inrJ of lM Ei,"rh AmtrictJn P,p'iMSympoliwrf. eds. V. J. Hrub)' and D. H. Rich. Pierce Cbemical Co.•1981.pp. 269-271;J.J. KnÎtlel. T. K. Sawyc:r. V.J. Hrob)' and M. E.Hadlry,J. Atrd. CIttm.• 1981,26. 125; D. F. Vebcr.R. M. Friedinger.O.S. Perlow, W.J. Palevedl. F. W. Holly. R.G.Slachan.R. F. Null,B. M. Atilan. C. Homnick. W. C. Randall, M. S. G1itur. R.5.tperstein and R. Hirschmann. Natwu, 1981.192.55.

• 1. Damell. H. Lodish and D. BaltimofC. Molu~14r Crll BW/Off,Scicnli6c Arnt:ric:ln books, frmnan, New York, 2nd mn.• 1990. p.53.

oS J. P. Meukli. V. J. Hruby and A. I. R. Brewster. P,or. Natt. iteM.Sei., 1977,14,1373; H.I. MosberJ. R. Hurst. V. J. Hrub)', K. Gee. H.1. Y.lmamur•• J. J. Galligan Ind T. F. Burin, Proc. Nall. Acad. Sci.,1983.10.5871.

6 R.S. Morpn, C. E. T'lIh. R. H.Gushard,J.M. McAdonandP. K.Wanne, 1",. J. P",. P'Olti" Rts., 1978,11. 2fI7.

7 H.I. MOlberlo R. C. Huxth, K. Ramalinsam,A. Mansour. H. Akiland R. W. Woodard./tJl. J. Ptp'. Prol~in Ru., 1988,32. 1.

8 M. Svoboda. 1. Sicher. J. Farku and M. Pankova, ColJ~er. C:~eh.

Ch"". COM"""'.• 19S5.lO, '426.9 D. Q. Holland and P. M.malis, J. Clttm. Soc.• 19S8, 4601.

10 H. T. Nasua", J. E. Elbl:tlin. and J. C. Roberts. J. M~d CJvm..1987, JO, 1373.

11 O. Plou•• M. CaNlO, G. Chassainaand A. Marqucl. J. Or,. CJwm..1988,53,3154.

12 U. Na.ai and V. Pavane. H~ltroeycltl. 1989, 18. S89.13 B. H. Nico5et. J. 6iol. Chnn., 1932, '3. J89.i4 H. E. Caner, C. M. Slcvmsand L. F. Ney.J. Biol. Cbtm.• 1941, lJ1t,

247.15 A. K. Mukerjœ. Ht'rocycltl.1987, 26,1077.16 D. Dmskr and H, Poucr, Diseowtin, En:f"I"l(Sl:imtific American

Library serin), Frecman. New Vork, 1991. p. 19S.17 R. Chenevtrtand M. Uloumeau, Cbmr. Lm., 1986, 11S1.18 T. W. Grctn and P. G. M. WUls, Prottrliw Group in Orrtmie

S.l'nlbtsis, Wiley. New Vork. 2nd cdn., 1991, p. 438.19 W. S. Fanes. J. Biol Chtm.• 19SJ, ... 323.20 J. P. Grœn.'ein and M. Winitz. Cbtmulty of ,ltt Amino AeUh.

Robert E, Kriqer. Malabar. F1orida. 1984. p. 2650.

,. CHEM. soc. PERKIN TRANS. 1 1993

21 D. D. Keilh, R. Yan,. J. A. Tonora and M. Weigclc.J. Or,. Chnrt..1978,43.3713.

22 F. WCYlo;;nd and R. GeiFt. Chrm. &r.• 1956.14.647.2J S. Vanariane! M. A. M'ilZ.J. Am. Cb,,". Soc.. 1957.19, tlSD.24 IUPAC-IU8 Joint Commission on Biochemical Nomenclature,

Biorbtmutry. 1970.9.3471.2S V. Cody. in CMmutry and Bioebtmu,r.v of lM AmiltO Arw, cd. G.

C. Baren. Chapman and Hall. New Vork, 1985. p. 6)6.26 V. Cody. rd. 2S. p. 640.27 G. Villeneuve, M. Drouin and A. Michel. unpublished ....ork.28 S. S. Zimmennan, M. S. Panic. G. Némethy and H. A. Sc.:herap,

Mucromoft(1lltl, 1977. 10. 1.19 A. Michel.G. Villcncu\·eandJ. DiMaio,J. ComPWI. Aid. Mol. DeSIgn,

1991.!5. "3.30 H. Bunte. &r;rhlt, 1874.7. 646;T. S. Prioeand D. F. Twiss,J. Ch~m.

SOC., 1909. 172S.31 R. M. Herbst and D. ShemÎn. in Orlanie Syn,btsis. cd. A. H. Blalt.

Wiky. Ne.... York. Coll. vot. 2. 1943. p. 1; p. 11.32 V. LePase. P. S. WlUte and E. J. Gabe. NRCCAD. An Enh~noed

CAD-4 Control Prosram. Annual mccting or American Cry­stallographic Association. Hamillon, Ontario. Canada. 1986.

J3 E. J. Gahe. V. LePase,J. P. Charland and F. L. Lee. NRCVAX. AnInteractive Program Sysltm ror Struclure Analysis, J. App1.CrJ's,allolr.• 1989.11, )84.

J4 A. C. Lanon. Aera Crys,aIlO!r.• 1967,13, 664.J5 D. T. Cromer and D. T. Waber. lnlrrno,;onol TGblts for X.ray

CryslallolraplrJ'.cdl. J. A.lber and W. C. Hamilton. Kynock Pms.8inningham (present diltributor Klu..-.'Cr Academie Publisher.Dordlt'Chtl. 1974. vot. IV, pp. 99-101.

J6 SYBYL Molcr;ular ModelinB, Venion S.S. Tripos Associate lne.,Fcbruary 1992.

37 H. Bnthod and A. Pullman, J. Chim. Phys.• 1965.61, 942.J8 M. Vilquez.G. Némethyand H.A.Sl:heraga,Ma~romoltndts,198J,

16,1043.

Paper 3/011821-tReeeived is, Mar~h 1993Accepted22nd April 1993

115

116

Supplementary remarks

Sorne points in the paper may deserve further comments

1) According to the initial velocity of the enzymatic reaction determined byHPLC, we evaluated the reaction rate to be about 0.05 \LM/min/mg protein,

roughly 1000 times slower than with the reference compound N-hippury­phenylalanine for carboxypeptidase A

2) No comments were made conceming the difficulty that might beencountered for the removal of the sulfur protecting group. Erickson andMerrifield41 have shown that this group is very stable to the condition of acidolysiswith 50% TFA in DCM (the usual condition for N-Boc c1eavage) but is completelydeprotected by 50% anhydrous HF in anisole for 1 h at O"C. On the other hand,Ploux ~ll have shown that the P-phenyl group of p-phenylcysteine is resistant

to a treatment with neat HF at O"C for 1 h. Consequently no problem :. to beexpected when this S protecting group is used with this particular amino 'acid inpeptide synthesis.

3) Since the publication of this paper it has been possible to perform the X­ray diffraction study of erythro and threo diastereoisomers of N-benzoyl-s-a­

naphtylmethyl-p-phenyl-DL-cysteine. Fig. 5 presents an ORTEP view of the

respective L-enantiomers. The side chain conformation adopted by the erythrodiastereoisomer is indeed the one that was predicted from the molecular modeling

study when the backbone is in the D conformation (Table 1) and corresponds to

the t rotamer for 6b in Fig. 3 of the paper. Similarly for the threo diastereoisomer,it adopts a conformation such that the number of gauche interactions is minimized

and orients the protons on CC" and cP in a trans relationship giving rise to the largecoupling constant observed in [2HelDMSO.

4) Fig. 4 shows how the P-phenyl substituent may be accommodated in

position 5 of the putative cyclopeptide. Oriented in such a way it shouldcontribute to increase the selectivity towards 8 opiate receptor as we specified at

the end of chapter 2. This was not explicitely mentioned in the paper since work isactually underway on tOO point.

a)

Fig. 5 ORTEPviewof a) threo N-benzoyl-S-methylene-a-naphtyl-P-phenylcysteineand b) erythro N-benzoyl-S-methylene- a-naphtyl-P-phenylcysteine

117

• Supplementary references.

118

39. H. E. Carter, OrganicReactions , R. Adams ed., John Wiley and Sons, New­York, 1946, Vol 3, p 198.

40. J. H. Bradbury, Biochemistry, 1958, 68, 475.

41. B. W. Erickson and R. B. Merrifield,l Am. Chem. Soc., 1973,95,3750.

Chapter 6

Insertion of the methylene-m,y surrogate to the amide bond into t-Boc-Val-Leu­

OH: X-Ray crystal structure, solution conformation and molecular modeling

study.

Authors' contribution

The following paper was written by myself with the kind help of Dr. DiMaio

and Pro Michel. Ali of the compounds were prepared by myself except 6 and 2

which were prepared by Dr. DiMaio. The X-Ray crystallographic work was done

partly by myseif with the precious collaboration of Mr. Marc Drouin under the

supervision of Pro Michel. The NMR and molecular modeling study were

performed by myself.

J. CHEM. SOC. PERKIN TRANS. .2 1994

Insertion of the Methylene-oxy Surrogate of the Amide Bond intoBoc-Val-Leu-OH: X-Ray Crystal Structure, Solution Conformation andMolecular Modelling Study

Gérald Villeneuve.t·· John DiMaio.b Marc Drouin e and André G. Michele, Department 01 Chemistry. McGill University. Montréal. Quebec. H3A 2K6. Canadab Institut de Recherche en Biotechnologie. Montréal. Québec. H4f 2R2. CanadaC Département de Chimie. Université de Sherbrooke. Sherbrooke. Québec. J7K 2R7. Canafla

1631

120

The conformational features associated with the introduction of the methylene-oxv sUllogate of theamide bond were explored by studying the crystal and solution conformation of the related modelpeptides Boc·Val-Leu-OH (1) and Boc-Val'\II(CH,O)-Leu-OH (2). TW9 independent molecularconformations were found for 1 in the crystal state whereas one wlls found for its congener 2. Incompound 2 the dihedral angle defined by C'-CH,-O-C' (w') adop,s'a value of -, 65.7(3)",close ta the situation encountered in the classical amide bond. 20 NOE NMR studies suggest thotthe prefelled backbone conformation of 2 in [2H,lDMSO correlates with the crystal structurewhereas the prefelled backbone conformation of 1 in [2H,lDMSO showed a departure trom itscrystalline conformation. Molecular mechanics computations demonstrate the effect of the shortC(Sp2)_O(Sp2) bond in compound 2 in dictating the preterred dihedral angle values adopted by 11)'

and fPs...'

A major goal in the quest toward understanding the inherentfunctioD or mechanism ofregulatory peptides such as receptorligands or enzyme inhibitors relies on the elucidation of theirbioactive confonnation. To this end. the sludy of conslrainedanalogues ofagonist or antagonist molecules has bec:n useful indcciphering the confonnational requirements at the le'Yel of theacceptor macromolecule. 1 Constrainls can he global such as inthe bridging of two remole amino acids l or more confined toindi'Yidual amino acid modifications. J Local constraints havebeen obtained through C' inverted configuralion. N and C'mc:lhyl substitutions. insertion of proline or related imino acidsand by the use ofmodified amino acid side chains.

More reœntly, local modifications ha'Ye centred on theamide bond itself and se'Yer..1 surrogale structures have beenproposed.· Amide bond replacements ha'le become attractivelools in efforts direc:ted al conferring proteolytic resislanœwhile olhers impart transition stale character to the nonnalscissile amide bond and therefore have produced powerfulprolease inhibitol'1. ~ Amide bond surrogates which do nolpossess the double bond character can he e~pcc:ted to be moreflexible compared 10 the original amide. 1 This mighl be anadvanlage when a fine·luning of the binding properties isdesircd bUI the new stene and electronic properties must heconsidered with care. In this regard. several studies on theconfonnational consequences of subslituting the amide bondwith various 'isosteres' have been reported previously.' As acontinuation of our work in this field we have Sludied theconformational consequences stemming from the replacernenlof an amide bond by a methylene·oxy group in a modeldipeptide. We report the comparative X·ray structures of Boe­Val·leu·OH 1 and the related .,-(CH 20) analogue 1: Wehave alsa investigalcd lhe confonnation of lhese compounds in[lH,JDMSO by IH NMR spcclroscopy. In addition we have

t P,,,sn" add"ss: Dtp.Inemenl de Chimie. Univenilt de Sherbrooke.Sherbrooke. Quebcc. JIK 2RI. Canada.: Boe: - ,,,,-Bulolyc:arbonyl; Val - valine: Leu - leucine; DCC ­1.3-dicyc:loMlybrbodiimidt; nOMS _ ,,,,,.bulyldirntlhylsilyl;DMAP _ 4-dimeth~laminopyridine: TEA _ lriclhylamine: HOBT ­l.hydrolybcnzolrWole; OMSO _ dimelhylsulfolldc. 1cal _ 4.184J.

•HOY~OHOI~OHO

'~Y..,.).,.)l"î(~..,.)...)lOHI("l.- ~ o-{ H"-{

PtiPllalin A

performed molecular mechanig computation based on thedetermined X·ray crystal stnlctures in order to assess therelalive confonnational mimicry.' The particular dipeplidesequence was chosen sinee it is related to Ihe sequence Val'.Sta J

of the potent .spartic protelsc inhibilor pepSlalin A. ~enin andHIV·I prolease' are Iherapeutically importanl enzymes thalare inhibited by pepstalin.

EXperl....I••Cllrmutry.-Compound 1 was prepared by the 1,3-dicyclo·

hexylcarbodiimide (DCC) mediated coupling of Boc,VII.()Hwith H·leu·OBz followed by hydrolenolysis of Ihe benzyl esterll'0up. Compound 2 was prepared based on the procedurereported ~y TenDnnk' for simillr compoundsand win. in ourcase o-Ieucine and L.valinol as slartinl malenall. The Itepsleading 10 compound 2 are shown in Sc:heme 1: ail inlermedialnwere characteriz.ed.

Equipmrn,.-M.p.s were recorded on a Gallenkamp c:apillaryapparatus and are uncorrected. IR speetra were recorded on aPerkin.Elmer 1600 fTlR and were taken in KBr pellell or l'nealliquid. 1" NMR speetra were rccorded eilher It 300 or 200MHz on Varian XL·300 or XL·200 lpectrometm. respcclively,

121

1632 J. CHEM. soc. PERKJN TR....NS. 2 1994

• •SdwtM 1 Rra,rnlf and conditimu: i, NaNO,. HBt (4lrl.), o-c. 30min; ii. 0.3 cquiv. DMAP, 1.1 equiv. TEA, TBDMSO. CH 2C Z' 0-<:­room temp., 8 h: iii. I.Ocquiv. HOoT. 1.1 equiv. OCC. CHtO,.O·C­foom lcmp.. 2 h: i.... AmbcrlYl1') S. MtOH. room temp.• 2 h; v, 1.I equiv.NaH, THF. room temp.• 2 h: \i, HO(6 mol dm'), 110 ·C. 2Q h;vîi. 1.1cquiv. 8oc; JO. dioline-walcr(S%). Et]N. room lemp.

o-leu l'YaIinoI

J' J. 0 'Vo .

~OH H"'~OTBOM5 • ~.OH•• - 1/'0... • H

ten·auloxycarbon.vltKJ(vlltllciM 8nI:yl Ültr.-L·Lcuciniumbenzyl esler p-toluenesulfonale (1.971. 5.0 mmol), l·hydrolY"benzolriuole (0.67 g. 5.0 mmol), It,,·butoxycarbonyl·L·valine(1.09,. 5.0 mmol) and N-elhylmorpholine (0.60 an', 5.0 mmol)wtre dissolved in dry THF (5.0 an'). The mixlure was cooled laoDC and DCC (1.081, 5.0 mmol) was addcd. The reaclioo wuallowed 10 proœed (or 1h al 0 ac and 1 h at room tempo Thedicyclohexylurea Ihal precipiuled was fillered and Ihe orslniephase was evaporatcd to dryness. The yellow oil obtaincd wudissolved in ethyllœllie (25 an') and lreated sequenlially wilhsaturated NaHCO, (10 cm') and duit acid solulion Uo-/.;10 cm). The orpnie phase WIS dried (MgSO.). filtercd. andevaporaled under reduced pressure. Chromltography OD s.i.licagel using eth)'1 acel:ue-heune (1 :3) gave the desired produe:t asa dear colourless thick oil which slowly solidificd on slanding(1 .68B. 80"/,). M.p. 8l.s-84.5 OC (heune): [.)~' - 52.5 (e 2.0.MeCH): TLC Rr 0.37 (eth)'1 aœlale-heune 1: 3); v-,'an· 1

m8 (NH). 2955 (CH). 1716 (CO). 1682 (CO). 1660 (CO).15.7. 1524. 1306. 1250 and 117l: 6,,(200 MHz: CDCI,) 0.85­0.95 [12 H. m. Leu (CH,),. Val (CH,),). 1.411 (9 H. s.

Bu'). 1.45-1.78 (l H. m. Leu C'H,. C'H). 2.07 (1 H. oct. J 6.6.Val C'HI.l.87 (1 H. dd. J 8.8. 6.4. Val C'H). 4.65 (1 H. m. LeuC'HJ. 5.04(1 H.d.J8.9. Val NHJ. 5.ll (2 H. s. OCH,ArJ. 6.22(1 H. d. J 8.2. Leu NH) and 7.l2 (s. 5 H. Ph): Jo{CDCI,)172.45. 171.50. 155.85. Il5.ll. 128.54. 128.l6. 128.23. 79.89.67.0l. 59.97. 50.79. 41.l8.3O.77.28.25.24.72.22.75.21.80.19.17and 17.87; ml: [electron impact (Er)] 421 (MH·). 420 CM·).l78 (MW - C,H,). l64 (MW - C,H.l and 347 lM' ­C.H,O).

tert·Bulox)'carbonylvaly/leucint (1).-10% Palladium overcharc:oal catal)'si (0.17 g) was introduced into a three·neck f1.askpreviousl)' f1.ushed wilh a stream of nilrogen. ll'rI-ButOl)'''carbon)'lvalylleucine heoz)'1 ester (1 .68 g. 4mmol) was dissolvedin methanol (25 cm') and carefully addcd ta the catal)'sl. AhydrogeD stream was Ihen introduced and allowed to f1.ow atatmospheric pressure for 4 h. The rcaction milture was filteredover Celite and the fillrate evaporatcd ta dryness under reducedpressure. The residue was dissolved in eth)'1 aectate (25 cmJ

)

and eXlracted with Iwo portions (25 an') of aq. NaHCO)(5%). The free acid was recovcred b)' addilion of citric acid(Congo rcd) and extracted with eth)'1 aectate (2 x 50 cm)).The elh)'1 acelate phase was treated with water (2 x 25 cm)and dried (MgSO.) followcd by cvaporation under reducedpressure. Rccrystallisation using dieth)'1 ether-hexane providedpure 1 (1.04 g. 76%). M.p. 156-157 DC (diethyl ether); [œ]:'o-41.2 (e 2.0. MeOH); TLC Rr0.19 (eth)'1 acetate-henne 1:3+ 0.5% acctic acid): v_..,'cm· t 35OQ-2400br (CO:t{) 3318(NH). 2955 (CH). 1723 (CO). 1687 lCO). 1641 (CO). l54l.1297. 1250 and 1166: J.[lOO MHz: ['H.)DMSO ldim.thylsulfoxid.)) SC< Table 4: Jo{CDO,) 175.6l. 172.29. 156.25.80.12. 60.15. 50.65. 41.ll. 30.59. 28.20. 24.72. 22.79. 21.71.19.0l and IB.2': ml' (El) III (MW). 275 (MW -C,H,).2S7 lM' - C,H,O) (Found: MW. llI.2228. Cak:. forC16HllN:Os: M, 331.2233).

(R}-2·Brom04mtlhylptnlanoÎc A'cid (3).-o-Leucine (2.00 g.15.2 mmol) was dissolved in aq. HBr (48%. 16 an)) and waler(27 cm'). Crackcd iœ was added to give a total volume of 80cm). The solution was stirred YÎgorousl)' and NaNO: (3.1 S,45 mmol) was addcd in smaU portion. When the reaClionmixture had reached room temperalure il was extractC'd wilhether (5 x 15 an'). The elher phase was treated with water(2 x 20 an'). dried (MgSO.) and the solvent wu evaporatedunder reduccd pressure. The yellowish oil (2.39 g. WI.) wasdistillcd (b.p. 138-139 ac/20 mmHg) and gave a c1car colourlessoil (I.9l B. 65%); [.)~. +54.0 (ncal): TLC R, 0.50 (ethylacetate-hexane 1: 1); II_Jcm-' 3500-24OOvbr (CO:"),1716br (CO). 1422. 1287. 1172 and 922:J.1200 MHz: CDCI,)0.91 (l H. d. J 6.l. Mel. 0.96 (3 H. d. J 6.'. M.). 1.79 [1 H.nonupk:.. J 6.5. CH,CH(CH,),). 1.91 (2 H. t. J 6.6.CHCH,CH). 4.28 (1 H.~J7.I. BrCHCH,)and4.5-5.5(1 H.br..ch. D,O. CO,H): Jo{CDCI,) 175.58. 4l.98. 4l.l5. 26.21.22.22 and 21.44: ml' (El) 181 (M' + 2 - CH,). 179 lM' ­CH,). 140 (M' +2-C,Hs). Il8 lM' -C,H, ) and 115lM' - Br).

Q-Uen·BulyldiIMlhylsily/)PtJlinol (").-Valinol (1.00 g. 91mmol) wu dissolvcd in dry CH:Cl2 (10 an) and tteatcd w.lhlriethylamine (1.18 g; Il.7 mmol) and 400dimclh)'laminopyridine(0.045 g. 3.4 mmol). The miltture was cooled 100 ac and It,,·butyldimelhylsilyl (TBDMS) ehloride 11.61 B. 10.7 mmol)addcd. The re&dion mixture was allowcd to rcach roomtemperalure and was slirred for an additional 6 h. Thelrielhylamine h)'drochloride salt was filtered and the filtraleWIS treated with waler (2 )( 5 cm)) and with aq. ammonium

. chloride (2 )( San'). Dryinl (MaSO.) and evaporation underreduced pressure gave Ihe erude producl. Distillalion of Ibis

1••

Uc NMR spetlta were recorded al 15 MHz on a Vanan XL·300 speclromclcr. Ali NMR spcclta werc rcferenced 10 thercsidl1llsolvcnt peak (CDCl, IH 7.24, "e 77.0; C.D6 • IH 7.15,Ile 128.0 l'pm); J.values are given in Hz. 1HNMR assignmcntswcre based on selOClive decoupling and 2D homoscalarcorrelalN experimcnl (COSY), Spin simulation. performedwilh Ihe SPINS prosram, was uscd to dctcrmine sorne of thecoupling constants and chemical shin values. Thin layerchromatography (TLC) was carried oulon glass·backed Mercksilica gel 60. 0.25 mm thickness. Optical rotalions weremeasured on a JASCO DJP·I40 instrument at sodium linewavelength at 20 DC in a 0.5 dm œil and the values wereintegrated over a period of 60 s. [1J]o·Values are givcn inunits of 10·' deg cm: S'l. Mass spec:lra and exact masses weredetermined with a ZAB·I F mass spcctrometer.

R~Qgtnl$ and Solvtnu.-AII reagenls were commerciallyavailable and were used without funher purification. Tetra·hydrofuran (THF) was dried by distillation from sodium­benzophenone and diehloromethane from calcium hydride.

1. CHEM. SOC. PERKIN TRANS. 2 1994

material under reduced pressure (b.p. 102-103°C/20 mmHg)afTorded the pure TBDMS alcohol 0.43 g, 68%); [.2]&0 +5.S(neat): TLC R, 0.40 (ethyl aœtate); v_.fan- I 3374w (NH).2956. 2858s (CH). 1471. 1388. 1362. 1256. 1095s and 837;_.(200 MHz; CDCI,) 0.05 (6 H. s. SiM,,). 0.83-0.94 [6 H. d.CH(CH,hl. 0.87 (9 H.s. Bu'Si).1.5(2 H. brs. NH,). 1.63 [1 H.OCI. J 6.7. CHCH(CH,hl. 2.56 (1 H. ddd. J 6.1. 7.7. 4.1.NCH). 3.38(1 H.dd.J7.7, 'J -9.7.CHCH.H,)and3.64(1 H.dd.J 4.1.'J -9.7.CHCH.H,):_<lCDCI,) 66.18.58.29.30.45.25.&4. 19.41. 18.27. 18.19 and -5.43; ml: (El) 218 (MW). 217(M"), 202 (M' - CH,). 174 (M' - C,H,) and 160 (M' ­C.H'l)'

N-[(2R).Bromo-4-m~lh>,JpentanoY/J-O·(te".blllyldimtlh>,J­

sjJyJ)-L-uaJinoJ.-{R}2-Bromo-4·methylpcntanoic aad (3)(0.72 g. 3.1 mmol) and I·hydro)(ybcnzotriazole (0.56 g. 3.7mmol) were dissolved in dry CHzClz (3 cm l ) and cooled atooC. DCC (0.82 g. 3.7 mmol) was added and the milture wasstirred for 2 h. O'('~rl-Butyldimethylsilyl)-L-valinol(..) (0.80 g.3.7 mmol) was Ihen addcd and Ihe mixture mainlaincd al 0 oCfor anolher 2 h. The dicyclohelylurca was filtered and thefiltrate was washcd with sodium hydrosen carbonate (1 moldm- l • S cm]), cilric acid (2 mol dm-]. 5 cm l ) and water(5 cm]). The organic phase was dried (MgSO.) and evaporatedto dryncss under reduocd pressure. The residue was dissolvcd inhexane and another crop of dicyclohelylurca was rcmovcd.Chromalography on silica gel (ethyl acetate-heune 1001.)yieldcd the pure compound as a lhick oil which solidificd onstanding (0.65 g. 45%). M.p. 58-60 oC; [II]bO -11.9 (c 12.CHzClz); TLC R, 0.35 (clhyl acetate-hexane 10"/0>; v-lcm' I

3297 (NH), 2958. 2930 (CH). 1649 (CO). 1551. 1258. 1113 and838; _.(300 MHz; CDCI,) 0.046 (6 H. s. SiM,,). 0.881 (9 H.s, Bu'Si). 0.902 (3 H. d. J6.4. Me). 0.912 (3 H. d.J 5.1. Me),0.94O(3 H.d.J 5.0. Me). 0.950(3 H.d.J6.3. Me), 1.78-2.03(4 H. m.CHCH,CH. CH,CHMe" CHCHMe,). 3.543 (1 H. dd. J 3.7.'J -10.0. CH.H,O). 3.643 (1 H. dddd. J 2.8. 3.7. 6.1, 9.0.NCH). 3.731 (1 H. dd. J 2.8. 'J -IO.O.CH.H,O). 4.329(1 H.dd. J 5.1. 9.5. BrCH) and 6.570(1 H. d.J9.0. NH);_<lCDCI,)168.62. 62.50. 56.23. 51.08.44.98. 29.11. 26.47. 25.90. 22.72.21.13.21.09. 19.05. 18.23 and -5.48; ml:(EI) 396(MW + 2).394 (MW). 380 (M' + 2 - CH,). 378 (M' - CH,). 352(M' + 2 - C,H,). 350 (M' - C,H,). 338 (M' + 2­C.H.) and 336 (M'" -C.H,).

N-[(2R)·B'om04m~th)'Jpen'anoyJ]·L·valinol (!I).-To asolution of N.[(2R}brom04-methylpcntanoyl]-Q-(ttt,.butyl.dimethylsilyl)-L-valinol (0.6 B. I.S mmo!) in melhanol (15 cmJ )

wu added Amberlyst-I 5 (3 g) and the milture stirred at roomtemperalure for 2 h. The resin wu fillered ofT. washcd withrnelhanol (10 cmJ ) and the fihrate evaporatcd under reducedpressure. Chromatography on silica gel (elhyl acetale-heune30010. then ethyl acetate-hexane SOOIo) afTorded compound 5 asa solid (0.38 8. 90"1.). M.p. 92-93 'C; [.l~· +2.5 (r 0.22.MeOH); TLC R, 0.12 (elhyl aœlale-heune 30'1.); •...Jem"35fJO-31OObr (OH). 3420 (NH). 2960 (CH), 1654 (CO), 1560.1458. 1388 and 1032; _.<200 MHz; CDCI,) 0.89~.99 (12 H.m. 4 M,). 1.77-1.97 (4 H. m. CHCH,CH. CH,CHMe"CHCHM,,). 2.12 (J H. br 1. exeh. D,0. OH). 3.66-3.76 (3 H.m.CH,O. NCH).4.340(I H.dd.J5.7. 9.1. BrCH)and6.46(1 H.br s. exeh. CF,CO,O. NH); -<lCDCls) 170.05.63.77.57.65.50.83.44.82.28.95.26.45.22.67.21.02. 19.58 and 18.68;ml:lEI)250 (M' + 2 - CH,OH). 248 (M' - CH,OH), 238 (M' +2 - C,H,). 236 (M' - C,H,). 196 (M' + 2 - CH,OH ­C.H.) and 194(M· - CHzOH - C.H.).

(2S.SS)·2.lsobllt.~·J·S·isop'opyJ.I.4-oxQ:inan·3·on~ (6).-Sodium hydridc: (0.072 g. I.S mmol; 5,"~ in oil) was washcd withhexane. 1be suspension wu added to dry tetrahydroruran CS

1633

cm l) under argon iJlmosphere. The .Icohol! wu addcd (0.38 1.

1.35 mmol). and the milture stirmi al room tcmpcratUrf rOt 2 h.Water and brine wtre addcd and (he phases \\~re scparllcd. Theaqueous layer wu further eltraclcd wilh ethyl aect_te (10 cml ).

The organic c:xtraets werc combincd and dricd (MaSO.) andevaporalcd ta dryness under reduœd pressure. Chrom.atography on silica gel (cthyl acclale-hexane w;.) .rrordcdcompound 6 as a white solid (0.24 B, 89%). M.p. 83-84 ·C:(lI]tO -16.6 (C" 0.28. MeOH); TLC Rf 0.31 (cthyl aect'Ie­heun, 50'1.); '..Jem·' 3446 (NH). 3214 (NH). 2960 (CH).1665 (CO), 1466. 1424. 1139 and 1108; _.(200 MHZ; CDCI,)0.914(3 H.d.J6.2, Me), 0.933 (6H.d.J6.5. 2 M,). 0.972 (3 H. d.J 6.8. M,), 1.60-1.93 (4 H. m. CHCH,CH. CH,CHM".CHCHM,,). 3.070(1 H. dddd. n.8. 4.0, 7.2. J••", 3.7. NCH),3.714 (1 H. dd. J 3.8. 'J -12.1, CH.H,O),l.76O (1 H. dd. J 4.0.'J -12.I.CH.H,O),4.093 (1 H.dd.J4.5. 8.8. OCH)and6.oo(1H. br s. NH); _<lCDCI,) 172.15.75.76.63.39.57.68.39.93,31.23.24.42.23.45.21.29.18.87 .nd 18.54;ml: (CI) lOO(MW).156(M' - C,H,) and 143(M' - C.H.).

& ..Vol.,,(CH,O)Ltu·OH (Z).-<:ompound 6 (0.24 8, 1.2mmol), was suspendtd in HCI (10 cm l ; 6 mol dm- l ) Ind themilture waS mainlaincd under rcOuI for 20 h. The solution wasevaporaled under reduced pressure affording a solid which wasdricd ovemight undrr high vacuum. The solid was swpendcd indioxane-water (5%: 10 cm l ) and the pH of the solution wasadjuslcd 10 8-9 using triethylamine. Di-un-butyl dicarbonlte(0.30 S. 1.48 mmol) was added and the pH of the solution wasmaintained al 8-9 by addilion of trielhylaminc. 1be rcactionmixlure was concenlratcd and redilulcd wilh waler. Themixlure was eltractcd with dielhyl elher (15 cm) and Iheeltracl was discardcd. The aqueous phase was acidified 10 pH 2with solid cilric acid and ellracted wilh clhyl acelAle (2 )( 25cm l

). The combincd organic extracts were dried (MaSO.) andevaporatecl to dryness under reduœd pressure. Chromato·graphy on silica gel (ethyl acelale-heune weI. + 0.5% accticacid) alTorded compound 2 as a while solid (0.23 1.6O'/J. tA .p.115.S-116·C (heaane); [.l~' -66.4 (r 1.08. MeOH); TLC R,0.17 (ethyl aœtate-hcxane 200;' + 0.5% acelic acid); w...Jcm- 1

3363(NH).2958(CH),1733(CO).1663(CO),1539,1367,1207,1175. 1133 and 1117; d.(2oo MHz; CDCI,) 0.910 (12 H. ni, 4Me). 1.42 (9 H, s, Bu'). 1.51-1.85 (4 H. m. CIICH,CH.CH,CHM,,, CHCHM,,). 3.48-3.57 (3 H, m. NCH. OCH,l,3.90 (1 H. dd. J 8.6, 4.4, DCH) and 4.72 (1 H. br '. NH);_<lCDCI,) 177.33. 156.67, 79.46, 77.99. 71.59, 55.79, 41.61,29.33.28.36.24.52.23.15.21.58. 19.53 and 18.72; ml' (CI) 318(MW), 274(M' - C,H,). 262 (MW - C.H,). 244 and 218(Found; MH ' • 318.2278. Cale. for C"H"NO,; M. 318.2280).

X-RDy Cry""J S,r"c'"rr Delrrmina';ofl of 1 and 2.­Prismatic crystals of 1 were obtained by slow evaporation of adiethyl ether solution at rcorn tcmperalure. PrismalÎCtryltail of2 were oblaincd by slow coolinl to room tcmperalUle of • holheune solution. For bath atructureslhe NRCCAD prolranu 10

were used for centrins, indelinllnd data colleclion. The unitccII dimensions were oblained by a Ieast-lQuares fil of24centredreftections in the range of fJ:r < 20 < SO-. Rdlectionl weremeasurccl wilh a conslant scan spced of 2.7" min· l

• Durin,data collection, the intensities of 2 standard reftcdionl weremonilored e'Very 60 min. No sisnific:anl dec:ay wu observed.Intensity data were collec:ted on an Enraf·Nonius CAO'"ilutomalic difTractomeler. Delails of crystal data and dallcolleclion are given in Table 1. 80lh strutiures were IOlvcd bythe applicalion of direct melhods and refincd by full matrillcasl squares usinSlhe NRCVAX pro.ram ,II Weipt balCd oncountinl slatistics were used. A sec:ondary Cllinclion coefficientwas inçludcd in lhe retinement,lZ the final values were 1.38('7)and 0.41(3) ~ for 1 and 2 rupcc:lively. Atomie ICIllcrin.

122

1634

Table 1 • CrysLlI dala and details of sUuc;lute dClcnnmalion forcompouncb 1 and l:

Ct)'tlal data

,Formull C,.HJDNjO, C•• UHNO,M 330.43 317.43Syslem Orlhorhombic MonoclinlcSplIœ p:roup P2:!,2.[18] P:!.(4]iJ:A IO.9546fJ) 6.0)41(10)b,A IU044(J) 15.]457(161"A 20.4474(7) 10.63080 Il

/II"' 101.064011VIAl 41n.5121 966.09(21)Z 8 ,DU1c"cm-' 1.065 1.089f(OOO)/(' " 1440 J4b#feu-KIllem" 0.61 0.62Cryslal size/mm G.ZO le 0.15 le 0.10 0.25 )( 0.25 '" 0.35

Dilla colleclionT,'K 293 213Radiation Cu·KIl Cu·KI9lmm·mul/t·J 1.0.71.7 1.0.71.7Scan type w,20 w/2BScan (', 1.0 + 0.141an10'l 1.0 + O.141an(8)Oal.III('1 0:13:0:22;0:25 -7:7;0:18;0:13Total uniq. /ht. .... 1974Obscrvcd rcfteçlion 2189 [1 > 2.Gail)] 1880 [1 > 2.5 atnl

RC'finemenl

Nu•• N", 2189.408 1880. 199R. R. 0.068.0.061 0.039.0.040GoF 2.41 3.41Mu. shifi:error 0.104 0.001Min. rn. denl..!e!" A,.J -0.16 -0.20Mu. ru denl.le- A-J 0.27 0.22

factors stored in the NRCVAX prograrn were those ofCromerilnd Waber. 1J HydroBen atomic positions wcre calculated for Jand 2 but nol refined for 1. An orientation disorder on the Valside chain was round in one of the independent molecules of J(lb). Ihe oa:upancy refinemenl showcd a 50:50 ratio. Bolhorientations were refincd usins riBid bond lensths and anBh:ssel al 1.54 A and logo. hilh thermal motion preventcdconservation of 1000 geomelry. Ali non-hydrolen atoms weresel a,lisolropic for refinemenl uccpt for C(ll) in 1. and C(IO'),C(ll') and C(12') in lb. Details of slructures refinemenl areliven in Table 1.-

NMR Solution S.udy of 1 and 2.-Solutions 0.01 S mol dm-J

of 1 or 2 in ['H.lDMSO were delassed by two cycles ofsuccessive freezinl and Pumpinl under hilh vacuum. The NMRlubes were fillcd with ArBon and scalcd. 10 And 20 spcclrawere rctordcd with a Brùker AC-F 300 HC spec:lrometeroperllÎn. al 300.13 MHz for IH and equipped wilh a BVT·lOOOE variable lernperature controller:t ID Nonnal andseleclive proton decouplcd spcctra were rccordcd wilh 32 Kdata points. The FIDs were zero filled 10 64 K dala points andresolulion enhanced by mult:~;tcalion with a gaussian functionhavin, ils maximum Il 0.3 s.

Assignmenl of Ihe 10 speclra wu performcd by seleelive

• AIOmM: coordinlla, bond 5enaths. bond ",n,)es and IhcnnaJ para·mcltn ha~ bœn deposilcd al the Cambridae Crystallo.raphM: DataCtnlrt: for dctlib ICC 'InllNchoni for Authon', J. CMm. Soc., p"ltu.TrtuU. ~, 1994. iDue 1., ID And 20 sl*lra. ha~ becn daposilcd undcr the SupplernentaryPublgllonsSchtmt: for dtlails ste 'Inllructions for Authon'. J. CMm.Soc.• hrku. TfGIU. ~. 1994, iuue 1(Supp. Pub. No. 57013 (5 pp.)].

J CHEM. soc. PERKIN TRA.NS. ~ 1994

prolon dccoupling and multiplicity analysis. One difficulty washowever encountercd: Leu CYH and Val e'H were overLappingin compound 2 which pt'CI:luded the assignment of the mcthylresonances br selectivc irradiation. A 20 relaycd coherencelransfer e.periment'· (which allowed lhe obstrntion of·Jcross-peaks) indicalcd a correlalion betwecn Val C"H and thetwo doublets al around 0.79 ppm. no cross-peaks were detectedwith the two doublelS around 0.86 ppm. This was furthtrchce:ked by irradiating the two doublets around 0.86 ppmlsufficiently scparau:d from those al 0.79 ppml which resultcd inIhe collapse of thc Leu C'H manance to a doublet of doubletsat 1.746leaving the Val C'H muhiplella quasi ocluple!) intact.Similarly when the Iwo doublets around 0.79 ppm wereirradialcd. th: Val e'H resonancx oollapscd to a doubkt al1.707 ppm lcaving Ihe Leu C'H complelt multiplet at 1.746 ppmintact. Accurale detennination ofchemical shifts and couplingconslant for complex and second order spin system wasperfonned with the PANIC simulation program. Spin systemswere rcduced to the minimum number of nuclei by suitableselective prolon decoupling in order ta minimize the numberof paramelers to he optimizcd in Ihe simulation. Once thechemical shifts and coupling conslants were delermined for Iheproton selective decoupled spectra. the undccouplcd spec:lrawere analysed using the previous values as starting paramelers.

20 NOE spectra were m:ordcd at 296.0 K using the standardsequence oflhrec 9()0 pulses. A relaxation delay of3 s was used.The experiment was repcalcd with Iwo difTerenl mixiDg limesCO.3 and 0.5 s). In cach eltp:riment, the mixing time wasrandomly varicd by ±4% in order to slrongly redua: the Jscalar crosspeaks. 15 Minimal speclral widlhs were used. 1 KDaia points were acquired in F2 dimension and 12811 in FI. 32Transicnls were accumulalcd for each t l' The increments for thet1 values were 418 ~s with 1and 452 Jl:i with 2. and the initial 'I

value was 3 ~. Zero filling was accomplishcd in F1 bc(ore 20Fourier transfonncd. This resullcd in a digital resolulion of4.67Hz/point with 1and 4.32 HZlpoint wilh 2. The 2D spectra weresymmelrizcd. NOE crosspeak intensnies and posilions weree5limated. from cross seclions laken at muimum diagonalpeak intensily. Relative cross-peaks intensity were similar forexperiments conductcd with mixinslime of0.3 or 005 s but weresignificanlly less intense Wjth the shorter mixinB time. Due 10importanl II noise at Ihe Bul resonance, cross-peaks found atthis posilion werc not considered. Cross-peaks Ihat were 100close ta Ihe diagonal peak! were also not considered.

Mo/tnt/or Modtllin, S.udy of1 and 2.-lnput structures forthe molccular modclling study were base<! on solid-statecoordinates of 1. and Z. HydroBen atoms were addcd al idealgeometric localions. Molecule la was minimizcd usingMAXIMIN2 unlillhe convergence erileria of 0.001 kcal mol-'was reachcd in order to reduce small distortions resulting (romthermal mOlion in the crystal (vilk supra). For each molccule,the SEARCH module o( SYBYL 16 wu used 10 explore theallowcd conformalions by syslemalically varying the torsionalangles with specified increments. 9',w-Backbone lorsionalangles (udefined in Table 2),:,1 7 wcre varicd from 0 to 3600 by200 incremcnls. Side chain torsional angles Val XI •1 and LeuXI.xU were varied 10 adopt values of 60 ± I(r'. -60 ± 100and 180 ± 10'. The amide and urethane torsional ansle wand Wo 'Nere held fixcd at their cryslal structure values. ln thecase of compound 2. the w' lorsional angle was first fixed atthe crystal slruclure value but Ihe SEARCH procedure wasrepeatcd with w· adopting dihcdral angle value of +60° and-W. Rigid bond lengths and bond angles 'Nere assumcddurinl the 5EARCH process. A leneral eut-offof0.85 times the

: The principal dihcdral an.~ arc useeS unn otherw!~'!stlted.

123

• J. CHë.M. SOC. PERKIN TRANS. :! 1994 1635

124

Cll3)

Boc·Val·\II(CH:O)·Leu·OH (:lI is shown in FilS. 1 and :!respectivel)'. Table 2 provides scleelcd comparative tonionalanlles.

The crystal structure of 1 consisls of IWO indepcndentmole<:ular conformations. la and b. which dirrer mainl)' in theLeu side chain conformation. The leu side d.ain adopls Ihe g-'confontlation (defincd by Xl and XU dihcdral anllcs) in 1.while it is found in the 19" conformation in lb. Il is notewonh)'thal the Leu side chain conformation obsel'\'cd in Il is the onemost frequenu)' found in reportcd cryslal structures while theother (/g·) is the nelll frcque(lll)' observed; ail olher con·formalions heinS rarely reportcd. JO As a result of thesc IWOdirrerent side chain conformations. the crystal struc1ure la iscompaet and characterizcd by a shon C(9)-C(l6) distance[4.775(12) Al compared to lb which has a lonler C(9)-C(l61dislance [9,597(15) A),

The Yal side chain conformation as defined by the principaldihcdral ansle li. a is found in Ihe' rotameric slale in la whilebath the 1 and ,- rotamers are observed in slruclure lb inapprollimately cqual populalion.11 has becn reponed thallhe ,rolamer occurs mosl often in Ihe Val residue incorporalcd inpeptides (WI.) while each of the IWo olhers (,. and ,~) werccquaUy populated (25%) in the crystal llructurcs. JO Theoccurrence of the,- rolamer for Ihe Val side chain is uluallyasscx:ialcd wilh a local", dihedral anale value of 155' % lD-lo

as is Ihe caIC in slructure lb.ln balh confonnalions. the amide bond adoplS Ihe usual

'rans confiluralion wilh a deviation from 1&0- of S' and r rorla. and" respcclively. The N.Boc urelhane ",oup wu alsoobscrved in Ihe trafU conlisuralion (dihcdral anlle wo). Jill isnotewonhy Ihal the urethane bond waS obscrvcd in Ihe cuconfiBuration in the reporlcd cryslal struclun: of Boe·V.I·OH.u

The crysl.1 slructure of Boc·Val·",(CHJO)·Lcu-oH con·sists ofonly one moleaJlar comormation whteb is slmilar 10 Ihecompact Siruclure of la. The Leu and Val side chains Idopllhesame confonnation as was found in 1. and similarly the ItomsC(9) and C(l6) ln: <1"", [5.060(61 Al, The dihcdral ar.,1e ....,and "L... values show sil"ificant depanure ftom the valuesobservcd in la or .. and Ihis can he a consequence or lhesurroSlle CHJ-Q aroup. The dihcdral anale ""_1 \/Ilue of

FiI.2 ORTEP plot Ind Itoms numberin" or2 oricntcd luch thllthe'Boe·Yal moiety coincided with thll depiC'lC'd ror 1. For t1;r,nly. thehydrogen atoms on lhe methyls and the methylenc ha~c becn IJmiUro.

OIS) ';X-"iJ

~ c(a)

"C(3) ,~

i51a)

C(l2) 1''-') CI").,/,:, =.'',' \\--./'{~"::...',jC(10l

~..'.~. C(2) 0

Nlj/~r~c-r-) ~ :.,.r-:r"-\_ \J0(3) N(2)

'':-4-.:~0(1) AI~ ::'-.,0(2)?if -C(5)

\)1\ C(7) c~IV <~Clla)

~,\~.,. C(16) \\~q C~ f,.~ ....../ C(15)

C(B) C(9)

(b)

C(B')

A,. 1 ORTEP plot and Itoms numberin, of the Iwo indepcndmlmolecules of 1 oticnted such thallhc Boe·V,1 moiel)' comcided [Ilia);lb lb)]. For clarit)'. the hydrogtR Itoms on the ntelhyl and themcthylcnc have becn omiUtd and only one rolamer for Val is dcpicledfor lb.

van der Waals radius served 10 rejcci conformalions with badatomic contact.'1 For each allowed conformation. the con·formational cnergy was computed. This enere was calc:ulatedusing the molecular mec:hanic: approach using the standardparameters of the Tripos force field. An cletlrost.lic conlri·bUlion was includcd. The partial Ilomie charges were compuledon the initial conformation from a Mulliken populalion analysisof molecular orbital calculalions using the AMI Hamillonian(MOPAC).19 Ali computalions were performed on an IBMRISC 6lX)() model 320H. each searc:h requinn, about 2.5 h ofCPU lime. The results are iIIusuated in the form ofRamachandran plot for each pair of t'...·backbone 10rsionaJangles. Confonnalions that were within 10 kçal mol· a of theglobal minimum energy were includcd in Ihe map. The sidechain confontlalions that Icd to the minimum energ)' for aparlicular 'W' values were used for ploning the maps.

Result'iX·Ra.,· MoltC'Uiar ConjormDltofU.-A vicw of the crystal

struclure and alomiç numbering of Boc·Val·Lcu'()H (1) and

(a)

1636 J. CHEM. soc. PERKIN TRANS. 2 1994

Ta"} xlttlcd loriional angles ,-, rOt 1and 2

An.'e Il I~ 1...CI6l-0(l}-C(Sf-NIII 8, -177.1(7) 168.8<71 IJS.H3}0(1 Kill-NII Kil) w, 1'•.2(7) 175.6(7) 172.9(3)

Val l

ClS)-N(1 )-C(I )-C(2) • -82.4(6) -100.9(6) -121.7(3)N(I)-C(I)-Cm-N{:!) • 121.0(7) 149.6(7) 58.)(2)CIIl-Cl2l-N(2)'-<:lll w.w·· -172.1(1) 178.1(7) -165.7(3)N(I)...C(I}...C(JO)-C(II) XLI 179.8(9) 175.2UW -174.7(4)NOK(IKOOK(12) II.: -64.9(7) -62.1(8)· -Sl.7(2)

l.<u·

Cl2l-NI2KIlK(') • -133.2(6) -99.2(6) -68.l(21N(2Kl3K")-O(') .,' -ll9.l(7) -".0(5) -28.4(2)NI2KIll-CI')-O(5) .,' 42.7(4) Il8.9181 152.2(3)NI2KIlKOll-Cll') X' -62.6(5) 179.2(9) - 56.8(21CllKOlKO'K(5) 12•1 170.8(8) 52.5(7) -179.3(llC(ll-ClIlKO'K06) , 1•1 -66.5(6) 178.5(10) -5'.4(2)

• Atom N(2) in 1corresponds 10 ()(]) in 2.• r 1•1and,": talr.e lhc valLlCs - S6.2{9) and 59.3(9) mpcctively in 1he clher rolamer•• For distinction. lhedCJi,nalion w' wu uscd rOt Ihis torsional anale ror compound 1.

125

~. }~...~t0'· -1

...........N ~ l ...··O /'. i~HD, ~ 1. J~o..H-N ~oWO b ~o. /-o....-..N

\ ;V\. \. + )'9X .. 1 • H

0'-(0 -f--,...;" i,

~ N O~-""OO....H~~O....... i H'

o if : O-HN 1. >-.l ,. .

H;.\ \->I..."'~-LJ01.)<···0-r- H,.

''1" ~O0.• H

Fil. 3 Schema." repracnlltion of the hydropn bondiR' networtinvolvcd in the ars••1pacltin, of 1

58.3(2)- is probably 1 eonsequcnœ of minimwna 1-4 inter·actions belween Val C. and the ether OlYJCft [lomonal ansieClIO)-C(I)-C(2Hl(J) - -17S.7(J)']. Similarly. lhe dihedrolIn,le ~•• value of -68.3(2)- il probably a consequence ofminimizinl 1-4 interactions btlwœn Leu C' and the surro­piC InClhylene [lonion.1 .nsie C(2Hl(J)-C(J)-C(l3)­171.'(3)'].

The w' tonional anale assumes. value of -165.7(3) incompound Z. Il c:Iearly adoplS • IrlllU orientation. allhou'"wilh laraer ~i'lion from 1so- than Ibe unslraincd IrDIU amidebond. OaKr cuminalion orthe SUITOSI,e seometry çompared10 the amide revellslhal: (i) lhe C(I)-C(2) bond lenglh mnainsessentially the lime for bath compounds: (ii) lhe CH1-o bondis 0.09 A 10nlOT lhan Ihe .veral" omide bond [C(2)-N(2)]: (Iii)lhe o.-c- bond is 0,04 Ashoner Ihan the l\'traBe C'...(."I bond[C(3)-N(2)]. Valence .nales oround Cl 1) and cm wcrc nolsi.nificantly difTemu belwcen 1 and 2 elcept ror C(2)-C(1~

CUO) which was larger in the surrogate compound 2. As aconsequence of the tetrahedral nature of the atoms forming themethylene-oxy bond and ta the quile short C(Sp3)-O(Sp3)bond. the (."'Ire•• 1 distance is 0.12 A shorter in thepseudopertide compared to the regular peptide: 3.667(4) Ain 2.J.806(8) A in 1. and 3.762(9) A in 1•. In molccule 2. Iheurethane group adopt the trans configuration and the Boegroup geometry is in agreement wilh reported mean values.~1

X-Roy Moltn4/ar PQ('kings.-ln the crystal of 1 ail potentialhydrogen bonding donors and acceptors are involvcd in inler­molec:ular hydrogen bonds. Fig. 3 shows schematically thehydrosen-bond nelwork for compound 1 and Table 3 sivesthe respective h)'drogen bond geometries. Eaeh independenlmolec:ule is hydrogen·bonded with a two-fold related molec:uleIhroush the amide N(2)H. These hydrogen bonds are orienlcdin the direction of the c e:rystallographie: axis ror 1. and in Iheb e:rystaUographie: axis ror lb. The two-fold relatcd dimers arehydrogen-bonded 10 lheir c:orresponding independent molec:u1ein both band c c:rystallographie: axis direction. The hydrogenbonds are ensured Ihrough the c:arboxylie: group of 11 in the bdirection [0(5)H and 0(4)]. and through the c:arbolylie: groupor lb in the c direction [0(5)H' and 0(4)']. Suc:h a h)'drogenbonding sc:hemc gives rise to layers or molccuies paranel tothe he plane. The pae:king alon8 a is achieved throuBh van derWaals interae:tions with the Val or Leu side e:hain and the Itrl·but)'Igroup.

Crystal pae:kinl is quite differenl in compound 2: owinl to theabsence orthe N(2)H donor. The hydrogen bonding pattem is amore typical head-Io-tail network.1.J The terminal c:arboxylie:group is hydroaen·bonded to lhe head of IWO molecules. 0(4)Aœcpll the hydr08en from urethane N(I)H of a moleculerelated by lranslation along Q while 0(5)H donales to 0(2) oramolecule relaled by lhe Iwo-fold screw axis (Table 3). This sivesrise 10 an infinite nelwork of h)'drogen bonds in lhe abc:rystallosraphie: plane. As is the case or 1. the e:ohesion in cdirection is ensured by van der Waals interae:tions involvinl theh)'drophobie: Val and Leu side chain and the Il!'rl·butyl group.

SoluliOlt ConformaIiOlt.-Table 4 shows the 'H ISSiJRmentof NMR spectra of compounds 1 and 2 in [~H.]DMSO at296.0 K. The prcsenc:e or aboui 10";' of âs urethane bond (wo)was detected rore:ompounds 1 and 2 al 296 K in (~H.]DMSO.This phenomenon bas been fmluentl)' enc:nunlered in Boc·amino-ac:ids~· owinlto lhe small enerlY difTerence between the

J. CHEM. SOC. PERKIN TRANS. 2 1994 1637

TabieJ Intennolecular hydrogcn bond distances and angles (or compoundll and 1

D' A' ~O···AII'" OlD-H ••• A)!I") Symmttry'

1NIIlH 0(4') 3,048(7) 146' (.... ,11,:1N(2IH 0(2) 2.993(6) 170' (x, -y.2 -:)0(5)H Dm 2.651(6) 16)' (-x.~-y.! +:1Ntl)H' 0(4) 2.921(6) 155' t-x.i-y.-!+:lN(2)H' ()(2'j 2.920(6) 170' (x, -1, 1 - :)0(51H' 0(3) 2.576(6) 157' l.t, 1.:)

2NIIlH 0(4) 2.969(4. 147(2) (-1 + X,1,:)0(5)H 0(2) 2.633() 156(31 (2 - .r,l + y. 1 - :)

• Donor. 1 Aa:cplor.• oralom A.• Hydrogen ;,Iom positions 'ftt'C calculaled but nol refincd.

l?ii

Val

6..J"'H-e-K6""J""....6""JcJH •..Je ••6~., '......Leu

'"")"'H-cH

6""Jl'"N~.

J"""",,,6"",6"",Jc'K.<'HJe-H-c'HJc"M..-c'M.6""Je ••Je•H ••

d"."d".'.

1.363

6.6389.183,7657.321.9067.046.'.0.8400.803

7.9687.924.2249.785.261.S361.4774.668.75

-1).611.650·j.S76.52

0.8740.B12

2'

1.361

6.5259.123.3586.091.7076.916.850.8070.780

3.7649.653.951.4991.3674.728.67

-13.991.7~

6,576.700.8740.862

1.)

lb)

Fit." Mosl probable solution confonnllion for J (a) Ind 1 (b) thltarc consislent wilh NMR dltl in ['H.]DMSO. The mOi' intmlCNOEs oblcl'\'Cd inside the backbone Irc shown wilh an Irrow.

ds-trcuu urelhane configurations. Consequently. an exchansccross-peak wu observed betwcen lhe downfield r,tJIUurethane NH and Ihe upfield cil urelhane NH in Ibc 2DNOE spcc1.ra.

Acc:ortiinB la Ihe NMR dala. Ihe ",CCH10) pseudodipcplidc(2) sccms la have a backbone confonnalional prefercnc:e insolulion Ihal is relalcd la Ihe crystal slruclwe. A J!ronl NOEcross-peak wu observed betwccn Val NH and the upfielddiastereolopic melhytcne-oxy prolon CH.-o white only a ....caksiBnal wu dctcetcd ....ilh CH...-o. Morrovcr. CH.-Oexperiente$ stronser NOE wilh Leu C'"H lhan CH...-o does.Takcn lOBether wilh lhe fKt Ihal JC'H<tIrO is sipificanllysrcater than JC"tt.Q.~ ....hich slems from a prcferred "lUISorientalion of CH.-o relative 10 Val C"H.··u and lhal the8)'slrov relalionship suualS valucsof -100- or -140" for Ibc

'''.1 dihcdral anBIe,le. the cf)'llai conformalion may indccdrcprcsent lhe mosl prefcrred conformalion on in solulion. Fil.4(12) sho....s a probable confonnation of 2 lhal ma)' explain Ibcprcc:cdinS observalions. OOIe cuminalion of Fil. 4(12) revealslhat lhe diaslereolopic CH...-0 lies on lhe lime side a. Ihccarboxylic sroup which may explain ilS deshieldinl comparcdtoCH.-O.

Frce rolalion probably oecurs for Ihe Val side chain of 2owinB 10 lhe value of JCW'CH' and 10 lhe sironi NOE delcclcd

• Rowncr populltion anal)'lil around N~-C-o utin. Pachlef,approach wilh patlmclm dcrivcd b)' Abraham and GaUI provided"9'1. for lhe " rotamcr (lhc ct)'ltalJoplphic: onel. ))% for Il,, , anat~l.rorlhc,·.

127

1638 1. CHEM. soc. PERKtN TRANS• .2 1994

fil'. ~ Newman proJC'Clion iIIustrlllting the rcLalionship bctweenprolons ror lhe two possible Leu side chain conronnations in solution:Il'1 Il,z,.1 .. ,-,: (b)XI.x:.J .. '~'. Compound 1. X _NH: Z.• -O.

• The X.ray dilTraclion study or Z wu first condueted at roomlemptr<llurc bul the hi.h thermal molion associalcd wilh the Val sidechaIn pre~ntN Ihe obtention or 1000 R even wilh risid bodyrcfincmcntlllnd IIllempllo locahleolhcr rolamers by Fourier difTcrcncc.The nperimcnl wu Ihcn conductcd a121) K.

bclwccn Val NH and Val Mes which Îs indicative of theoccurrence ofg. or g- rot.1.mers for Ihis side chain,'

The important chemieal shift difTercncc:s bcl\lo'ccn the Leugeminal diastcreolopÎc et H1 CâlS = 0.13 ppml along ....ithIhcir considerable difTercncc in vicinal coupling WÎlh Leu C'Hand C'H (Al .. 4-6 Hz) indicalcs conformational homo­(llcncity P for the Leu sidc chain. Assuming tbat the larsecoupling constant values are associalcd with prcfcrred trDIIS

orienlalion for the involved protons and that the small couplingconstanls arc associalcd \l,ilh a gaucht- orientation. IWO sidechain conformations for Leu arc possible. Fig. 5shows Ne\1o'manprojections of the 1\10'0 possible side chain conformations.Ellpreued as Iheir principal dihedral angles (xl and 12.1

)

Ihey correspond to the g-, and IR· conformalions. As

...~r~H("").., ~ •..:qx.Co,H

H(poR)

(al

....HI.....). 't"~

.~CX>} ..,­(0)

mentioned above thC\C are the two mosl often oceurring sidechain conformations ciled for Leu ..nd it is not possible 10 rcject('ne oflhem on the basis ofunfavourablesteric interaclions.lt isalw nol possible 10 rejcct oneofthem on Ihe basis of presenceor absence of NOE (sinee the usual NH is replaccd by 0).Inlercstinsly.lhediaslereolopic H' which is Irans relative to H­can he eXpcc1ed 10 bc more dcshieldcd compared to Ihe onewhich is gauC'nr relalive 10 H· (Fig. 5): Ihis is indccd the caseITable').

The imponanl NOE obscl'\'ed between Val·NH and Leu­NH for compound 1 is indicative of a solution backboneconformation in [2H.]DMSO Ihal departs from the bad:bonecrystal structure where Ihe IWO NHs were pointing in opposiledireclions. Funhennorc Ihis is accompanied by a very slronSNOE hetwccn Val·C-H and Leu·NH (CHjNH; .. d. Thcscobscl'\'ations along with Ihe facllhallhe ..·alue of Val J NttCH isquite high sUBScsts values of -ISOC' and tU for Val,."dihcdral angles rcspcclively as dcpicted in Fig. 4(b). Thepossible existence ofa stable C:.. intramolccular hydrogen bondbclween the urethane carbonyl and Lcu·NH. although nolsupported from NOE dala. was invcsligalcd by studying thetemperalure efTccl on Ihe NH chemical shifl in [2He]DMSO(!tI5llin. A constanl slope of -5.1 ppb K- I was obtaincdbctwcen 296 and 346 K which rules out the presence of thisinlramolccular hydrogcn bond in solution_

Similar 10 compound 2. Ihe Val side chain of 1 is frce 10 rotaleowing 10 Ihe Iypical J",v value obscl'\'ed for JCOHC"tt. The LeuH's are diaslereolopic in compound 1 bUI the chemical shifldifTerencc is smaller than obscl'\'ed wilh compound 2. ThecouplinS conslants involving the Leu side chain protons suggestthe same possible side chain confonnalion dc:scribcd for 2(Fig. SI. The obsel'\'alion ofa small NOE bclween Lcu·NH andLc:u·C'H SUggeslS Ihat the g-I confonnalion ma)' he dominanlin solution.

,""",

•00

"....••

Lau

::~:::o••••• 00•••••• 0••••• 0.. ... .•••••••••••••••••••••••••••••• 0••••• a 0••••• 0•••• 0•••••••••••

o

180

•••

•........•

Val

-1800 180 -'80 0 '110

,(°1 ,,(0)(1)

VII lIu180... DIO'.'... 000 ••••.. 000 ••••.. 000 ••••.. o•••••• •.. o••• •... '00 •.. ;;- D'D'

0 DOD"i D'III'"D•••••• •••••••.. 000 ••••• ••••••00.1'

000'000' •

-'BO0 '80 -'80 0 '80.(0) ,,(0)

(0)

00

'!~HO....... 0a .o•••••••••••••••••••o.".....

o••••••••••ODDao

o••••••••••a •••••••••:~:::•••••••••......o•••••••••••••••••••••..

00••••••

o

o

'80

'BO

-'80-'80

fla.. Ramachtnclrlln mapl t"..,1 for lhe Val .nd Leu raMi. for hl) 1 and lb) 2 obtaincd by s)'slcmalic conformaliona) se.n:h. The full dolsrtprntnll confunn'llOfIllhtl wcrc .ilhin SItcal mol- I orlhe liobal cnero minimum. lhe emply dol rcpratnts conformations thlt wcrc bctwœn Sand 10 Itcal m:3I'\ of the 110bU cntrJ)' minimum. The smalllcllcn a.nd b in ((JI indicate lhe , ... vllues obscrved ror Il and Il fCSpcclively in lhecrys,al stllr. l'be small numbrT 2 in lb) indicaln the , .• values oblervcd in the crystallllte:.

l. CHEM. soc. PERKIN TRANS. 2 1994

CQn!orma,iona/ Mimia)'.-Ramaehandran maps obtainedfor bothcompoundsaredepicted in Fig. 6.The mapsdepieted forcompound 2 are with the w' lorsional angle filed at thecrystallographic value. When the syslematic SEARCH wasconducted with a/torsional angle filed at + 6QO or _60°. theallowed conformations were more than 7 kcal mol· ' higher inenergy comparcd to the minimum energy conformalionobtained using w' filed at - 166°. Moreo...er. the plot ofRamaehandran mapin thiscase (data not ShOV.l1) re...ealed only anarro\\' populated area. These...ere gaurhl'sterie interactions thaloecur bc:twecn the Iwo tcniar)' C's when w' :::: +6W or -()OO

could account for the highcr conformational cncrgies obtainedin Ihcsc runs. The resulting low population and hig.h sterieenergyofgallcherotamers forthe w' torsional angle is furlhersupponedby our NOE measurements sinee no NOE was dctected belweenthe two e-Hs for 2 which should ha...e becn obsc:rved olherwisc:.

Comparison of Ihe Ramachandran maps for the Val residuein 1and 2 shows thallhe number of allo"'''ed conformations islarger for the pscudodipeplide. The replacement orthe carbon)'1by a mClhylene increascd the number of ...alues a...ailable 10 1/1.particularl)' for the negati...e values of "". Rotation around""....1 for 2 is indeed weil rcprescnted b)' a three·fold rotationalbarrier with thrce low encrgy minima .lit 600. _60° and 1800.

On the other hand. the Ramachandran maps of the Leuresiduc show that the number ofallowcd conformations within5 kcal mol- I for 2 is smaller compared to 1: Ihe replacement ofthe amide NH by an oxygen rcduced the number of'l......aluesavailable. The preferred ~ln ...alues are concenu3ted around- 60° ± 300 in 2 while values from - 6()0 to -180° wcre allowcdwithin 5 kcal mol- I for 1. As mentioned earlier. the preferreddihedral angle value for !Pl... stems from a tendency 10 minimizcunfavourable interactions betwccn Leu·C· and -CHJ which areamplific:d owing 10 the short C(spJ)-O(spJ) bond length.

DIstus5ionOwing 10 the double bond character of the naljve amide. thisstructural clement is recognized as of prime imporlance in Iheelaboration of the three dimensional conformation or poly·peplide chains. Il is weil known that peplide bonds usually donot deviate from planarity by more than 24°.JI The substitutionof an amide bond by a melhylene-olY group within peptidestruclures would he elpected to introduce substanlial rotalionalflcxibility. Our elperiments demonstrate Ihat the methylene­olY surrosale confers unelpcc:ted rigidification ractors in amodel hydrophobie dipeptide.

The X·raycrystal structure of2 and the molccular modellingstudy revcaled a preferred "allS orientation for the C-e-o-cbond (where C is an Sp' carbon). In the pscudopcplidc contellit directs the preferrcd ...alues of balh the w', and "1+ 1

10rsionaJ angles. This is corroborated by a previous study onolo-erown ethers whic:h ha...e dcmonstrated the antipathy forgauch~ orientation of the C-e4-C bond. Jt This isal variancewith theC-e-s-c bond (where C is an spJ carbon) whic:h haveheen shown to adopt a gauch~ orientation in Ihia-erown ether.Z9

This reversai or preference has bcen elplaincd in terms orfa...ourable or unfavourable 1-4 laucllr interac:lions. Owing tothe short'C-O bond length in the sequence C-e-o-c Ihe1-4 gr1llCM intcracts are unfa...ourable while in the C-C-S-Csequence the lonl C-S bond renders the 1-4 ,auchr interaetionsfa...ourable. Rec:cntly. the crystal slructural of Boe·Ala·\lI'(CHJS)-Phe·OH has becn reportcd JD and indeed Ihe w'angle adoplS a value of - 71-.

Our NMR sludy SUUC$IS that the preferred bac:kboneconrormation or 2 in DMSO solulion miShl he c:losely relalcdto its crystal structure conrormalion and Bives support th.t anune.peelcd rilidificalion elemenl is introduœd by Ihe methyl­eneoQlY sUTrOsale.

1639

ln summaT')'. owing to internai conformational constrainlS,the methylene-oxy su"ogate adopts il preferred orientationsimilar 10 the IrQ.ns amide bond and cau~ the ".1 tonionalangle tO adopt a preferred ...alue of -600. Based on molecularmt'Chanic ~Iculation. the replacement of Ihe carbonyl by amethylenc group allows. howcver. more rotllional ftelibility for!p,. The ether ox)'gen is a weak hydrogen bond acceptor andalteration in biological aclivilY upen substitution with thissurrogate can bc associated wilh 3 lou of il h)·drosen bonddonor or acceptor in...ol...ed in a crucial inlerOlClion with Il

rt'Ccptor macromolccule. although lhe conformalional efTctts(t'ide supro) must also he ta!ten into account

AcknoM'lfdgementsG. V. thanks NSERC for a doctoral scholanhip. Our Ihanksalso go to Gaston Boulay for mass spectrometry measurementsand 10 Françoise Sauriol and Normand Pothier for judiciousad...ice in NMR speclroscopy.

References1 For rteenl reviews. sec J. Rito and L. M. GieralCh. A"" Rn'

Bj!Jcnml.• 1992.61.387; A. Giannisand T. Koller. .•IIA't.' Chtl" .• 1",.Ed. &,1., 1993. 32,1244.

2 J. DiMaio and P. W. Schiller. p'M. Nad. Acad. Sel. USA. 1980.17.7162.

3 For tlample. let G. Villeneuve. J. D.Maio, T. H. Chan andA. Michel. J. Chtm, Sile.• Ptrkin T,GlU. 1. 1993. 1897.

4 For review. sec D. T. Elmore. A",."o AClds Ptp,.• 1991. n. 83.SForeumrle.sec M.C. Allen. W. Fuhrer. B. Tuck.R. W.dcandJ. M.

Wood.J. Mtd. C1rmI.• 1989.32. 16S2;J. F.lkllaria. R. G. Maki. Il.H. Siein. J. Cohen. D. Whillem. K. Marsh. D. J. lIorrman. J. J.Platiner lInd T. J. Ptrun. J. M,d. Chmt.• 1990.]J. 534.

6 A. F.Spalo\a.A. L.Rockwelland L. M.GieralCh. BioptJJ.I·mm. 1983.22.147; M, Goodman. BjopuJymm. 1985, U. 137: B. HalUn and M.Goodman. BjocMmu"J·. 1986. 15. 1596; N. J. Mamm. and M.Goodman.Biuthtmu".~·.19B6.25.7607;A. F.Spalola. M. K. Anwer.A.L. RockwellandL. M.Gierasch.J. Am. Ch,,". 5«.• 1986.1011.825;P. Van der Elst. M. Elscvien. E. DeCadI,. M. V.n Manenille, D.Tourwê and G. Van Binsl. In'. J. P,p'ith p,ol,in R,s.. 19B6, 17. 633;G. lanoui. C. Tonialo. T. J.~ ilnd A. F. Spaloll. Ac'"Cr.l's'DJJo,'''S«" C. 1988."'. 1S76;C.Tonolio.G. VIUe.M.Crisma.J. S. Kahenbronn and J. T. Repine. P'P'''' Rn, 1989, 1. 332; C,Toniola. Bjopol)'lMfS. 19119. JI. 247: D. B. Sherman Ind A. F.Spaloll. J. Am. CI"m. SM.• 1990. 111. 433: M. K. Anwer. 0, B.Sherman Ind A. F. Spalola. 1111. J. PtplUk Proltitl Rn. 1990.36.392;D. F. Mierke.C. Panaroni. N. Dellet.A. Toy and M.GDOdmln. llIt.J. P,p'iM Pro"in Rts.. 1990.36.418; A. O. Michel. O. LaJoie andC. A. Hassani./tu. J. P,p,Ult P'O'tin R'J.. 1990.36.4119.

7 J. Zlbrocki, O. D, Smith. J. B. Jr. Dunblr. H. lijim. and O. R.Marshill. J. Am. Chmf. Soc.. 1988. 110, 5875.

8 N. E.Kohl.E.A. Emini. W. A.Schlcif. L.J. Oavis.J.C. lieimblth, R.A. F. Dilon. E. M. Scolnitk and 1. S. Si,.l, 1"0(. Nall. AcQl/. Sc;.USA. 1988.15.4686.

9 R. E. TenBrink. J. 0". Ch,,".• 19117,51.4111.la V. LeP'Je. P. S. While and E. J. Oabe. NRCCAO. An Enhanœd

CAO... Control Prolram. AnnUlI Meelinl of Amencan Crys.tal10lraphit Auocialion. liamilton. Onlario. Can.dI. 1916.

Il E. J. Oabe. V. LePait.J. P. Charl.ndand F. L. Lee. NRCVAX. AnInteractive PrOlTam Syslem for StNtlurc Analysis. J. Appl.Cr>'sIQII~r .• 19119. n. 384.

12 A. C. Lanon. A(,o C,ysI"'lo" .. 1967.13. 664.Il D. T. Cromer and O. T. Wlber. /,lImw,iUNlI Tabl" /0' X·,uy

Cn·s'allo"QPlly. cds. J. A. lber.nd W. C. Hamillon. Birminlh.m.KYnoth Preu (presenl distribulor Kluwcr Academie Publisher.Oordr«hll. 1974, vol. IV. pp. 99-101.

14 S. W. Hom'M.R.A. Dwek. O. L. Fem.ndcundT. W. Rademlthcr.P,oc. Nad. AtIJd. Sci. USA. 1984.1'.62116.

15 S. M.cura. Y. HUinl. D. SUler.nd R. R. Emsl. J. MIIIII. 1f'l1Nl..1981.0.219.

16 SYBYL Molccular Madelin" Vcnion 5.5. Tripas Auociale Int..February 1992.

17 The dtfinition is in 'Irecmcnl wtlh the IUPAC·IUB JointCommiuion on Bioc:hcmial Nomenc:laturc. 61oclwmu,ry. 1970. J.)471.

128

1640

18 A. Michel. G. Yil'eneuvcandJ. DiMaio.J. Comput. AUJ, MDI. 1k.Ji,,,,1991,5",553.

19 M.J.S. [)n.'.r,E. G. Zocbish. E. F. HealyandJ.J. P.SlCWan,J. Am.Ch"". 5«., 1985. 101, 3902.

20 E. Benedetti. G. Morelli. G. Némethy and H. A. Sd\erasa, 1",. J.P'P'IM Prat"" Rn., 19113. n. 1.

21 E. lknedcUi. C. Pedane. C. Toniolo. G. Némcthy, M. S. Pottle.ndU. A. Schctap. /111. J. P,priM Prot,in Ru., 1980••6. 156.

22 C. Tlmiolo. M. Palumboand E. Benedclli. MOrromolulll,s. 1976,9,420.

23 C. G. SureshaRd M. Vijayan,l"t. J. Prpt/iN Prortin Rrs.• 1983. U. 617.24 M. Br.nik and H. Kcs!lcr. ClIrm. B"., 1975. 101.2176.25 K. G. R. Paçhler. Sprr'roc:/r",.. Acta, 1961,10, SSI: R. J. Abraham

andG.Calli.J. CMm.Soc. 8,1969.961.

J. CHUt. SOC. PERKIN TRANS. 2 1994

26 v. F. Oyllro.... V. T. hanov, S. L. Portnon, T. A. Ba1&shava andY. A. Ovchinnikov, Tt/rahA/fOrt, 1973.19.873.

27 H. Kesskr. An,",. c.vm..r",. Ed. &,1.• 1982.11.512.2S 1. L. Karlc. J. Am. CMm. Soc.• 1978. 100. 1286.29 R. E. Wolr.Jr.• J. A. R. Ha"man.J. M. E.SlofeY. B. M. FOlmlon and

S. R. Cooper. J. Am. CMm. Soc.• 1987. 109. 4)28.30 G. bnoUi. C. Toniolo. T. J. Owen and A. F. Spalola. A('ld

C'YJ,allo" .• SHI. C. 1988.". 1576.

PlJp~' 3/07609AReuived 30th Du~mber 1993

Accrpted t5th Ma,cI.I994

129

Chapter 7

Conclusion

A topochemical model was derived to account for the diverse selectivity ofopioid ligands using a computer molecular modeling approach. The opiatepharmacophore was used as a minimum prerequisite to align the peptides HTyr­c[(D)Om-Gly-Phe-Leu] (c-ORN, IL selective) and HTyr-(D)Pen-Gly-Phe-Cys-NH2

(c-PEN, Il selective) onto the semi-rigid alkaloid 7-a-[(lR)-1-methyl-l-hydroxy-3­

phenylpropyl]-6,14-endoethenotetrahydrooripavine (PEO, unselective). Usinglow energy conformations for the cyclopeptides, it was possible to find a commonlocus for the aromatic rings of Phe4 for both peptides with the ~l aromatic ring of

PEO while still keeping a fit with low deviation for the tyramine moieties of therespective ligands. Assuming that the opiate receptor binding site is finite, the

superpositions that gave rise to maximum mutual steric volume occupation of themolecules were retained for possible model candidates. The steric volumegenerated by the pendant ~.~-dimethyl group of Pen2 of c-PEN extruded

significantly from the common volume and can be incompatible with the topologyof the IL receptor. When the aIkaloid naltrindole (one of the rare alkaloid showingpreference for Il receptor subtype) was fitted onto the previous model, the extrabenzene ring present in the a1kaIoid was fl'und located in the vicinity of the ~­

methylene of CyS6 of c-PEN. On this basis we proposed that a putative accessorysubsite present in the Il receptor may interact with the supplementary aromatic

ring of naltrindole and also with sorne structural elements of the terminal amino·

acid of cyclic disulfur enkephalins. This subsite is presumably of different nature,or differ significantly in its location, at the IL opiate receptor.

If our hypothesis hold, we reasoned that an aryl group added with theappropriate stereochemistry on the methylene of CyS6 of c-PEN could reproduce

the role played by the supplementary. aromatic ring in naltrindole, thereforecausing significant increase in potency for Il receptors. To do so, we decided to

prepare the special amino acid ~-phenyl·cysteine. We engaged in the preparation

of this amino-acid using a method based on the addition of mercaptan to

azlactone. Owing to the presence of a P·phenyl group, the S-benzyl protecting

group was not suitable for selective clean sulfur unmasking. Use of an a·

naphthylmethyl protective group was found ta he suitable for clean sulfur

131

deprotection but unfortunately, no resolution could be achieved with the enzymecarboxypeptidase A on the corresponding trifluoroacetamide substrates becauseof the bulkiness of the new protecting group. The sulfur containing amino-acidpreparation was then adapted to allow the introduction of a S-p-methylhenzylprotecting group whose dimensions were shown to be tolerated by the enzyme.The actual method for obtaining ail four isomers of p-phenylcysteine had the

advantage of being suitable for large scale preparation. Ail purifications andseparations were performed by extractions and crystallizations. Enzymaticresolutions were efficient and highly enantioseleetive.

On the other hand, further structural support was sought for Il selectiveopioids. The solution conformation of the model cyclopeptide NIXCbz-c[(D)A2bu­Gly-Phe-Leu] was studied by lH NMR in [2He]DMSO. The observed intemuclei

distances, coupling constants and assignment of non exposed NHs were shown tohe compatible with a structure characterized by a Pn' tum centered on Gly'PheC

locked by an hydrogen bond between Leu5 NH -> OC A,Bu2. In addition, the

temperature dependency of chemical shifts agrees with the presence of anhydrogen bond involving HN A2Bu2. Using molecular dynamic simulation, the P­tum was shown to be quite stable while two possible acceptors were observed forHN A,Bu2(OCA,Bu2 and OC Gly'). By the same token, the dynamic aspect of theendocyclic side chain of A,Bu2 was unrevealed: the possible values for the side

chain dihedral angles Xl.x' were -60",-60" and 180",+60" with approximately thesame residence times. The macrocycle conformations involving an A,Bu2 side

chain dihedral angles Xl,X' =180",+60" were found to be highly compatible with

the topology of the computer generated opiate receptor mode!.

In a side project we investigated the conformationai consequence of the

substitution of an amide bond by a methylene-OllY group within peptide structures.

Although substantial rotational flexibility would he expected to be introduced, our

experiments demonstrate that the methylene-Ol'y surrogate confers unexpectedrigidification factors in a model hydrophobic dipeptide. The X-Ray crystalstructure, NMR solution conformation and the molecular modeling study revealed

a preferred trons orientation for the C-C-O-C bond (where C is an spi carbon) inthe model t-Boc-Val-['CHzO]-Leu-OH. In the pseudopeptide context it directsthe preferred values of both the <al'; and 4li+l torsional angles.

•132

Contribution to original knowledge

We have proposed an heuristic model conceming the divergent receptorselectivity of sorne cyclic enkephalin analogs and narcotic alkaloids towards opiateIl and .s subc\asses. Up to now, several models of the opiate receptor have been

proposed in the litterature but, for the first time we proposed that the spatial

locus of residue five of opioid peptides may correspond to the locus of the extraphenyl ring of naltrindole in the opiate receptor microenvironment. On the otherhand, we have studied the solution conformation of the model 14 membered ringcyclicenkephalin NaCbz-c[(D)A2Bu-Gly-Phe-Leu] by IH NMR and by moleculardynamic and deduced the presence of a relatively stable p-tum type Il' centered on

GlYSPhe4• The conformation proposed in solution for this cyclopeptide iscompatible with the shape requirement of the opiate receptor as proposed by us.

The four enantiomeric forrns of the unusual amino acid p-phenylcysteine

were obtained using an original approach based on enzymatic resolution andsuitable for large scale prepartaion. During the course of this preparation, wehave explored the mechanism of benzyl thioether c\eavage promoted by metallic

sodium in liquid ammonia. The results that we obtained favor a 2 electronprocess (anionic). Also, the resolution steps have perrnitted us to explore thesteric tolerance of the protease carboxypeptidase A Branched p-phenyl groups

are allotted for erythro or threo diastereoisomer while the main side chain cao

expand up to three carbon terminated by a phenyl group. ortho substituents are

not tolerated on the phenyl ring while a p-methyl is. The side chainconformational preferences of p-phenylcysteine were studied by X-ray diffraction

and molecular modeling study and a simple rule was proposed for determining il.

The conformational consequences of inserting a methylene-oxy surrogateto the amide bond into a model dipetide were investigated. We have

demonstrated the unexpected rigidification element introduced when doing this

substitution in peptide using X-ray diffraction, solution NMR, and molecular

modeling study. The geometry of the surrogate relative to the correspondingnormal amide bond is almost identical in terrns of O"I"C"I+I distance and torsional

angles.

Supplementary Material

Al

~

~ .r-.•"

- a~U~HB~ ~~.

LeU--=O==H::;;::::::::~=::::J:: ~ 0

r-""r~r •r-""

(D)A2Bu N"'H (cis)

(D)A2BuN&H

(D)A2Bu N"'H (trans=)~~.~ ~ 0

Leu N"'H - =---:J f"

[2HslH]DMSO

PheC~HB

PheC~HA

Leu C~HA

(D)A2Bu C~HA (D)A 2Bu C~HB

Phe CH aromCbzCHarom

IH 300 MHz spectra of N"'Cbz-c[(D)A2Bu-Gly-Phe-Leul (3.1) at 297K in[2HelDMSO

H20

------=---------------CGH.\y;Cca;;jHH:"B---::~=~s=;~:(D)A2Bu OHA ::

Gly caHA ---=~;i~: ~:PhecaH =1F ~

(D)A2BuCUH { ~:

__Cb_Z_C_H_2 Le__uca_H ~. _~ ~~~

GlyN"'HPhe N"'H

• IH 300 MHz 2D NOEof NaCbz-c[{D)A2Bu-Gly-Phe-Leu] (3.1) at 297K in

[2H6]DMSO

8§ " 3.0000000

" 1i.00"0112 5 " .0000030

CVU6H23.Sf'lX 1/0111 5 P3 6.00AU PRDO: SSII2 0 " •sooooooHors y • AU 5SBl 0 RD •••

O""E 23-1O~O '", " o••

PLI" RDIt: OE 232.50

'" 1024 " 9.31lP NO ..5" "2 F2 .335P os 2

"2 2117.391 AND CDLUI'lN: " '"'" 13511.U6 " 9.311 P IN .0003680

"'. , F2 .335P " •

GVU6 NOE 23

- 1 ~

Q ,. ... If, Il'' "''o.{?,' ~ ,..Q ~d~ .~ {).ol;} .-

2••

0

·Cl ..

.~~• 'lI!l:> .. 08 3••0 '.' .. . llIl- ·f'O

,.,; ..~: ..... . ~... "!

l tJ :"~o'.0.. (J '.. '0'..

~ .<;' 0

d. .~;. -;e ... s.,

•••

• *? /!} ,..~ , "', '. . ZlO " :III:lCl 0 fJ

~o.•

•o'

10 .... ,..-• • •.. .l.r

,1 1 1 1 1 1 1 1 1

pp,

,.. 0.0 ,.. •• • s.• ••• 3.0 2.• 1.0pp,

A2

• lH 300 MHz spectra of NaCbz-c[(D)A2Bu-Gly-Phe-Leul (3.1) in[2HalDMSO

A3

301K

306K

1.' 1.' •.•

,,

,',1 1

.J, ~L ,L .. Iii i. u.l~~~~~~~~~~"~'"T'"'-''' 1•.• 1.1 I.t t.t ••• 1.1 ~: 1.' t.1 ,.. 1.' 1.1 1.' 1.1 1.1

A4

IH 300 MHz speetra of NttCbz-c[(D)A2Bu-Gly-Phe-Leul (3.1) in[2HelDMSO

311K 1

Il ! I~ '1Iii 1 1

:ru'lJl, lfiLAa..--JI Wi!

1 1 ,.........,.. ,', ... ... ... ... ... ù ... ,:, ,:.•• •• .. o., o.• ...m '" ...

r

316K

IluWI]\---J JAll u , J~jlJ~..... •• ... ... o.• o•• ... ... ... ... o.• o•• ... ... o•• ... o., o••.~

t,t 1.' •.• J.' ',1 1.' 1.1 '.' 1.'mt.I 1.' 1.1 1.1 >.1 '.1 '.1 1.1 1.'

. ,

321K 1

1

- ~W U~ I/~ fi U' Il III1 --

1 1 1 ...-.,.... ............, 1 1 1 1 1 1 1 1 1

326K1

1J.j~_.JI j JLol.J\!

1.' '.1 •.• 1.1 1,1 lof 1.1 •.• 1.1 :.:. '.1 f.t 1.' 1.' 1,1"T" i

1.' '.1 '.1

AS

Graph of chemical shift of NHs proton with temperature for NIlCbz-c[(D)A:Bu­

Gly-Phe-Leu] (3.1) in [:Hs]DMSO

(D)AzBu NIlH

7.0,-------- ...,

â... 6.9...--$1.<:l., 6.8-'".~a~ 6.7o y = 9.1998 - 7.6814e-3x R-2 = 1.000

330320310

Temperature (K)

6.6 7--""---J-.--....---JL...--~-...L... _.J~90 300

(D)A:BuN3H

6.62 r---v.;---------------,......a~ 6.60......$1-=., 6.580;.lOIel~ 6.56o y = 7.2909 - 2.2800e-3x R-2" 0.997

330320310

Temperature (K)

6.54 L..- ...L..._-'-_--L_---''--_.L...._.............;:,..J

~90 300

A6

GlyNH9.1......------------------,

y = 10.454 - 4.7428e-3x R-2 = 0.979

330300 310 320

Tenlperature (K)

8.9 '--_-'--_......._ ........_-'- '---_.....,>..--J~90

PheNH

9.1 ......-......,---------------....,

330320310

Temperature (K)

3008.9 '--_-'--_......._ ........_--L_--'......._J...... ~

~90

âj:l,j:l,.....$:i~ 9.0«l.lolEl.,5 y = 10.654 - 5.2986e-3x

LeuNH

y =7.0964 - 3.6000e-4x RA 2 =0.986

300 310 320 330

Temperature (K)

-- 6.990El

""j:l,.....:El 6.986~.,-'".!olS 6.982.,~

0

6.97~~0

A7

eU446 111 ilI'lSO-c16

~8 7 ,

H','",

H65536

r

DEC. '" VT

_H

,

JI.-_

d.1 2eedp..r Jeb.p 2".e

PROCESSINC-1.34e.568

lIol lla.c1

d,d.rd.d..

lb,r,r....Ull.procr,••t.h

..,

,,,"

293.e4366.3

JO'8

25817.4?JJ.5?

51e4.1361S.9

71. ...

SAMPLES.p a6 93

D"SOd.l.aol .... lllIll.

ACOUJSlTIONdrq 499.S43lfI Hl.l 1.S921Ip 3e212a.. seee.efb 44eeba 32lplolr 61'poo 6.edl •loi e" ." 8.lock 11IfUIl 1l0llla'c1

FUIeS

DISPLAY

....d,ho

.,-,••P<.,hz••..'"","...al cele ph

_-,~,'Ill..__-" 1 J'UJ~ ...IU, 2

AB

!•1,

~ ~

:jl.. :c

"':c <JI(") '68crN =:=:(") ~:c:c o N~ '"~

"0(1)n

= ....~

~

0;c ...

zJo'

Il(")

tl:l cr':'= nq ~......0;c .....)-

"'I:l ..:r tl:l(1) =,q (;)

.:c ~~ ~::1 (1)

Cl.,

(;) ~-< =-~

......!"':c ........

> ~..N

l , '0-..J~

6"

:

.............•....._­l......

t::::'.0'-_......_....._.•..

~.O

!•1, )-

'3..~=z.:c)-..tl:l=Z.:c~::1Cl.)-..tl:l=Z'":c

••

i.,.~!"~~~~~~~~~~~~~~ .. !"~~~~!"~!"!" ~ .. ~

~1!~ii~;;ii:=i=i;~.1~2~1!!~!I;;!5;1!

~~~~'~~'~~I~I~~~~~~~~~~~~~~~~~~ ;.. ~..~.~- _ - - -

---_0_-.0o

."-"

~~~~~•.o-_.=--t••

'-".11 ....~,-"".-----......" ....

~.o -.....

--r-'.___.0

.... .,. '!" .. a!" a'!" .._ a _ ..

:~~l!l~l~I~~~.!!!!!~~~:I~i!!I!lSi~il!!!J:il;ii!5

j

~. _.

cro... ,a_'"

1_' ........,........._._.....', '''·1. •. 01' ..• .om ,- "0 'H_ '.,1'1 ..· IJI.Il' 1.

0" II·'

• ~Jl."J , H' ..• 'n- , •. ".• 'II ".

,~ ..• ~I'·U' 1.... ..•• " ..'" ,..• ..."

Il•._1.•' Il.'

" Il•.•n ..- 1.·1

" -- I·:PI ".U ••• 'H' "." 111'1 w. I.,n ,•.," Il''.''' 1.:tOl II·'

" "'.''Il ... '.0

" .U.''Il :~~JI~.I

" U. ,.> ...," .'11.'" • ln ••.. .11.'Il :.:J U'.I.. .... JIIl "'.1U ••• ... .... .,.01 .- .... ",.,.. 1.'", ,.n ,.,.- •• ,.

'-'

..,....n,n,".n.u .•

·.-.cc.,..........0•••...'H'

,_. ,_ 1 ••• , .. 1 .... 1. 1 ....

Phe NH and Gly NH.!!I i

pj!m!rj'jj!i~

Ilfllll r~i(ill-" ,."

••• ,...co.... ,1 ~J>"J

• os...J ot.n U'• .11 .....'os,, ni

_06,a_...

..

IH 500 MHz spectra of NaCbz-c[(D)AzBu-Gly-Phe-Leu] (3.1) at 297K in

[2HeJDMSO.,­...'"n.n.H'••H'.,...."..••H'.,"..'_.1U.,••••..-.

Ill.'u •.•....1•. ',.-.Il.'H'.,.,H'H.'O •H'••II.'

,......,-,J 'n0­,.11o.·,~

0­o.o.o.,.-.-..,~

0­,.­J.JI',~

0­o.,­OM•.•n'.M..­,..,1."".-..-•.m•.-..-•.,..'.'"'.m

~-. .Il ..

_ _..., cY' ,

1 ..,. ...1 n .. _, ,_.,· .-...· ,...-l ,,._

, l' ...

• u ..• un_Il _•.11'

Il 'l'It...1. UlI.11'1'l' .,..•" ,_.-Il ...,"'Il ,..,._

U IUI.raIl ',".'JIIl .."._N un .." , .tz If .,.1:) 1

t. "'"'.tI." nll._. ,.,."," ' •. tUIf 'Mn." llI'I_.'" lm,""Il lm'"11 U".llI

n'"·'''

AzBu CtHA• Gly caHu •water. Phe dlHA• Phe dlHA• AzBu DHu

~

" " " ,'. ..Leu CtH. Leu dlHu• Leu MeA' Leu Meu

• e

A10

IH 500 MHz ofNQCbz-c[(D)A:Bu-GIy-Phe-Leu] (3.1) at 297K in [:He]DMSO withselective irradiation

j 1

~

ICI""n .•.,..1'.'Il.!l'.,

' .....II:WI... '1.110..~'.m..,..'.?ll

'.' II:W".I

I UI1 .UIII.".lm....1:I'N.ltI

,•.•".."..

'.

Il

"

,j 1 j j

of A:Bu CIlH:.

of A:Bu NH.

,._1_' ICI""1.. Il.'1.'" l'.'1." Il.'1." l'.'1.'" ....1." ".J1." ".11." III.'1.'U lU.'I.DI " ••1.. ...,l.tI. ..11.. .,.•1•.., ' •• 41." M.I1." •.•1.," ....1.'" •.•1.'" Il.'1." Il.'1.'''' M.l

of A:Bu CXH mainly

OCIIlO'

l'.'U.l.•.•K.'n.,lUor.,K.'K.'K.'Il.'.,..U .••..,K.'K.'K.'K.'", ..

1....

Il'.'Ill.'

.....,1..1..,.,.....1

"'.111..1.1"m.'"m._m.11<I_.­.,.­",.,""'...._.-_.'"m.__.­_.­_.­..•.m._"..,.."I.NI.,,.,Il.'U.J

1'.'Il. ,n.l1'.'n .•K.'Il.'U.IIl.''~.Jn .•U.'U.'Il.'t1.1tI.l

n.'U ••...Il.'...Il.1.......U ••.... lJuLl'.•..._.....tI ••U ••

o .• ::::~~:::;::=~~:;,=~~n.'::: L' Il Il ......

_..-

'IIJ_'_'•.n'..~'.1"'.m'N..•.•'.10'••111

' ••J.......1.•• IM..UI•. Ut...••• 111

'.11''.1.••W

•.11'...,,.-

.......-....,... , ,

1 , 11

r Il•. 'U) Il.....

• "n._• 'In,l.• IlU·I., Il•. _· "".,"• Il•.•n\. _.1.Il _ ••

It ,.,.....

U "",JI'\0 _.1"

" ".hl1. _.111

U '"'.11'" ~.t1t.. ., ...· _..." ,•.•• llU.•

,­,•,·••,••""""""""""KWO.......... Il

..."IhIWtn.f""' '_', Il...... ...1 Il•.'' t.mJ h... '.1111 IIn.11t t.IU· .11.... ..131• '111.'" •.a, _.411 '.1.• _.," '.111• _.. '.1".. _..... '.1)1Il "'.NI •. UIIl _.101 '.IUU _... ..11''0 ...... '.111

\1 ".171 '.1"1. ....... M'I, _.1" 0.'"

_... 1.1Il

"",," J••1,",'" J.*"IW.. J.",,.... J.'"1•.1. J.'"....... J.'"nll,." J.IIIln... ,...U'Mft '.411IM••n , .•lM.... , ••

Im.ll' '.)l'IM.IM •••1... J ••I1...... J••

lIA." J.'"lin.... ,..,1... '.'1'l'II." •.Inl".eu •.•UJ'l.'" 1.'"

lH 500 MHz of NaCbz-c[(D)A2Bu-Gly-Phe-Leu] (3.1) at 297K in [2He]DMSO with

selective irradiation of A2Bu OHA

AIl

IV

-. .. -..twP'HItIIbèt""'" l11li__'_'1 1..... 1.'"1 lla,. l,",J J'ft .., 1."l ' ••:hl •. ",, IlIN •.., '.111• IM.I. ,.,.\7 ,_.:111 1._1 lai.. I.nt, 1... '.r.lI

" III'••, ,Il IIU,II, 1 .Il la... 1.•" Ia'!·... • ....It lDl••' I.n,,. u»·.n ....

..

"'CM'Il·'W·.W·.tl·lIl._W.,....,1'·'".,".,1).'

Il.'11·'·'.1

1-::

CIH_ .. -..

fIftl"'IhDtèt..... ,.,,-,,_,

.. ..

ClIO••• __

tlIIP'Httlibètf.lr '."_1_' .. ,on1 ~._ ' ••J n .•• JoII,1a .... ,..&J _.. '.Il' n.,'-.'11 •.• _.•• JJM.. •..., ".11 ~.m '.MJ n .•

,•,••••••"""u.."""""ft•ft

"J.•m ....hi••,.....................,Il'.••n.ll'N ...•.':11.,.,"•.lnM ._.........,.....­..............

,...l,'"1.'",.."....,.~,....\.,..1.11'1......•,...1••J

1.'",........,...,..,..,.",....1,11'

..

.. ,go,Il,'11.'It. ,

""• •Il.'W.H.]...,,_.IL'Il. ,

Il.'10.',..n •...,n .•...n .•Jo.'••

A12

l,

1:

Il1

IH 500 MHz of NUCbz-c[(D)A2Bu-Gly-Phe-Leu] (3.1) at 297K in [2HelDMSO with

selective irr~i,~:'n . ~\fIIft"'I.ll8tJblLl"',...on,_, .. ,..... ~

1 ....... 1.•' 11.'1 _.01 1.•' ".1 ,

o D.~ ,.,. •• \• 1Df.710 1." Cl.'

• "'.11:1 l.tA ".1• _.~lt l,tu ....f l·nt 'J.'• ..... In.'• '11.1n 1." 110.2Il ....., I.n. ln.'Il 111.11' 1••' 10.'1. •.• I.IU n .•u •.•• 1.. '0.'U ..._ l,. ".J" •.• 1.. Il.'Il ••• l,", '1.'If ••". 1.,. ".1Il m." 1.1'11 ..t.'It R.. 1.?l1 M.'lOI ..._ V," .fi ••131 I.TlI .

... -

ICI;'1

H'•..n,'H,'••>n .•n .•

_..-tIMP'IIh&bhJL"",.....,t_,

, _.. '.In• _.. ' ••JJ )lM•..,. 1.11$

• ,....111 1.111• ..... 1."1• _.C1 I.U?, ~.,.. 1.'.

.,~

.,'31.1

.,'Il.1a,'n.'.,.n.l·••

2."1>,.2••J>,~,.Wo."o,w>,.>m

'''.$111_.•'''.I.~1"'.•l''._~, .... '11

"".lI)t'....'f1'" III'n.:_j o." a .•

.. .. o, .. .. ..u...'" u" a .•Im,•• f >.- n.',n,.• >.~ n,'ItI.... >,~ n,>

ofA:BuN6Hn•.111 ,.tl. n",..... >,~.,.

lin•• 0,", n.'lin,'. J.)II' Il.1

'UI.'" 0,* 1-.'1..... o." .,.....- >,- .,.IW.U' >,- n"'''."1 >,. ,...lUI.'" >,. IOJ.'1••1" J.•' lU.'lM." ". l".?u.... .,- IIJ.'l''.N 1.''10 lIl.t

•••• ". 1•••1••1.· ..,,, .,'1".• n

.,.,. n .•1••• I.nl n .•

,i':.4.ta .,,,. n"1•••'

.,. ....1IJ'I.... ',", .,>

1:rIt.Ui I.n, $1.,['lm.• 1.'" n"

1••'".,,,. .., U

Il ~ 1

'II

U~~

1J \J~ • .~ J..... -..,0 .. .. o. .. .. .. .. .. .. ..

•a

,•>•••••,..

u..u......""..•hna..

-_ ..­"""~"_'_I

IH 500 MHz ofNŒ

Cbz-c[(D)A2Bu-G1y-Phe_Leu] (3.1) at 297K in [2He]DMSO withselective irradiation

_0'_''''_....-,•. '11.__" .. ,

UU'I.IO;>, ... , OOlIClo' of Leu NŒH, ruo,"" '.l'l'l l\.'• rul.'. '.ft. JJ.',Illl.'" ..,-. ...· 1111·"3 '.IU •• • •• "_.•7 '."' '0.'• _.on .... ,7.1

1J

11

• _.~

'.IM ".' ,• "".ID '·1" H.'

1 i" 1• _.~ ··,It H'

Il li

"_.~

'·11\ 1'.'" "".ll' ·.Ift $O.'

i~~i" nn.'" '.lU 10_'" l'IIII.n. '.101 Il,1

" ltIs.~. '_Ili II.]

" lNJ.... ..." 'J.'" ~.... 0,111 ll.7

il" m..nl 0.111 1'.'"

_.~ ..,. ...."

_.~

'.- Il.'H "It.D .- s.," nu." ..- o .•u .•.... ,.- ...a 'M.... ,.- B.'

AB

,.......-.._ .•. ft ....

,...,•,••••••"""""""""H..Ua..aa..aHB

"B~

'••'lIC,,,,,,1:IlI.a."".lt.Q.'"w._IIU.O'Itll....

Il'.'''Il','1'_.­_.~

"J.IlImol."'.,N",. 'IH".111'".nllOl.70,

'01..,.f1I.IU

"'.10.?JI.,,]

'JI.nl?II••,_.­'.0.­_.m'1........,_.­".JII_.~_.­,,..•u

'.~,...,\.'"1•.",.­,.­1.1"

1.'"I .•n,.-..1.1I.IU,­,.­,­,.­1.117l.oV

1......-\ .."\ .."..­'.N..............­I.U'l,.­,.­'.m..­......-

oClCIOIIl,l..,\ •• '1Il.'1n .•~1.1

B .•a .•n,'IIl.'..•H.'n.~

)1.'o.,..,1'.1•..lM....".,ijJ,l'I.t..,U.I.........".X;~:;

of Leu C"'H mainiy

Mt,. da ...

"'1 ..,JI/ 'l' i

r

•

_.---

'\

1\;\

/\i\..) \,..//

1 IR 500 MHz simulated spectra of A2Bu OlR2

(3.1) when A2Bu DRAis decoupled

; 1

ilil

Il: \

IR 500 MHz observed speetra of A2Bu OlR

2

(3.1) when A2Bu DRAis decoupled

A14

1.gei

1.85i

1.se 1.15

•AI5

lH 500 MHz simulated spectra of A2Bu CilH2

(3.1) when ~Bu CtHBis decoupled

i •

lH 500 MHz observed spectra of A2Bu CIlH2

(3.1) when A2Bu OHBis decoupled

\

'.N.."

i'

1

1

1!1 !\

1 \ ! Il 1\

V ~ \\~

..•,..•...,•• ft

• 1 IH 500 MHz simulated speetra of A2Bu CilH2

i when A2Bu exH is decoupled

,1Il

,)i

i'l: '.'

liliII1:1:.1

'Iiii

IH 500 MHz observed spectra of A2Bu CilH2

(3.1) when ~BuC"H is decoupled

A16

•

"1 •

1

t·..j

Al7

l,

1Il IH 500 MHz simulated spectra of A2Bu CilHzj\ ! , (3.1) with no decouplingl "l i

i"V V\

\\

\/,\.

--'

\, \' .'v'''.i ~"--

IH 500 MHz observ~d spectra of AzBu CilH2

(3.1) with no decoupling

1

1

1 1

1

•ï

~,\

~ '.,1,/.1:" -, 1

U\------~-_ ..

1." 1.• 1.. 1.. I.U I.~ I.V 1.• 1·" 1."

e e

IH 500 MHz simulated spectra of AzBu OHA and AzBu 0 Ho with no decoupling

M~) ~~

)-'

IH 500 MHz observed spectra of AzBu OHA and AzBu OHo(3.1) with no decoupling

t...- . ---~-'-~-..--

~. ,~ J.. J.' ).' ,., J, ~.

:>....00

e •IH 500 MHz simulated spectra of Leu CPHA• Leu OH. Leu cPH

Bwith no decoupling

IH 500 MHz observed spectra of Leu cPHA• Leu OH and Leu cPHB (3.1) with no decoupling

J Lil, l , .-- -------- ---,

Lt 1.1 1.1 1.5 1.5 1.4 1·]

»....\0

e e

IH 500 MHz selected area of 2D NOE spectra of NUCbz-c[(D)A,Bu-G1y-Phe-Leu].,(3.1) (correlations with Phe NH and G1y NH).

~

ef

fi (PP.'

1 ,•

I~~I , a

I~~.'dlh

9

9.18-

9.15

9 2S; ·~""'ï-"-"----'--'''''''''''''''-'-''-----'-r-.--T-• '1 1 1 1 ~ ~ J 2 1

8.95

....

..(pP·

CV."S Ln D!'tSO-dSHI_dat.... III 111. HIPULSC SEOUEI'ICE J1D••1IOISERIK HI

fJ1EOUEI'ICY 499.IU Nt::t:Sf'[CTRAL UJDTH 526]. 9 H~

ZD SPfCTRAL UIDT" 5263.9 HzIlCOUrSlfJOf'I TJM: '.195 ••cli!ElAXAUort J)[LAY J .818 ne"1'IUC TII'I[ '.588 ••C'.

PULSE UIIlTH 8 •• lIalKTEl"ft:IfATURf 24•• da;_ C.HO. ~PETIfI0f'I5 :re1'tO. II'tCREMIUS Z5& lia

IMY....LE PRtCISIOf'I IlCOUISlfIOrtDATA PfroCESSI/"tCCAUSSIM Af'ODIZATIOf'I '.898 ••t.

H SIlE 28••F'1 DATA PROCESSll'IC:

CAUSSIAtt flf'QDI2ATlOIt '.822 UI;.FT SIlE 1~4

TOllll ACOUJ51TIotI TInE 21.2 houu

~.

a PheNH LeuNH

b PheNH PheCUH

c PheNH PhecllHB

d PheNH PheAr

e PheNH G1yCUHA

f G1yNH AzBuCUH

g GlyNH G1yCUH B

e e

~...•,,•s

FI l~f.1

•1•,11

"Cbz-c[(D)A2Bu-G1y-Phe-Leu]

Bu NBH).

1 " . ~II " 1 li, Al ,1

·,

•

s

g h, Ç) U0 f ~ 1 ~ 1

, 1

1

.' Cj(JO ~ d, ~je, , ~ 1 ·C

IC 0 [Ç! 1

lb•

1

r' Jo,, 1 1 ~""""""---'-r-"""""""""",-·,---'-·-"·T·

,.

,.

••

••

..

..

..

..

.."(pP'

a Leu'NH ~BuNBH

b LeuNH PhecaHc LeuNH PheNHd ~BuNH A2BucaHe ~BuNH A2BudlH2

f ~BuNBH A2BuOHAg ~BuNBH ~BuOHB

not seen at this levelLeu NH Phe dlHBLeu NH Phe caH

h ~BuNBH A2BudlH2

~BuNHci. ~Bu NBH1ren•

(exchange)j ~BuNH ~BuOHA

IH 500 MHz selected area of 2D NOE spectra of N'

(3.1) (correlations with Leu NH, A2Bu NH and A2

e e

~

Al .1

~i. , f

g~ "'()1,\XJ~ C~~'D'Il" 0

, .", .~~. Ci Cl 0 ~,.. tl' 0 cr ''o., ofF 0000 /) 0 a~~ ~

he

1 1 1 1 1 1 1 l '-'-~-"'-~--"---T"'-·-·-'--T .-.. -.... ......,.~.-J 2 1 9

..(PP·

J.8

J .•

1

4.1; Ih4.1-1

1:"'1'.J

•••">-

l ..,

• ,•••8 7

fi Ipp.)

10 •

Leu OHLeu Mea

A

Leu MeaB

GlyNH

PheNHPhecllHA

PheAr

LeuC"HLeuC"H

LeuC"H

~BuC"H

PheC"HPheC"H

PheC"H

e

ab

d

ef

g

h GlyC"HA Phe NH

i GlyC"HA GlyC"HB

not seen at this level

~BuÜ<H AJBu NH

~BuÜ<H ~Bu cIIHz~BuÜ<H AJBu OHA

~BuÜ<H AzBu OHB

IH 500 MHz selected area of 2D NOE speetra of NttCbz-e(D)AJBu-Gly-Phe-Leu]

(3.1) (correlations with Leu C"H, AJBu C"H and Phe C"H, and Gly C"HA).

_1 L L, .,lll.1

e •

~

~

lm

•

~.

III1 .Il li. 1 n Al ",..

g'1

kil'

~n

~J

•

i ~

f~

AzBuN<lHA2BuN6H

AzBucPH2G1yNH

G1yC"HAS =PheAr

AzBuOHA

AzBuOHA

AzBuOHA

G1yC"HsG1yC"HsPhecPHA

b

cd

ef

IH 500 MHz selecled area of 2D NOE speclra of N<lCbz-c[(D)A2Bu-G1y-Phe-Leu)

(3.1) (correlations wilh A2Bu OHA• G1y OXHs •waler, Phe dlHA• Phe CflHA• and

AzBuOHs ).a

g PhecPHA PheC"H

h PhecPHs PheNH

i PhecPHs PheAr

j AzBuOHs AzBuN6H

k AzBu OHs AzBu C"H

,,,,~~~~~'~h~.'_"/~.~~~~~-~~

el lUI

1 AzBu OHs AzBu OHA

m AzBu OHs AzBucPH2<n PhecPHs Phe C"H

COSY

nol seen allhis level AzBu OHA A2Bu C"H

J.S

).6,a~ lb

IJ, r

i 1 , ~'l"'" .." • • 1 , S • J • 1 •

FI <fP.1 ~....

e e

IH 500 MHz selected area of 2D NOE spectra of NlICbz-c[(D)A2Bu-G1y-Phe-Leu]

(3.1) (correlations with A2Bu (jlHA• A2Bu (1IHo• Leu dl HA' Leu OH. Leu dl Ho

...OH. "'d'H..... MUh ...... Mu,,). 1 ~l~",==

~1 --'.6

a ~Bu (jlH2 A2Bu OHAb Leu (jlHA Leu CPHoc Leu (jlHA Leu Me~A

d Leu cPHA Leu Me~o

e Leu OH Leu CŒHf Leu OH Leu Me~A

g Leu OH Leu Me~o

h Leu cPHo Leu Me~A

not seen at this level

.~~~~o

~BucPH2

~BucPH2

~BucPH2

~Bl1CŒH

A2BuOHo~BuNlIH

-.-5~I

t42€~"

le

oa

~

~

,1

~;

~b ~~1

~

A25

The rule of thumb to predict the direction of cleavage of thioethers

"cleavage occurs to produce the least basic anion of the pair having the greatest

difference in basicities" W. E. Truce, D. P. Tate and D. N. Burde, 1. Am. Chem.Soc., 1960, 82, 2872.

Alkyl or aryl substituted thioether RSR' can be cleaved according to Iwo differentpathwayi: a 2 e- process or a 1 e- process. Thus when the R' carbanion is

thermodynamically more stable than R carbanion we have

or

RSR' ----- > RS- + R'-

RSR' -----> R'S- + R· ----- > R'S- + R-

(1)

(2)

Since the same reagents are involved in eq. (1) and eq. (2), the reaction that will

be favored is related to the relative thermodynamic stability of the respective

products. On the other hand, since the stability of anions is related to the values

of theirs respective pKa's we can write

AGi = pK,. RSH+ pK,. R'H + K

AG2 =pK,. R'SH + pK,. RH + K

(3)

(4)

where AGi and AG2 are the respective free energies of equations 1 and

2.and K is a constant that take into account the reagents contribution. NeglectingK, the value of AG is positive since this process does not occur spontaneously in

absence of strongly reducing species, therefore if AGi > AG2 reaction (2) is

favored. More explicitly

if pK,. RSH + pK,. R'H > pK,. R'SH + pK,. RH

reaction (2) is favored. Rearrangingif pK,. RSH - pK,. R'SH > pK,. R'H - pK,. RH

reaction (2) is favored

(5)

(6)

CQFD

1. The possible reaetion RSR' -----> RS' + R'- ----> RS- + R'- was notconsidered because the first cleavage involved the formation of a radical sulfurand a carbanion whereas the formation of a thiolate and a carbo radical isdefinitely easier.

A26

Observed and calculated structure factors of threo-N-acetyl-S-p-methyl-benzyl·~·COlUMIi are lOFe lOFe lOOOOSlg. for InllgnH1canf. phenylcysteine methyl ester"0 " S" >Fo Fo SI, , >Fo Fo S"

, >Fo Fo S"

-', 0, 1 -', " , 2 12J 127 ISla ," 60 6683 , 61 :li 6750 , ". 213 2918

2" '" l'59 1 " JS 7lU 5 " 65 5051 6 '" 179 3238 -6. 16. 1 6 " 69 4112

" " 7909 6 lU 135 4no 6 60 6C 6038 ,'" 156 3639 1 " 100 1883 ,

" 85 4264

-', 1,1 -', " 1, " '7 7215 • 121 126 4157 , 142 143 3656 • 106 98 4219

42 J3 1671 1 '0' 98 422. -, ," 1

-6, " 1 • '0' 19 4219 • '" 134 4253

63 55 5892 ," 42 !!i.e!! 1 " " 7038 1 163 169 2689 6 .. 62 6221 -" " 1

'" 128 4068 • 6O 59 5616 2 63 " 50J8 ," 66 4197 -6, 17, 1 1 " 39 5281

-', " 1,

'" 116 40292 , " 75 UO] , " nUai 1 " 40 6562 , 11J 112 2795

111 106 U29 6 42 JO a410 • " 77 462' • " 79 U09 , .. 81 4637 , 176 180 2670

'" 128 41011 -e, 10, 1 , 63 54 554:6 ," 4 7168 , 61 56 5692 , '0 l!! 5596.. 81 5212 " 81 5091 , 110 114 4724 6 211 211 3215 • '" 154 3795 6 U n 70se

-', " 1 '0 50 7360 -,. O. 1 -6. '. 1 6 .. 61 629. ," 79 4aOJ, '" 129 3998 112 109 3991 1 6O " SUC , '" 122 2886 -6, 19. l • " 45 6325

• llJ 114 U91 " 27 7548 , '" lU 3305 , 1JS 133 3210 1 " 73 4892 • " 56 7928

-o. •• 1 " 56 6220 , 116 117 3726 , IJJ 137 3!l41 , U !l8 81'5 -'. ., 1

2 ",. 6911 -l, 11, 1 • '0 78 !lU8 6 "0 168 33!l3 , '06 101 66416 63 66 U2!l, " 59 5915 • " te 6652 , " 62 5200 ,

" 29 6553 -6, 19, 1 '" 157 27U

• 110 1104713 , 6. 7060U ,'" 113 41382 • '0 !l8 5391 .. 88 41877 , " 66 3792

-o. 6. 1 -8, 12, 1 -7, ID, 1 -6. 6. 1 " 82 5280 • m 220 2765

'" 168 3668 , 121 Il' U96 2JO 231 3181 1 '" 276 2592 -6, 21, 1 , U 54 6679

" 67 5910 • " 71 7!l56 .. 92 410412 ," 80 60!l0 .. 97 5531 6 111 108 31411

-0, '. 1 -8, 13, 1 " 28 8050 • " 82 4558 " 56 7967 ," 47 7053

" U 64138 1 '0' 10fi 41239 -7, 11. 1 , ". 293 2917 -6, 22. 1 • " 71 641151 " 57 6519 , 62 60 7512 , 128 129 3506 6 ISO 153 3350 ,

" 54 68251 • " 69 7666

• 8J 82 5401 • 6O 78 7013 , " 60 533a -6. '. 1 -'. o. 1 -s, 10, 1-o. '. 1 -s, a, 1 ,

" 56 5571 1 '0 fi4 5559 • .. 90 3362 '" 231 25801 " 62 6992 1 6O U 6378 • " 41 6695 , 6. 7l U87 6 '0' 210 2894 '" 381 2573,

" 65 521!l ," 65 5954 , .0 73 C92fi , "0 165 30CrJ -'. 1. 1 "0 ua 3256

• 65 52 5717 -8, 15, 1 ," !l9 7686 • '0. 208 01917 , 103 1041 281a .. 66 6577

-o. o. 1 1 " " 5191 -7. 12, 1 , 10J 182 3177 ,'" 295 2371 '0. 1041 41078, 63 49 5578 , " " 7086 " " 5686 6 80 7J 4871 • '" 111 2691 -s, 11, 1

-!il, 10, 1 -8, 16, 1 1J2 1J6 387a , 10. !il1 4042 , '06 108 3496 1 61 63 41248

" 68 6268 " 66 7136 -7, 13. 1 -6. '. 1 6 " 36 60410 , 6O 55 4U3

-'. o. , " 70 6118 ,'" 118 3!i167 , 00 9B 3!i11!i1 , ., B5 38Ba ,

" 62 5UO, 182 1114 3287 -a, 17, 1 , " 64 5425 • 106 108 3aoa • 112 lU 3987 • 63 60 "a236 .0 U 60'3 " 36 7399 ,

'" 118 3U5 , 11 7J 4!i151 0 .. 70 5882 6 JO 15 7373

-'. 1. 1 -,. o. 1 • " 41 7220 6 42 51 8037 -'. '. 1 • '" 125 601811 " .. 5809 ,

'" 2!i13 2788 , 128 126 41052 • m U3 4339 1 '" 3U 2206 -s. 12, 1, m ". 3159 • '0' 91 4634 -7, H. 1 -6. '. 1, 142 UO 2558 , 1JJ IJJ 3071, 8J .. 4613 -,. '. 1

, " 66 !l!il12 1 128 132 3110 , ,go 28a 2363 , '0. ". 26417

• 11 77 5151 1 '" 2Ci6 2737 , '0 416 7123 , 1Sl 150 2988 • '" 266 252' , '00 107 3569," 59 6373 , 211 214 2955 6 " 57 5819 , ". 279 2805 ,

" 25 7081 • " 71 51116 " f,2 6109 , .. lOf, 38J5 -7, 15, 1 • '" 137 3U8 6 42 25 5741 ,

" 60 6074," 47 8351 • .. 88 3189 1 100 116 3606 ,

" 45 !l7S7 , 111 109 3713 6 ". 122 3798

-'. '. , , ., 99 41215 , "6 lU 3752 , 163 161 J151 • " ., 6472 ,'0' 103 f,550

1 " 74 6127 6 " 52 6222 ," CS 7166 • " 341 6536 -,. '. 1 • 8J 78 5558, .. 36 6196 , ., 91 f,521 -7, 16. 1 -6, 10. 1 1 " 43 4668 -S, 13, 1, 185 184 3372 -,. '. 1 .. 8J 4"3 , 1JI 139 3257 , JO 24 5577 , ..0 201 2903

• " 45 7U4 1 '" 197 21166 -7. 17, 1 , .. 65 U60 ,. 1J1 132 2771 ," 99 3847, " 68 5UO , 116 112 339J " " 5732 • " 71 U73 • 1JJ 135 2985 • .. 91 3981

• " 57 7216 , 210 220 J002 63 " 5511 ,"0 128 3642 , m 213 2731 ,

'" 171 3460, lU 1'0 4161 • " 76 f,12f, -7, 18, 1 6 " 57 6764 • ., 94 f,572 , '0. lOf, U71

-', '. 1 • 10. 11!i1 U02 , 120 111 f,005 • " 67 6123 0 ., 91 4743 -5. U, 11 " 70 4506 ,

'" 132 un -7, 19. 1 -6. lI, 1 ". '. 1 1 8J 79 39712 " 52 S09f, .,.

'. 1, 59 57 6255 1 63 78 4921 1 m 126 2608 • " 83 f,845,

" 81 5034 1 '" 239 2119 , .. 53 8262 ," 97 3656 , m 379 2259 • " 65 843f,

• "0 174 3489 ,'" lU H5f, '6. O. 1 , 250 2'3 2934 , 180 186 2513 -5, 15, 1, 59 52 6114 ," 53 6211 • m 261 2749 6 " 62 6155 • '" 212 269& ,

" 57 48106 " 78 5438 • " 89 UO!iI • ". 158 3643 ,

'" UO 3982 6 '" 163 JI7) ,'" 126 3617-.. '. 1 6 ". 138 3599 -6. 1. 1 -6. 12, 1 • 6O 565U2 , 1SO 142 3271, 16O lU 3579 , .0 95 5690 1 " 59 4567 1 11S 118 3404 0 " 38 7119 • .. 89 f,116,

" ., 4216 -,. '. 1, '" 169 2815 , 106 183 30S9 -'. 5. 1 ,

" 52 8244

• lOI 158 3542 1 .. 84 U39 • '" 180 2n6 , 121 123 3651 1 '42 157 2585 -5, 16. 1

• '" 150 3912 , '0. 306 2119 5 a. 152 3242 • '" 110 3713 2 " 68 3499 1 ,.. 209 3106

-'. '. 1, 10. 95 3655 • .. 86 3670 6 '0. 108 41039 , l8J 183 256' , '0• 214 3127

1 " '0 U66 • " 644916 , U1 130 3521 ," 78 5516 6 ,. 53 5029 3 " 57 7H9, U " 6727 5 .. 97 4255 • " 74 5359 -6, 13. 1 , m 125 3807 • 81 88 4552

• " " 5161 • 130 137 U75 0 " 645546 , ., 98 3923 • 42 37 7796 6 111 113 4098

-'. 6. 1 .,.'. 1 -6. '. 1 • '0 38 7164 • .. .3 5615 ,

" 59 61251 '" '" ]274 1 m 212 21U 1 " 67 f,026 6 " 13 6567 -,. 6. 1 -s, 17. 1, .. " U69 , m 235 3067 , 121 127 2995 -6, 14. 1 1 100 117 3024 1 " 70 5103,

'" 125 3711 • '108 109 3156 , 63 70· 4"2 , 116 116 3619 ,'" 151 2563 , 100 104 403]

5 .. 17 4&05 , 8J 73 f,676 • '" 146 31.. • .. 52 fi594 ,'0' 1"9 3032 ,

" 69 63856 " 50 6559 • 81 91 5793 ,

" 74 4042 , lU 138 3689 ,'" 120 33" • ... 174 3576

-'. '. 1 -, , 6. 1 6 111 121 3609 • " 35 7427 , .. 92 4230 , '0 80 565111 " 94 '228 , ISO 161 317. 0 110 91 4519 , 11 65 5747 -'. '. 1

, U 41 852f,,'0' 107 U50 ,

'" 112 3666 -6. '. 1 -6, 15. 1 , .. 77 3268 -s. 18. 1," 62 5594 6 63 66 6995 1 210 215 2671 , 211 207 3U6 2 63 6' 3908 1 JO 32 75U

• " 'f, 6415 .,.'. 1

, '0. 215 2681 ," 56 5651 , .0 102 3285 ,

" 65 6239, 162 U1317' ,.. 199 J06. • '0' 306 2736 • " 50 6654 • JOO 373 2548 , 00 87 4262

ColUlllnll ",1E! 101"0 lOFe 10000519•• for Ir..19~lUcant.

1 kFo Fo SI. 1 kFo Fo ". 1 kFo Fo S'. 1 kFo Fo S" 1 kFo Fo SI. 1 kFo Fo Sl.-5, 18, 1 , 'DO 199 2269 J lJ1 129 Hli5 ,

'" 2DJ 2e9_ • 110 122 ]J5' , .. " 588fi, .. Go( !l584 J 72 54 3529 • 116 123 lH9 • •• " UO" , 16' 163 JUfI • '" '" 3102• .. 56 6546 • 35' 355 naD ," 61 5352 • 6J " 6026 , .. .0 4968 • ., U 599•

-5. 19, 1 , 116 140 2921 -ol, 19, 1 10 1O. " 470' • 87 112 5697 -2, 1, •1J4 132 J6U • m 130 3301 ". '" J043 -J,

" 1-3, 15, 1 1 m '" 1683

" 48 6691 • .. 67 5719 '0 ., 533. 1 .., '" 2122 1 J26 326 2461 , 070 .., 1511-5, 20, 1 • 7J 63 5576 " 71 '828 ,

'0' 'OJ 2016 , 116 317 25.6 J 5JO 5<18 1701," 66 '9'0 10 '0' lOf, 4975 -t, 20, 1 J '0' '0. 2157 J " n "060 • m 110 :nlJ

J .. J9 6803 -'. " 1 151 '" 3594 • ". '50 2252 , .. 29 6051 , m lJt :Zl5.t-5, 21, 1 1 31J Jll 2148 '0. '" 3233 , m '" 2.09 • '" 124 372" • " 56 J!109

l2D 112 40.0 , 127 lJJ 2650 .0 3 6963 • '" H7 4!906 ,'" 230 JJl!i6 , 51 "9 5052

116 10. '605 J '" '" 2606 • 11. '0' 3918 ," 50 5577 -J. 16, 1 • '00 92 3889

-5, 22, 1 • '" ". 2565 -4, 21. 1 • 41 62 8098 1 lJ8 '" JOU • " 50 6111], 71 '0 5837 • " 11 7042 • 120 11J 4081 • 110 126 406. , 7J " "'052 -', J, 1J " .. 5381 , 111 11J 3691 -4, 22, 1 -J,

" 1 J .0 56 US1 , Jl4 J11 1612-s, 23, 1 • 101 10J US4 10. '0' 3952 1 ". m 1932 • '" '" 3241 J 210 '0' lU8• " ,. 5605 • 91 '0' 5549 141 lU 3896 , 10. 86 2362 ,

" 11 5787 • .. 52 3631-', o. 1 -. , " 1., .. 6918 J 122 116 2479 • 110 136 3821 ,

" 36 5080, J06 JO, 2020 1 J75 378 2212 -4, 23, 1 • ". 147 2531 • .. 60 6886 • .., 153 2724• '" '" 2205 ,'0' 101 2880 " 96 4359 , JO 43 7017 -3, 17, 1 1 119 120 33lJ• " 61 5057 J .. 96 3497 '0' " 4332 • 121 124 3328 1 .. 79 4557 • 8l 17 4172• '" J68 3001 • m 266 2561 -4, 24, 1 , 171 166 3149 , 16 23 6971 • '0 71 n9&-', 1. 1 • '0' 107 3787 " " 7236 • 100 84 4070 • 91 93 4228 -', " 11 '" 279 1957 , :'14 115 3824 -4, 25, 1 10 '0 75 6529 • " U 6468 1 m .., lUS

J ... 452 2080 -4, ID, 1 1 " " 6679 -J, ., 1 -3, 18, l ,'" 181 1626• " 46 4715 l lJ5 131 2690 , 101 '0' 4508 • 158 155 2247 • '" 118 3391 J '" '" 17111• " 79 3928 , 8l 77 3361 J .. 41 8327 , 14 26 5385 ,

'" 259 3121 • m ... 1958• " 60 5342 J 111 321 2413 -4, :!6, 1 J JO 23 5088 ," 75 4792 • 116 326 2U2• " 90 4595 • 101 101 4121 " 57 8340 • ,. 86 3UO -3, 19, 1 • 87 79 4092

10 " 566n7 , 14J 150 3670 -J, 0, 1 ,'" 229 2739 1 128 133 3359 10 128 125 4081-', '. l • .. 38 7254 , 51J 50& 1722 1 '" lU 3U2 l "0 134 3323 -', '. 11 '" 236 2008 • 86 68 5304 • .., 462 20&1 • '" 234 3105 • " 41 7011 , .. .. 2963, .. 47 4051 -4, ll, 1 • .. 35 5û46 10 .. 71 5102 , m 1611 J456 J ... ." 1195

J ,U 556 2088 1 .. 69 3U9 • ,.. 230 300& -J." 1

, ., 57 6032 ,'" '" 2212• 24O 2n 2358 , ,.. 173 25&5 .0 " 82 55fS , ." ... 2060 -3, 20, 1 • " " n93, .. 87 3352 J 116 3102489 -J, l, 1 J '" 148 2450 , l1J 177 3128 1 .. '0' 369&, 9J 96 3676 • "0 265 2630 1 .., ,.. IBM • llJ 111 2889 J 56 51 6035 • .. .. 5567,

" 103 3821 , 14l 134 3295 , J" m 1757 , 61 ., 4809 • m 266 3224 '0 ., 7J 6&:lE'>• " 66 4766 , 10J 109 4304 J '" 253 1963 • " " 6013 , ., 70 6149 -', " 110 .. 50 51151 • .. 72 4924 • '0. 690 2023 ," '0 7477 -3, 21, 1 • no no 1619-', J, 1 • '01 98 5124 ,

" 23 4122 • " '0 6960 , 56 711 6511 , ... 518 11081 .. 79 3030 -4, 12, 1 • '" 237 25711 • " 87 5175 J 10. 102 41a4 • '" 291 2089,

16' 154 2204 • 16 29 5810 ," 54 5136 10 18 .. 15670 • '" 148 4071 , 11. 1192752

J 18' 191 2340 , ... 1152 2738 • m 221 3048 -3, ID, 1 5 " 30 8111 , 161 161 3150• 9O 94 3045 J .00 91 3470 10 " 52 66915 1 11 .0 54515 • .. 89 4707 • " 40 6910, ". 310 2508 • 110 127 3328 -J," 1

,'" m 2175 -l, 22, 1 '0 .. n 5215• .. 86 3739 • '" 160 3673 1 181 184 1814 J m 110 2713 , 128 129 1779 -', " ,1 '0' 113 3605 • 11 77 6946 , 121 116 21311 • " 101 3361 J '0 151 5117 l m '" 1712• " 35 6264 -4, 13, 1 J .. 64 3083 , m '" 2619 • 111 120 4345 , .,

" 2657• 151 158 39U , ,.. 18J 2762 • m 265 21152 , .. .0 5322 • " 103 4964 J '0. "J 190410 " 33 6635 • ". m 2795 , 410 Ul 2Z55 • 56 .. 70!il5 -3, 23, 1 • '" '" 2248-', " 1

, 19J 198 31411 • '" 158 2771 • 116 128 4091 1 170 112 3433 , 31J JI3 23001 '" 153 2216 • .. 51 6493 , 160 155 3070 -3, 11, 1 , .0 51 6214 • ., 62 4210, .,. 620 2009 ,

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" 17 1217 -', ., 1

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'" ". 3114 -3, 24, 1 , .00 m 1124," 82 3814 J m 326 2693 1 '" '" 1720 , 87 ., 4581 " 705U7 • 161 ,.. 2415• 51 4B 6407 • .01 102 4115 , 115 111 1115 ,

'" 19. 3301 ., 49 157115 • JO, JO, 2510'0 " 4B 7210 -4, 15, 1 • m 178 2332 • 112 112 4543 -3, 25, 1 , .. 61 5581-', " 1

1 176 179 2955 , 125 123 2871 -3, 12, 1 " 6J 7252 • " 54 79191 ,.. 287 2062 , 182 166 2917 ,

'" 145 2956 , .J 101 30U .. ., 58152 10 116 1104.14,'" 412 2073 J 8J 92 4n9 • '0' 101 1933 J "

., &909 -3, 215, 1 -'. " 1J " 49 402~,

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" 29 5540 -4, 16, 1 -J. ., l • '" 243 2908 1 " 56 6831 J '0' 202 2175• "0 136 3081 l ,. 75 4325 l ., 63 31415 • '87 194 3411 , 6J ~3 6384 • 176 18l 2U91 .. 56 4942 , ... 192 3027 ,

" 70 2734 9 .. 64 66';:0 -" 0, 1 , 118 140 2932• " 45 7290 J u. 123 3626 3 16O 331 1971 -3, 13, 1 , 881 '" 1411 • .0 14 6761• .. 565441 • " 55 4119 • JJl 335 2133 1 m 377 2281 • 101 '03 2593 , 141 141 333l10 " 80 5616- • .. 46 7052 , .. " 3292 , .. 59 3937 • 12' 106 2807 • .. 47 6421-', •• l ·. " 70 5589 • JJ6 3J5 2510 J 35 13 51194 • 24O 250 2940 .0 '" 161 1945, m 122 2519 -4, 17, 1 , lU .35 32154 , .. 4B 6760 10 '01 9. 4411 -2, 10, 1

J 14O 340 2249 1 m 271 21119 -J," 1 • " 35 7426 -', l, 1 1 ". .., 2061• m 177 2513 , ,., 163 31156 , 570 '" In6 , m 230 3203 1 1375 un 47154 , 190 174 2152, lU 231 2984 , m 148 3541 , m ,.. 1928 • 51 42 7417 , 1022 1035 1417 • U U 6179• ., 68 5072 • " 65 1113 J m '" 1981 -l, 14, 1 J .,. 680 1682 , JU 315 2.'....• 161 156 3682 -4, 18, 1 • '" .65 2404 m 112 2635 • ... 1592 1815 • " 71 ",~67-', l, l • 121 126 3557 ,

"., 3538 ... 110 3017 , m 250 2220 • 122 IV. 4159

10. 115 2685 , 100 104 3902 • 19. 191 2797 U, '" 2863 • ., 9J 3064 -2, 11, 1

A27

• ColulIIlll .,. lOfo lOfe 10000519, for In.lgnlt1cant

"0 " sl, 1 "0 " SI, l "0 " Sl, 1 "0 " SI, 1 "0 " SI, 1 "0 Fe SI,

*2, 11. 1 , 92 97 U72 , '" 4)1 1597 • " )5 7633 , '" 932 Jl5J , m 127 4129

1 '" 267 203" • 100 105 usa , ". 591 1741 -1. 16, 1 ," sa 2365 10 .. " 4982, 16' U2 2]11 , " 31 7151 • 120 117 l,ua 1 lB' 181 2500 • 131 135 1505 0, ID, 1, 100 100 2813 7 " 29 7901 • " J9 S052 , 116 120 3004 , ", 378 1441 0 '09 '" 1759

• m 3U 2143 -2, 23, 1 7 lOB 110 ]713 , '" 126 lU9 • '" 218 1680 1 ", '" 1764, " 108 ]SlI9 • '" UO 3852 • " 49 6121 • .. 597426 7 .. U J821 , 129 lJ5 :2188,'" 223 2877 -2. 24. 1 , " 407 7126 , 76 BD 6192 • 107 113 2802 , ". 2]4 2129

7 .. 6 6506 l5l 160 ]720 10 102 101 U'Q -l, 17, 1 , '" 242 2217 , '09 liB 2390, " 76 50]9 " 5] 111197 -l, 7, 1 1 lJJ 120 2850 10 "0 1402143 ,'" 191 2782

-2, 12. 1 " H 61" 1 ." 412 1555 • m 251 2199 0," 1

7 " 52 6499

1 III 124 2493 -2, 25. 1 , '69 165 2030 , 191 185 J021 1 1242 13n 26U • " 46 8392, '" 236 2290 10' '" 4237 • 270 278 2091 , '" 109 4074 , 281 ". '" , 210 213 3557, 71 11 3792 118 117 UOI 5 .., 466 2192 7 1!J 113 4310 , '" 261 1163 0, 11, 1

• 120 115 2983 70 71 6]5] , m fl23 2407 , 92 96 5467 , 177 188 1415 1 '" <al 190e

5 lJO 140 32811 ·2, 26, 1 7 '" wi 3305 -l, 18, 1 , :l::lR '05 1550 , "0 lU 2371, m 224 2896 " 47 8101 • 10. 97 4094 , 88 95 4099 , lJJ '" 1938 , '" na 2138

10 101 89 41119 .. 46 8317 10 100 94 4880 • .. 105 4164 7 205 204 1943 , U' ,U 2763

-2, 13, 1 -2, 27, 1 -l, " 1, " 52 5791 , 65 74 4140 , 150 l5l 2967, "0 142 2552 llJ 109 4419 1 185 187 1813 ,

" 64 6265 10 109 106 3033 • " " 7801

• '" 296 2477 -2, 211, 1 , 102 106 22115 -1, 19, 1 U 104 88 33J4 • " JO 7421, m 214 2752 , 50 54 7944 ,'" 2115 1936 , " 88 3617 0,

" 10, 12, 1

7 " 55 8372 -', 0, , , '" 173 2331 , '" 219 2791 0 '" '" ,., 0 " 15 45119,'" 122 4413 , ". 306 1362 ,

'" 22S 23113 , m 138 34117 1 768 1107 2929 1 '" 239 2304

-2, 14, 1 • '" 260 1906 ,'" 123 3259 , " 92 4412 , 76. '" .., , lJ7 135 2408

1 '09 286 2272 , .. 27 47511 , 101 lU 4492 -l, 20, 1 , ... 497 U43 , m 128 2675, lJ7 136 2812 • 16' 172 31)3 -l," 1

1 " 52 5514 , 165 159 1478 , '" 243 2433,'" 237 282!! 10 '" 145 3636 1 " 44 3111 , " 77 4785 , '" 457 1461 , " 52 4342

7 180 181 3504 -', l, 1 , .., 447 1810 , m 234 3063 , '" 242 1685 , " 106 4126

• Ul 124 3922 , J6' J6' 1085 , 20' 215 2130 • ". 146 4170 7 m 163 2032 • 155 157 3440," 57 5613 ,

'" ". 13H , J07 304 2172 -1, 21, 1 • )JO 331 1993 , 117 139 4376

-2, 15, 1 , 157 U, 1856 , ,.. 269 2396 1 15' 150 3186 , 187 195 2337 10 .. 30 78n, lU 108 3066 , m 561 1791 , 156 149 3013 , " 98 3975 10 " 26 6188 0, 13, 1, 101 106 3266 ,'" 220 2150 7 '" 128 3522 , lJ5 137 3514 0, " 1 1 67 77 4458, .. 105 J753 , 192 203 2504 • .. III 6239 , 82 76 4951 1 6J8 650 89. , lJ5 133 2654,

" 46 6230 7 80 77 3889 , 195 198 3531 , 101 103 4499 , 1245 1279 3609 , '" 268 2284, 75 81 4822 • '82 186 30n 10 105 109 4796 7 69 65 6262 , 191 189 1297 • '" 2S3 2471

7 '" 186 3439 , .. 97 4405 -1, ID, 1 -l, 22, 1 ,'" US 1904 , lJJ 132 3133,

" 60 6978 U " 35 8801 , 'u 119 2458 2 117 118 3561 7 20' 209 1978 , 182 183 3175

-2, 16, 1 -l, " 1,

'" 147 2398 , 102 911 4237 • 16' 161 2233 7 " 86 4018, l7l 171 2743 , 56. '" 1107 • lJJ 122 2709 70 69 5717 , lJJ 133 2547 , 82 80 5252

• " 71 4479 2 - 7n .02 1324 ," 45 7094 -l, 23, 1 10 66 66 4079 0, 14, 1, 16 78 U03 , m ,n 1561 7 5J 54 5914 1 79 70 4340 0,

" 10 1Jl 120 2398

7 " 57 6033 • '09 304 11169 • 85 83 4408 , .. 63 6555 0 101 100 1310 1 J07 315 2192, lCl 103 5276 , 20' 199 2212 , .. 42 8464 , !JO 132 3849 ,'" 984 JU4 , '" 323 22::5

-2, 17, 1 7 12' 131 3165 -1, 11, 1 , 104 102 4742 ,'" 243 1162 , 65 64 4217

1 121 116 2932 • .. JI 6427 1 76 72 2772 -l, 24, 1 , .., 454 12!3 , 69 58 4442, .. 7J 4544 , 112 117 407' , 76 78 30.5 1 51 414 618. • " 21 '537 , 160 159 3060

• m 121 3299 10 llJ 118 41499 , l5J 155 2461 ," 4S 6155 , 89 86 2207 , 50 52 7110,

'" 18. 3297 -l," 1

, 203 204 2US , lOB 92 4087 • J6 36 6000 7 '" 131 3522, 62 6. 5889 1 .., 702 117O ," ,7 5611 ,

" 73 5622 , 62 70 3865 • .. 52 6198

-2, 18, 1 , '" '" 1467 , m 219 2845 -l, 25, 1 10 .. 60 .525 0, 15, 1, 60 54 45.0 , 190 '89 1785 7 " 51 7297 1 80 8. 5075 0, 7, 1 '98 '" 2630," 25 6.58 , '07 204 1996 , lJ7 132 4021 , 62 50 6122 1 JO< 207 1168 2 '" 15' 3123

• 101 101 3692 ,'" 485 2059 10 " 47 8219 -1, 26, 1 2 " 32 2833 , 188 188 2660, m 125 H91 , .. 89 3331 -1, 12, 1 ,

" J1 8108 3 '" 13) 1500 , m 114 2809, .. 50 7676 7 " !8 6181 1 20' 205 2103 , 173 168 3635 • 29 33 )647 , l7J 178 30137 " 41 7869 • '" 249 2941 , 305 301 2094 -l, 27, 1 , " 43 3229 , J19 320 2895

• 90 9. 5241 ," 98 .52Cl , 26J 260 2405 1 65 68 6110 , 386 391 1706 7 ," 140 3537

-2, 19, 1 U " 91 5161,0 , ,., 168 3199 2 115 123 '572 7 .., 478 1841 0, 16, 11 98 " 3516 -l,

" 1 • " 80 4321 -l, 28, 1 • U' 117 2519 0 '" 188 2431, .. " 5681 1 1402 1459 4429 -l, 13, 1 2 " 46 8778 , lU 105 2749 , '" ua 2821J 11l 10' 3617 , 81 90 2497 1 '" 290 2127 0, 0, J 10 " 96 3376 , 51 .. 4882

• 104 107 "06 , 128 137 2027 ,'" 331 2167 2 6lJ '" ••0 0,

" 1, 155 ,., 3145,

'" 146 3695 ,'" 1251 2301 , 71 65 3U5 , '" 358 127" 0 J70 371 1097 , 122 125 H03

• 90 105 4923 , J68 364 2122 ,'" 182 2702 ,

'" 2li 1663 1 ". 246 1193 , 51 .1 7616

• 62 59 6112 , ". 13~ 2777 , 117 124 3454 • 6J 59 3721 2 '" 23~ 1273 • 116 III 4160

-2, 20. l 7 175 110 2927 , lJ2 140 3369 10 10' 113 2971 ," 21 4834 , " 668UO

1 126 132 3367 • '" 200 3031 • .. 92 4678 0, 1, 1 ,'" 130 1684 0, 17, l,

'" 126 JJ76 10 88 86 5141 -l, 14, 1 1 l2J m '", 161 172 2525 , 65 66 4182,

" as 4600 -l, " , 1 " 110 3341 2 156 161 1066 , '" 361 2487 , 59 66 4952," 70 5262 , '" 232 1616 , lOB 101 2806 , 665 654 1068 7 m 272 2772 , 121 121 3571

• 105 10. 4371 ,'" 251 1791 , m 216 2U7 , lJ7 lU 1490 , .. .3 7329 , .. 67 615.

-2, 21, 1 , 191 190 2321 ," 47 6551 , " 66 2659 10 " 53 7879 , m 212 3280

1 65 56 5111 , 87 79 3274 7 lBO 180 3241 7 155 158 2009 0," 1

7 165 160 3468, 61 63 5566 7 91 119 3759 -l, 15, 1 • m 239 2030 1 '" 150 2093 , " 49 7316, 61 69 6305 • " 55 6321 1 lJ7 '" 2637 10 .. 67 4017 , '02 195 1919 0, 18, 1

• '" 135 35:,) , m 132 3610 J !J' lJl 2853 U .. 86 3482 ,'" 428 1912 0 U' 124 3074, .. 73 .920 10 '" 203 3663 ,

" 71 3922 0," 1

,'" 421 209. 1 66 60 4516

-2, 22, l -l, " , , 157 ln 3301 0 '" '" 5Jl , '" 151 2714 • m lOB 3755

65 " 5131 ." '" 1U3 7 65 60 5378 1 .. .. 1762 7 91 105 4360 , lJ5 126 35451

A28

ColUlMlI lire IOFo lOFe 10000519. for Inlllgn1fle.nt.1 "0 Fe 51. 1 "0 Fe 51. 1 "0 'e SI. "0 Fe Sl. 1 "0 Fe 51. 1 '" Fe 51.

0, 18, 1 • .. e3 SlS~ • " 17 75'• 1, 22, 1 • :105 210 2:00 0 " '0 3211• .. " 7874 7 '" 250 2667 , 1>1 Ut 3931 0 16' m JUG , lU lU 2'HZ , 10' 10. 32687 " 63 7597 • m 260 2928 1. 12, 1 1 .. " U77 • '" 2U 261' , 19) '" 21160, 19, 1 ,

" S9 8123 0 193 181 2I'n , .0 52 5559 7 m 221 2913 • ". ". 1019.. 96 3646 10 " 36 6836 1 '" 297 2036 ," H U09 10 '0 82 5JU • '" ,.. 3237, 167 163 2968 " " 1

,'" 355 20112 , 98 99 4525 " 7, 1 • lJ9 m J909,

" 79 39ô5 0 '" 691 1177 • 120 110 1068 • 7. 77 SBU 0 " 511 24]9 ,'0 '0 Sn]• 163 164 3385 1 190 '" 1452 , 117 119 ]280 l, 23, 1 1 162 167 1919 2, 16, 1,

" 102 "'260 ,'" '" 1495 , ,.. 150 JIn 1 " 62 5U5 , 120 112 2305 0 " 31 61067 " 75 5245 ,'" '" 2050 10 " 75 5590 • " SB 66..5 , 100 n 260] 1 '0' :197 2422

O. 20, 1 • '" 441 189] l, 13. 1 , 98 89 4001 • 89 79 2928 ,'" 210 2697

0 '" 26' 2177 ,'" 281 2157 0 ,,, JO" 2120 1. 24, 1 , 107 98 ]090 ,

" 96 HBl• m '" 3184 • '" tH 2321 1 '" 164 2400 0 '" 158 ]516 , 112 109 J'04 • J1l ]20 2799• " " 5065 • " 85 5276 , 78 70 3276 ,'" nt 4090 7 10. 112 ]1149 , 93 99 U56, 89 87 5255 , 1J6 136 3729 ,

'" 241 23!l9 ," 78 4821 , 69 fi!l 4923 • " ta 7U.9~

0, 21, 1 10 91 91 4!l63 • '" 226 2604 • 61 !l1 6867 ,'" lU 3683 7 .. t3 7988

1 115 119 3921 " " 1 • '" 237 2839 l, 26, 1 " " 1, '0 U!l4!l4,

'" "0 339!l 0 '" 360 1320 ," 60 6048 0 .. ta 7393 0 lBO 179 1161 2, 17, 1• m 191 3!l651 1 '" 706 13!l1 l, 14, 1 " 0, 1 1 '" 149 2013 0 " " 4382, 125 128 3756 ,

'" 267 1788 0 m 224 22U 0 '" 888 4!l79 , 6JB 63!l 1137 1 " " !l3667 77 " 5816 • 220 222 20U 1 ,., 249 2258 , '01 201 1676 ,

" 95 29U , '" 190 28251O. 22. 1 7 190 190 2908 , 126 126 2931 • '" 530 1930 • ,.. 137 253!l ,

" 70 6202m "0 3498 10 10' 101 U21 • '0 90 3707 • '" 1!l6 2620 ,'0' 207 2642 • ,.. 269 2911

100 10' 3877 l," 1

, '01 206 2868 , .. 97 4090 • 1lJ 116 3340 , 7J 76 4188.," 7!l14 0 ... 464 1402 7 169 114 3423 10 m 139 3920 7 '0' 204 2978 • " 23 1473

69 55 5!l91 1 576 !l86 lU7 l, 15. 1 " 1, 1 ," 8!l 4841 , 61 63 !l213

BJ '0 !l3U , .., 558 1580 0 " 91 3296 1 '0' 4171404 " " 1 2, lI, 10, 23, 1 3 690 614 1748 1 lJ2 lU 2676 , .. U 4111 0 10' 10' 2334 0 9J ., 3770

1 ., 46 7750 • " 36 4051 , 117 121 3286 • 167 1!l9 2132 1 ,.. lU 2009 1 '" '" 3207," 86 4995 , 30' 309 2242 ,

'" 15!l 2687 , 179 179 23U , 111 99 2!le7 , 100 10' JII27• 10' 104 !l035 , 301 301 2480 • " 62 4642 , 9J Il!l 3292 3 71 6!l 3240 , lB' lBl 297!l0, 24, 1 7 10' 107 3620 , 126 127 3361 , 187 179 318!l • 157 156 2506 , 71 70 !'I216

0 m m 3204 , 61 77 !l602 • 10. 106 3807 10 ". no 3832 ,'" 251 2513 , m m 3326

3 '" 173 3710 , '0 37 66116 7 " 70 !l891 " " 1, '" 151 3189 7 126 ". 4215

0, 25. 1 10 " 53 8084 ,'0 70 usa 0 '" 272 1407 ,

'" 136 4041 2. 19, 1

"' 102 3916 " 7, 1 1, 16, 1 1 '" !l88 1407 10 91 92 !l1l0 0 '" m 3239

" 62 6458 0 '" 928 1467 0 ". US 2683 , J77 31U 1!l90 2, 10, 1 1 ., .. 3890.. 63 !l984 1 '" 496 1537 1 61 61 4103 3 lBJ 189 1911 0 71 67 2959 ,'" '" 3290

0, 26, 1 , .. 922U3 , m 417 2404 • 175 171 2186 1 .., .., 1929 , '0 " 741910' 97 46!l7 3 369 347 1855 3 127 124 3081 • "7 134 2810 ,

'" '" 2111 • '0 .0 7178.. .. 7964 , 1>1 157 2607 • ,.. 158 3030 7 '" nI 3023 3 " ~3 5102 , 56 !'I7 63280, 27, 1 • 237 2U 2564 ,

'" 122 3678 , .. 54 7103 • J7 9 5683 2, 20, 1," 75 5658 7 10' 96 3668 • ". 151 3557 , B1 77 4!l90 ,

'0' 208 2704 1 139 lU 33563 " 68 760J 10 56 57 5929 7 56 65 5784 10 7J 70 5613 ,

" 61 5429 , 99 110 U390, 28. 1 1,

" 1, .. 54 8165 " 3, 1 7 '" 224 3030 ,

" " 72400 .. 100 4757 0 '" 230 1694 ,

" 56 8875 0 1135 1172 4852 • '" US n01 ," BO 4832

1 " 58 5740 1 " 40 3719 1. 17. 1 1 '" 226 1578 2, U, 1 7 " " 7342

" 0, 1 , 510 !l18 1731 0 100 111 J197 , ... 567 1584 0 '" 4!l9 1955 2, n, 10 1135 1215 J196 3 m 134 2256 1 70 93 4716 ,

3" 346 1103 1 50> 591 1970 , 157 167 J5J4,'" '" 1325 , m 275 2325 , 237 242 2671 • '" US 1969 , m no 2076 1 "0 lU 3Jl1• 130 124 2210 , 30' 306 2522 3 '0 64 5505 ,

'" 141) 2505 , m 303 2595 , 136 13' J442, .. 34 6Jl51 , '0 65 S759 • 55 81 54J8 • '03 204 2592 , ., 95 5266 • " .. 709210 " 45 7768 , 51 47 7106 ,

'" lU 3375 7 100 90 J631 2, 12. 1 , 1lJ Ul 4037

" l, 1 10 BO 80 5374 ," 566644 • " 67 5859 0 " 70 3022 • " 61 7464

0 390 '" '" l," 1

7 '" 117 3974 " " , 1 m 25J 2173 2, 22, 11 '" J73 1107 0 61 47 2892 • " 58 6642 0 ". '" lUO ,

'" 241 2273 0 "0 161 3312,'" ,.. lUI 1 17' 179 1910 1. 18. 1 1 ." '" 1503 3 6J 87 4247 , 167 '" 3310

3 .01 '0' 1610 , 112 98 2355 0 .. 61 6313 ,'" 276 1701 • '" 128 2914 • 41 " 7003• m m 1123 3 '" 269 2028 1 m 282 2615 3 m 207 19311 , 6J 55 5256 ,

" " 69507 UJ '" 3161 • 320 318 21017 3 " 70 4857 • ", 259 2011 , 187 112 306li 2, 23, 1, m m 2918 ,

'" 163 2774 , llB 113 31167 , 70 64 3661 , 56 60 5377 " 01 5366," .. 6086 , 70 75 4592 • 10' 104 4253 , m 220 2612 , 103 100 45211 " .. 7133

l," 1

7 127 126 3U2 7 " 53 7229 7 106 95 J750 2, U, 1 " 67 57170 '" m 1002 , m 222 3U6 l, 19, 1 • m 3411 28119 0 191 203 2303 2, 24, 11 '" 391 1148 10 '0 60 8251 0 'o. 209 2846 " " 1

1 116 117 2744 0 " S9 5675, ." 478 1375 l, la, 1 1 '" 130 3495 0 J79 303 1521 , lBO lia 2474 1 101 103 4571, '" 549 1595 0 01 82 2709 3 '" 270 2913 1 m ". 16113 3 " 53 4191 , •• 90 4252• 'lB 626 1819 1 .., 487 1795 • '0' 193 3035 , 179 1711 1846 • 191 11~ 2709 • " 154 5573, m 330 2089 ,'" 151 2194 • " 33 71560 ,

" 41 un , UO 143 3333 2, 2~, 1• 103 101 3076 • " 54 4709 7 "6 73 5397 • 199 203 21115 7 " 41 7087 m 179 36617 " 68 4189 , m U4 2353 l, 20, 1 , 198 194 2409 ,

'" 142 4057 .. " 8494, 131 135 3!l23 , . 194 197 2782 0 .. 104 4965 , 161 153 2167 , 56 65 6587 2, 26, l, 7J 76 5399 , .. 44 6553 1 " 15 4311 7 '" 256 2184 2, a, l " " 6691

" 3, 1 l, 11. 1 , m 135 3315 , 07 91 45011 0 210 194 2342 2, 27, 10 ,.. '00 1140 0 ..,

4~6 1153 3 139 145 3397 , ,. 81 5037 3 10. 113 3235 0 " 36 6J1~1 110 119 1121 1 '" 332 1930 • " 78 ~5112 " " 1 • " 65 4218 1 " 51 675~, 10' 114 2081 3 m 220 2237 l, 21, 1 0 m 157 lUI , 16] 167 3153 , .. 20 7HJ3 103 106 2187 , 87 87 3635 0 '" 198 3361 1 '" '" 1652 ,

" !l5 7775 3 71 74 6046• 'O. 314 1917 • '" 153 3075 1 111 lU 3861 , m 2JO 1871 • ., 23 1162 2, 21, l, 07 81 25141 7 '" 155 32J3 • 168 176 J33? 3 .00 '" 1162 2. 15, 1 52 70 63J3

A29

Columnll are loro lOFe lOOOOSlq. ,,, l nil19nl Clc.nt1 kt·o " "0

, ",0 " "0 1 ",0 Fe SIO 1 ",0 Fe S,O 1 ",0 Fe S'O 1 ",0 Fe SlO

" 0, , , l2l 119 2~J1 • .. 78 5507 • ". 203 2503 • 109 111 .lU 0 " 65 3883

0 ." 504 160& , 277 264 2206 J. 18, 1 , '" 109 Jt73 7 102 183 3.76 ,'" 350 2284, !JO U9 2061 • '" 265 2369 0 '"

,,, 2912 7 77 '4 ..aoe ., U. 1 ,'" 196 2546

• " el 21171 • " 59 5017 , ". 200 2790 • " 76 SUl 0 " 89 35J1 , 102 180 2897

• '" 19. ;ZUt • '77 ut 3661 , m '" 2958 " " 1 1 '" 234 2686 • '" 273 2920

• '" 190 nu " ., 1 , ,.. 10. 3191 0 .. 70 39f>O , ". 279 2676 7 67 61 556'

10 71 51 5988 0 " 18 5236 • lJO !JO 34091 1 '" 193 2153 ,'" 211 2908 • .. 86 .'85

" " 1,

'" 728 1985 • "' l2J 4158 , m 263 2213 , .. oU 6999 " " ,0 '" ." 1627 ,

'" 125 2788 7 .. " 7419 • '" 379 2357 .f 15. 1 0 20. '" 2242, ... ... 1662 , 100 96 2999 J. 19, 1 , 127 119 3052 0 .. oU 6230 , " .. 3222,'" '" 1860 ,

'" UO 2877 0 "' 119 3716 , lO' 167 3222 , lO' 110 2877 , " " J745, 120 '" 2336 • " 57 6516 , 78 70 4925 " " , , 103 100 3604 , .. 87 325J

• .. " 2882 J, ID, 1 , 109 106 3865 0 !JO lU 2307 , " 95 11166 • J07 '" 25087 '" 137 3300 0 170 176 2207 , " 63 7228 1 '00 JOI 2091 • 01 39 7798 5 70 71 4397

• '" lO' JUS 1 '" U9 2088 • " 57 7125 , m Jll 2169 5 7J 78 5118 • " " 448910 lJO '" fol37 ,

'" 236 22S' 7 " 95 4601 , 270 258 2339 7 .. 65 5616 " " 13, " , 3 85 103 H69 3, 20, 1 • 1J1 130 2935 • " SS 7446 0 153 lS9 2.73

0 " " 26S. , 67 68 '777 Jl2 312 2883 5 " 96 3750 4, 16, 1 1 140 138 2628

1 ,,, 265 1768 • 70 73 .80' 53 .6 5S98 • '" 233 3001 0 '" 234 2776 ,'" 225 2.83,

" 69 28'S 7 " 77 .921 .. 36 6361 7 118 108 3741 , 110 116 .0811 • 85 85 3989,'" 291 1987 0 187 186 3802 .. 68 4826 • " 41 6383 • 100 109 4115 5 106 lOS 3631

• " 25 5216 3, Il, 1 " 62 6230 0 7J 68 !;392 5 77 Il 5207 • " S8 6462,'" 370 2323 0 217 217 2228 3, 21, 1 " 7, , • '86 188 358' 7 '" 125 389'

• 90 95 3745 , ... 475 2141 ". 1J1 3409 0 " 63 3276 4, 17. 1 5, 5, 37 251 2S8 2891 ,

'" 427 2200 100 139 3449 , 112 119 2S66 0 119 12. 3642 0 '" 205 2372

• 160 170 347B , 70 73 3826 " 54 6802 ,'" 29' 2230 , ,., 195 3094 , 128 12. 2713

• 8J BS '983 • 10. 103 3388 3, 22, 1 , 197 200 2'S7 ,'88 186 3259 ,

" 68 404323, " 3 5 '" 138 32S' 1 " 60 7101 • 117 115 2997 • '" lla 3603 , " 9' 3379

0 '" m 1707 • " 39 S633 3 UJ 118 4093 5 170 193 2949 7 .. 82 S630 • '" 287 26871 51. 515 1700 7 " 61 S562 , "8 130 4438 • ,,, 270 2867 4, 18, 1 5 .. 95 3967, m 751 1793 • 100 97 U57 • .. 18 8396 7 130 130 3688 0 160 162 3179 • '" 210 3158, "5 427 1969 3, 12, 1 3, 23, 1 8 '01 198 3U3 1 .. 30 5937 7 00 27 8013

• " 48 3674 0 '" 133 2597 0 71 63 4H5 " " 1 • 75 90 5635 • 8J 78 5.095 106 96 3035 1 " 80 3343 1 .. 107 4225 0 205 202 2251 5 12J 110 3984 5, " ,• 5J 48 S155 , 210 211 2509 • 65 73 6565 , lJO 144 2636 4, 19, 1 0 J7 32 54937 388 393 2725 3 00 49 6704 3, 24, 1 , 121 119 2828 0 61 57 5643 1 271 273 2341

• 190 188 3359 • 135 126 3024 0 " 85 5289 • " 57 5050 , 51 47 6222 , 177 182 272610 " 546U3 7 7J 80 52114 , 07 53 7545 5 61 62 S307 , 00 21 7739 • 10' 112 3578

3, ., 1 • " 94 5003 • 90 89 4775 • '" 155 3212 • 100 182 3366 5 20' 210 29780 512 '" 1695 3, 13. 1 3, 25, 1 8 '07 153 3891 5 70 91 5789 7 10' 133 3752, 70 65 2881 0 07 " 3265 0 " 69 6526 '. " , ., 20, 1 8 8J 30 80703 '86 '" 2040 1 .0 07 3223 1 105 101 4.58 , lJO 133 2793 0 '" 161 3447 5, 7. 1, 112 117 3049 ,

'" 321 2..01 , .. 46 7389 , '" 120 :'845 1 .. 92 &068 1 88 8.. 30 ..9

• .. 90 3864 3 '" 250 25&6 3. 26, 1 , 90 89 J386 ", 21, 1 , JO 30 62757 m 128 3632 • ". 153 30H 0 " " 5753 5 J05 302 2797 0 51 77 7299 , 51 ..6 51638 190 193 3211 • '60 IH 3315 ., 0, 1 • l2J 123 3651 , 81 68 4840.. • 75 80 4681

• 65 67 51189 7 .. 61 8301 0 lOI 200 196" 8 61 ..664045 • 81 80 5114 5 J7 13 1495,, " , • lJ2 137 U09 , '00 314 20U • " 56 8237 ", 22. 1 • 81 9.. 45400 51' 5" 1736 0 '07 97 40779 • " 711 3298 4, 10, 1 0 67 57 5490 • " 9.. 5087, '" 1" 1968 3, U, 1 • ,.0 197 2853 0 173 180 2463 ,

" 68 5622 5, 8, ,, '" S21 1866 0 " 65 3736 " " 1, SO, 55' 22U • .0 8651406 , 137 133 2832, 10' 100 2800 1 172 176 2601 0 257 '" 1958 , '" 178 2565 5 " 67 7101 , 271 282 2555

• J5 '6 63U , ". 245 2684 , 230 m 2117 , 67 69 U8.. " 23, 1 • 105 194 29535 10' 131 2788 • 135 131 3351 ,

'" 241 2211 • " 98 3584 0 " 16 5896 5 10' 106 4054

• '" 177 21406 5 '" 263 2943 • '" 212 2407 5 165 176 3181 1 00 20 7706 • 70 64 53467 .. 61 7680 • '" 119 "503 • "' 111 3393 7 80 76 4585 • .. 81 4973 7 .. 25 6713

• lU 109 ..022 3, 15, 1 7 116 lU 3710 • 61 56 6826 4, 24, 1 • 106 103 ..566

" " 1 0 5J .. 9 49110 ., ,. 1 4, lI, 1 0 .. 107 5088 5, 0, 10 m '07 1841 1 207 210 2616 0 25' 255 1972 0 " 68 4207 , UJ 103 40365 0 60 67 3925, lOS " 2333 ,

'" 270 2585 , '" 206 2073 1 71 79 U22 , .. 8 7394 ,'" 163 2768, 506 '" 1924 3 .. 72 5196 , .. 82 2606 , '60 169 2H2 4, 25. 1 , 07 35 5213

3 '" 249 2133 • .. 106 4214 , 60 19 3772 , .. 50 5393 ," 82 5576 , 130 133 3164

• 90 93 3065 5 8J 140 4f110 5 JOJ 349 2505 • " 50 5342 5. 0, , • 65 66 50727 .. 53 6942 8 .. 36 f1631 7 ". 159 3409 5 JO, 299 2839 0 m 223 22" 5 '" 254 29450 103 100 4622 3, Hi, 1 • 77 66 5193 • .. 60 7782 ,

'06 617 2226 8 .. ..0 74"3, 7, , 0 '" 131 30'9 .,

'. 1 8 60 62 6126 • 75 68 '061 S, 10, 10 '" 452 1835 , ". 232 2761 0 '" 232 2002 0 5J U 7743 • '67 176 3248 0 m 234 2575, 160 173 2089 , 10. 110 3701 , 90 119 2320 4, 12, 1 8 m 116 3933 1 '00 211 2669, ... '62 1982 , 100 104 '232 , U 29 4193 0 " 79 3436 5, " 1

, .. 59 5077, JOO 299 215~ • '78 174 3255 , m 346 2195 , '06 20. 2679 0 07 5040241 , 211 210 2811

• .. 52 SUS • us 158 3620 • J72 J71 2316 5 125 13" 3525 1 16. 160 2285 • .. 90 41325 " 81 3743 • '129 125 U74 5 .. 53 5966 • 250 2.. 8 3099 ,

" 11 6327 5 .. .. 0 7364

• 100 182 2923 3, 17, 1 • " 44 7055 8 .. 101 473• , '07 112 3017 • 60 56 58577 .. 8044028 0 " 50 5120 7 '" 248 3013 ", 13, 1 • '00 115 3302 • " 62 7196

• 101 110 '40401 1 178 170 '900 8 " 55 566" 0 m 122 3034 5 157 16.. 3075 S, li, 1

• 7J 79 S800 , 107 IS3 3364 ., " , , 190 201 2676 • 7J 64 4862 0 161 159 283210 '" 115 41408 l 170 162 3162 0 J07 3J2 1978 , '" 193 2750 7 " 86 40887 1 m 125 3076

" 8, 1 • 1Jl 128 3..00 , .. 101 2536 , 51 45 587O • 70 69 5722 , 01 .., 75380 ... ... 1901 5 " 19 4205 , m 5"( 2059 • 97 95 4026 • Il' 100 4329 • lU lU 3321, 30S '" 1913 7 10J US JI19 , 78 72 3537 5 .. ..6 6.65 5, 2. 1 5 150 146 3245

AJü

A31

CahlM' tire 10ro lOFe lOOOeS1Ç, for Inslçnlflc.nt."0 Fo '" 1 "0 Fo Sl, 1 "0 Fo Sl, ",0 Fo Sl, ",0 " Sl, 1 "0 Fo '"5, 11, 1 0 " ~O nag 3 " 70 5776 " U 6070 7, U. 1 , 10' lU 4;:JO• U " 7779 1 '" 207 2666 • " 56 6286 1. 3. 1 0 lU 110 3895 3 " al !lUaS, 1;:. 1 , JlB J)e 2540 • le' 107 4567 1 120 12. 3466 3 " .. 6S4S • " " Slll

0 m '" 26351 3 " 41 7058 1 .. 43 6980 • '" m 3279 , 10' 101 4732 8, 10, 11 " " 4220 • "0 UD ng) fio, 12, 1 ,

" " 5.3. 7, n. 1 0 " " l1U, .. " 6232 ," 74 4578 0 '" UJ 3195 • 13 68 S481 0 m 121 4018 1 Sl U 6lU

3 " " 403' • " 9S US) 1 " .. "696 1 12. 1101 4136 1 " Je 6702 3 '" lU 3778• " " 4275 1 " 56 S800 J " .. 6549 1. " 1,

" 80 ni. • " U 6571• 10' 10' 4202 " '. 1 • lU m U"8 0 "J '01 2913 • " III 6J35 et 11, 15, 13, 1 0 '" '" 2517 6, 13, 1 1 " " 3661 .,. 16, 1 0 .. " 6806

0 " .. 5J48 1 '" '" 2460 1 " 10 6980 3 '" '" J21Uo 0 " " .,96 1 " J6 76651 103 " 3549 ,

" " 16U , ". '" 1090 • " H 5317 3 " " 5794 ," " U9',

" " 4086 3 '" '" 2654 • " " 8063 , 11 65 5020 • " " 11310 3 " " S.OIl " " 4813 , m 100 3151 , 110 111 JiU • U 29 8067 ." 17. 1 • " " 4890,

" " 4797 • .. 87 4290 • " " 467t. 1. '. 1 1 " lO 7U6 8, 12, 1S, U, 1 1 " 28 8000 1 " .. 53J) 0 11 11 4519 3 110 10. U67 13 " 52)'.!

0 " m 3778 '. 3, 1 6, 101, 1 1 m m 3200 • 12. 10. 'HO '" " Ui661 '" '" 3320 1 116 179 2687 0 6J " 397~

,'" '" 314. 7. 18, 1 8. 13. 1, m m 3489 , 100 100 3371 J '" ln 3~93 3 lU ". 3.09 " !I!I 7.31 10' " U9!1

3 '" m 2892 • '" 178 3165 , 61 " 6026 1 .. " 66.9 7. l'l, 1 8, U. 1, .. 90 4190 , J1 38 7268 6. 15. 1 '. ., 1 , .. 56 !l96. 0 " " 72871 " 98 4842 '. " 1 " 38 7068 0 10 .. 45.0 7, 20. 1 1 60 .. 6830

5. 15, 1 0 m ". 2565 " 59 5795 1 " " 4009 0 " 91 5199 3 III 101 tri280 6J 81 Ua6 l 10 " 4135 " 84 5040 • '" 200 30103 a. o. 1 8. 15. 11 61 88 4062 3 m '" 2978 6. 16. 1 , m 118 3760 0 13 .. 4658 0 " " 019001,

" 90 01866 • '" 279 2830 0 " " 01195 7 " 57 6135 , m 230 1117 1 " " 470113 13 79 50loi2 • 111 108 01769 ,

" " 4859 1. '. 1 • m 118 01392 8, lIi, 1• 67 49 5315 '. '. 1 • 101 " 43201 0 " 60 6200 '. 1. 1 " " 5320," 103 01363 0 10' 170 2813 • " " 7354 1 19. l'loi 2938 0 100 98 3799 8. 17. 1

5. 16, 1 1 m 163 2864 6, 17, 1 3 106 101 3858 1 m 122 3758 0 " .. 729)0 100 120 1573 , lO' 105 3418 0 " 61 01866 • 6J 68 58n , .. 013 7041 " 0, 11 6J " 01397 • 10 72 4711 J '" 112 nu , .. 62 5958 • 110 117 40!l2 0 " .. 5801, 136 '" 3658 1 " 801 4873 • " 51 6405 • " 66 7176 , 6J 77 0loi77 • " 103 01996• " JJ 6167 a " 50 61129 6, 18, 1 7 106 99 4653 • .. 86 5478 ,. 1. 1

5. 17. 1 O. '. 1 " " 01503 1. '. 1 a. '. 1 1 106 11. 462010 116 165 3136 1 " 97 3487 106 115 0loi04 0 19. '" 3084 ,

" .. 7158 , " " !lDZII1 " " 4764 , JO 18 60156 '" 131 3608 3 " .. 6917 • " .. 7379 '. '. 1,'" m 3143 3 '" 268 2812 " .. 6121 • "0 131 301901 '. 3, 1 " " 70175

3 " 69 4936 • 160 160 3169 " 11 5993 • 16' 16. 39001 1 '" '" 3756 " " 01537• U 31 7837 '. 1. 1 6, l'l, 1 '. '. 1, 81 15 011001 " 6J 57015

5, 18, 1 0 65 fiS ua .. 54 6085 0 153 160 3202 3 " 14 4367 '. J. 1

" 68 5004 1 11> 106 3179 " 74 4775 1 100 98 3779 • lU 113 4197 0 m m 396600 801 4868 , lU 141 3135 6. 20, 1 3 53 46 60160 , .. 7660loi8 1 " 13 !l8UI

5, l'l, 1 3 '" 232 2874 0 " 36 01903 7. la, 1 a. '. 1 3 U " "421 " 69 4511 , 130 1010 30149 6, 21, 1 0 m lJ1 3415 1 " Il 01031 • .,

" 6488," 87 5119 • .. 61 6593 " 99 4779 1 '" 153 3315 , 103 97 41101 ,.

'. 1J 10 72 5642 '. '. 1 13 75 5S16 , 111 106 3726 • " 95 4501 0 " 83 01290• lJ1 125 4003 0 16' 161 2878 6, 22, 1 • " 010 8105 a. ,. l ," 39 7784, .. 82 4933 1 10' 106 30120 0 "

,. 77901 , 101 91 ...62 " 103 4176 '. '. 15. 20, 1 , 11> 112 3290 1 " " 49U • " 017 7929 ". lU H55 0 " " 78910 " " 4782 3 67 70 4913 '. o. 1 7, lI, 1 00 " 5382 1 67 .. 5589, ". '" 3687 • m 257 2964 0 J" JO< 2675 0 U 0loi 7016 " " 6458 '. '. 1

5. 21. 1 ," 36 70178 • 130 123 3679 , 151 168 35019 a. '. 1 " 0loi 5UI

1 lU '" 3875 7 " 66 6483 • "0 '" 3528 3 " 50 6607 1 10 72 5260 " 65 52953 " 013 6940 a 11 801 6681 1. 1. 1 • 116 111 3975 , .. 59 7052 '. '. 1• " 91 47901 '. '. 1 0 .. 87 4024 ,

" 76 5124 3 '" 152 36'01 0 " " 52115, 22, 1 0 JO 18 6702 1 150 150 3073 • .. 53 6533 • " 73 "'!l9 1 " 6J 67!13

" " 50loi9 1 JO 32 6921 ," 60 4337 7. 12, 1 , m 123 40017 , 10' '" 4102

S, 23, 1 , m 113 3485 3 .. 65 4616 0 .. 61 4923 a. 1. 1 '. a. 10 101 100 4145 • " 67 5807 • " 29 6049 1 U 011 6660 0 136 131 3669 0 " " 54861 " " 53801 , 10' 107 4191 ,

'" 121 4151 3 " 110 5375 1 U " 7579 , .. SI ~'lJ

S, 24, 1 • " 70 5162 1. '. 1 • 10' 106 4821 • lJ1 '" 3910 3 " 61 6UO

" 6J 6133 6, la, 1 0 ., 71 4589 7, 13, 1 , 130 m n!1l '. '. 1'. 0, 1 U 36 6297 1 ". 138 3178 1 " " 7000 '. a. 1 0 16 72 57120 JO J1 6342 " 53 58301 3 '" 149 3401 , 16. 167 3435 ,

" 56 4961 1 " 60 5700, m na 2596 6, li, 1 • 6J 87 4562 3 " 75 H08 '. '. 1,

" 57 60150• 6J 66 49601 0 m 155 3153 ,"0 129 4039 • m 96 01000 0 ln 1J7 4095 9. 12, 1• " 016 7742 1 110 112 3UO • m 133 3776 ,

" 63 6802 1 6J 60 5227 0 m IHI 01171

'. '. 1, 160 1015 Jln

• CF,f'0 0

HNfoMePh S

l..Ar

D Erythro

+ Eu(hrch

Il

A32

~_l,

....... '}f' l" .1: ,'------..;. , l'________/\._--:_=-=_~I \.__._.__~__ ....-----:-!_'J_,._.;..,._'--_~~'"':_~~,. \ 1 ,. Il \ 1 l' '1 . t <.1 '.' '.1 :.~ '0' • 1 0.1 '.' • 1 1 t 1.1 1.,' 1. ~ 1 1 l'

DL Erythro

+ Eu(hrch

i 1 i i Iii i J i Iii i i i i i ( 1 i i i1.' 1.1 1.1 ••• '.1 ••• 1.1 1.1 1.1 1.1 1.' •. 1 1.1 •. 1 •. 1 1.1 1.1 1.1 1.1 1.1 •.• ..1 1.'

'"

1 i i i 1 i j i i i Iii 1 i t i Iii 1 i1.' ,:. 1.. 1.' 1.1 1.1 1.1 1.1 1.' 1.1 1.1 •. 1 1.. '.1 •. 1 1.1 1.1 1.' 1.' 1.' •.• 1.' 1.•

n"

A33

Observed and calculated structure factor of t-Boc-Val-Leu-OHColulII\. U. tDro lOFe 100051;, for Indllnlt1r:an"kFo Fe '"

, kFo Fe '", kFo Fe '"

, kl'o Fe '", kl'o Fe '" 1 kl'o Fe '"0, 0, 1 n 10' " J5l , 'O, .. '0' ,

" " .., ,'" 'O, m ,

'" m m, 1052 1181 JO] " 'DO 1lO lU • 65 71 '", 100 '" '"

, ... m '",

'" 150 lU.. 21.7 2351 '" Il 65 " m '0 m m OU • m 100 .., 10 m '" ,.. • '" ." '"6 125. Il" '0' 11 '0 " '" n '00 '" m , 55 65 .00 " '" ". '" 10 m '" ,.., '0' no m '0 '0 " '" " ." 152 020 ,

" " '" " '" '01 '" n U5 151 '"'0 m '0' m 0, '. 1 Il 'DO llD '0' n " " '0' II 121 ... 100 " 121 ". ln12 " " '"

, 126 m m 19 " " '" " " 16 ,.. 15 " " '" II " " ,..10 " " .., ,

'" '" ." 0, ID, 1 0, 17. 1 Il m 121 ... Il .. .. OU'0 " " '19 , "0 Oll '" 0 " " m , 10 " ... " 111 '" '" li 10 '0 ,..

0," 1 • '" '" '00 1 m 'Dl ,.. • ,. 00 ". 11 " " ,.. Il '" '" '",

'" 50' III ,'" OU 20' , 11 " 061 • 55 60 020 19 " .. 100 " .. " 00', m m '" • " " '"

,'" '" m ,

" " 10. " 00 " m " " 1• 019 m '", 201 '" m • " 61 '" • li " '" " " 1

0 115 121 ,..,'" '" m ,

'" m m , '02 '" ]Il 0, 1'. 1 0 Il .. m 1 lU no '"• OU ,.,'" • '" no m • m '" m , 121 m '" 1 lU5 n'2 '"

,'" 521 m,

'".., 201 10 160 '" '"

," " 601 , 101 ... '"

,'" '" '"

,'" '" '", 122 n. m n 201 m ,., • " 10 'li , 50 " ,.. , 1171 1155 '" • '" '" '"• 050 '" '" " .. " "0 10 m m '"

," 51 ,.. • Il " '"

, ... '" ".10 '" '" '" 10 ., .. '" n 69 .. ,.. 10 .. , ... ,'" '" '"

, m m li'11 lU '05 '" li ., .. '" 15 " " '00 li " >6 m ,

'" '" 20' ,'" m ,..

12 020 OU '" " m ." '" li " " '" 0, 19, 1 , ll6 115 m , m >60 '"II '01 111 '" 11 ... 10' U6 10 " " m 1 " " 656 , 20. .06 '" • '" m ,..10 " .05 '" 21 65 00 '" 19 Il "

,., ," " '" , m '" li' 10 '" li' '"15 120 110 '" 22 " 10 116 O. 11. 1 , 00 " 621 10 ,.. m '" n III '" ".16 122 m '" 0, " . • "0 m ,.. ," 21 '" n lO5 20. '" " III '" ...

10 ... 115 '" 0 '" '" ... ,'" lU '" 10 55 .. ,.. 12 159 '69 '" n " 00 ."

10 " " III • 136 m '" ," " ,.. 0, 20, 1 10 " " m .. " " '"" " 61 m ,

'" 10' '" • 02 02 ." 0 01 " '" 15 119 .01 011 li .. " 51522 " .. '" • ID' 115 lU , .02 '" m ,

" II ". 16 150 150 m 16 52 " '"0," 1

,'" li' m ,

" " lU n " " 601 " " " '02 " " .. 6910 1117 119. m , m 10' '"

, .. .. m 0, 21, 1 li .. 01 '" li .. .. 55'• m 126 '", n, '" m " " 60 .., " " "' " " 1

19 " 19 "', 260 '" U7 , m 010 '" " .. .. '"., 0, , 0 551 '" .11 21 .. 60 ..., 1037 1098 '" 10 '" ." m 0, 12, 1 , 1166 1284 m • '" '" ,.. " " ,

1 1694 1511 ,.. 11 m 215 '" • m 110 "0 1 1415 1"' 'Dl , '00 m 110 0 .. " on5 1113 1281 m " " " ." , .. " n. ,

'" '" m ,'" '" .00 • '" '" ".

6 1002 1021 109 Il " Il m , .00 .90 m , 00. '" "0 • lOI '" 101 , ." 100 ,OO,'" m 216 " n 01 '"

, ,., 260 m 1 1034 1011 '", m '00 ". ,

'" m ,..• .. " '" a 126 162 no , .. 51 501 , '65 .., 111 , m ,oo '" • 155 a. m, m '00 m .. .. " 656 • 01 "

.., ,'" '" ". ,

'",.. m , Il Il no

10 m m 200 17 " .. '" Il " • on , 1" '" m ,'" '" 2~1 ,

'" '" mIl " " '" 0, " , " '" 155 '" • '" '"

,.. ,'" '" ". , m 261 2Il

10 li .. '" • '" ". 216 .. " 69 '" 10 '" >60 '" 10 .., ... 1O2 , ". '" lU15 01 " 110 , 50' '" '" 19 " 60 ,., 11 '0] 115 '" .. " .. ... • '" m 'DO11 " .. '" • '" 216 '" 0, 1], 1 Il .. .. '" n .00 ... ." 10 " " '"li 06 " '20 , li " '"

, .. " .., a " " '" " .. DO ,.. 11 115 102 60110 .. " ,.. ,

'li '",,. • 10. ." '00 17 51 " m 15 no '" '" " " " '"22 " 26 '"

,'" '" m , "0 m '" 10 " '0] 60. 16 " .. 110 " " " "0

" ",

'",

li' 115 ,.. ," " '" 10 " " m 11 " oo '" a .. " n,

0, " , 10 102 " '", 107 110 51. " 01 " ..0 10 " " '" " 50 " m

• 'DO m 155 " H' m m , 05 " '" " " 1 10 .. " '" 10 " "..,, ,.. '" ... " Il " m • .. 10' ,.. 0 '" ,.. 010 " " " III l, " ., H. 112 '" 11 " 61 ,OO 10 ,. " ,.. 1 1012 1117 "0

., " , 0 .. 02 no• '" m '" " .. .. ,.. H 50 " "0 , ,.. m m 0 '" ,.. '" 1 '" '" ,oo,

'" "0 201 " " " m 12 " 61 Dl] ,'" 20' .59 • m m '"

, '" '" '", ,.. 116 '" 0," 1

n .. .. m • '" '" 60' , m '" .,. , m m ".," " ,.. ,

'" ~11 201 " Il " '", "0 '" 116 , "0 216 ,.. • '00 10' '",

'" '" "0 • " 10 ]Il .. .. ",., , ". ,., 100 • ,OO '" :as , '00 115 m,

"' 112 '", ... .., "1 20 "

,'"

," 02 ". , ,., m '"

, 1" 110 ]Il

10 '" 60. m , 106 105 '" 0, 14, 1 • '05 192 '" • m '" m ," .. OO.

H '" '" '" • ,.. '" '", '" 112 Cli. , m ou '"

,'" ou '" • 150 no m

" " 60 '", no 10. '" 1 Il .. '" 10 06 00 ,.. , ". m m 10 110 m '"II m '" '", no .60 '"

," " '" H m '" ,.. • m '" '" 11 .. " ...

10 220 200 lOI , ". '" '00 ," .. '" .. .. " ,.. 10 lU '" lU " 05 " '"" '" '" ". , 110 III m , 02 " 501 n 'DO '02 "0 H m 116 l50 n " " '"li " " '"

, ,Dt .,' m , ,." '" " 51 DO 611 " m ." ,.. 11 .. " ...

11 " " ,.. 10 '00 '" '" 10 " " .00 a 111 .., '" n 162 151 'ID 10 01 " 00'

" " 20 '" H 150 1.. ,.. H " " m 10 m 116 .00 15 " Il '00 21 01 20 1100, " . n " " m n " " ." " " " ,.. 11 " Il on 1. 10, 1

0 1114 120. ,.. " .06 10'.,. 15 01 " '" 10 .. " m 10 " .. ." 0 m no '"1 1278 1310 '01 ., " " '" 11 " 12 on " " " '" " 65 " ,.. , .oo a' m, ,.. "0 16l 11 " " ,oo 0, 15, 1 " " . 21 .. " '"

, 'ID ". '00, .., '0' 111 0, " . • lOI " ,.. 0 9!l9 leu' '" " " 1, .01 '00 '"• ,oo m 195 • '" 52' ,.. , .. " '" • lOI m U5 0 '00 001 100 • " " '", m m '00 , ,.. li' '" 1 110 U6 m ,

'" 520 uo • 201 201 '", m ." ,.., .06 160 '"

, .,. '" ". , .. ",,. ) 1017 1113 ". , 205 21• '"

," " .", 120 m '" • '" m '"

," " ... • ". no .11 , Il " m ,

" " ..., 85:" ... '" • ID " '" • ,.. .00 50' ,'" m 100 • ,.. III '"

," .. m

• '" '" '" • '" '" ,.. 0, li, 1 , ." '" 1" • '" 12' '", .. "

,,.10 205 201 m , 110 no no 0 m '" m ,

'" 011 ,Dt • m ." '" 11 U6 12. ."

A34

tolulII. Ar. loro lOFe 100051'11•• tor Jn.lllnlUc.n~

". le .'. l ". le .'. 1 "" ro .'. 1 ". ro .'. , ". le al. 1 ". le al.1. 10. 1 12 .. " ". 12 lOI '0' ". " .0 " '" • " " ... 1 " " '"12 .. " .., l7 " " m 16 .. " 707 l7 " " '0'

,'" 119 m , .. 51 '0'

16 91 lU m 1, 17, 1 " " .0 m lt 61 " m • lU U1 616 ," '0 ,.,

" " " '" 1 109 n' 55' " .. 51 '" '. " 1 • " " m • " 51 '"l7 .. n '", ... n• m l7 10' '0' 51. ° '" 170 m '0 .. " m ,

" " '"19 " 51 ,.. , U6 162 '09 11 " " ,.. ,'" '" m 11 m '" m • ,. 56 '0'

1. 11, 1 • " .. '06 " ,. 1 ,'" m '" 12 59 " '" • " ,. '"° 160 165 '"

," " ." o 10U 1027 '"

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A35

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A36

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A37

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" " " '0] • 101 '0] '" " 0, l • ., 10' 'DI " ]. .. 'N 10, 0, 115 .. •• '" '0 •• .. 712 ,

" .. 772 • ,. .0 770 " " " III 0 or .. m

" " 111 ,. Il m • .. ., ... • " ]7 07J .,

" 1 • 60 .0 ,.,0 m 16. ,..

" 71 71 51', .. .. '" '0 '0 " .0' 0 Il 61 ... ,

" ]] '"1 ,. '0 m 13 " " ... 11 61 " 7J1 11 .. 76 '0' 1 " ., ,.. • " 49 1022,'0 .. '" " .. JI m " JI 17 167 13

]." '" • Il " '0' 11 .. ]] m

] ". '01 ,.. 15 JI ,. ..] ., l, 1 " " 1,

" or 712 10, l, 1

• ., 117 "' ,, " , 0 " " 71J 0 " .. '" • " 17 ... 0 ,. '0 56., m m ]" 1 ,. U 51. 1 Il 77 ,.. 1 .. 76 679 10 '0 " ,.. ," JI 770

• " .. '",

" " II] , ., 101 '",

" " ,.. 11 •• 71 ,.. ] 17 " 910," ]0 m ] Il " 517 • '" 171 127 • 61 " 71. " " U '"

, 17 " ,.,• .. " '" • '00 m <Il , .0 ]0 ,., , .. " '" " " 11 ... • .. '0 '0'

17 ,. " ,.. • 'O, ., ". • .. .0 '" • 17 " m ., ], 1 15 '0 • 7J117 JI " ... • '0 .. 712 • ,. ,. '17 • 71 '0 '0' 0 10. Il m '0, " 117 " •• 9J1 • '0 71 ... 10 77 " ... 17 ]] 17 692 1 96 .. '"

," " '"15 " 72 ,., n .. 60 .00 n " " 711 " ., 1 , 122 10• ... ," " ."

17 71 " .0. 17 " 62 .,." " •• m 0 " '0 671 ] '01 'O. m ] ,. " '"" ,. "

..," ., 1 16 " " ... 1 " ]. 110 • " 77 ... • •• 51 121

" ], ] 0 ", 'N " " 1

, 61 " m • ]7 " ... ," '0 617

0 151 J71 J61 ] " 56 ". 0 '" 10. '" ] 56 " ,., ," " Il< '0 .. • m

] '" 100 m • ,. 51 m 1 '0 •• '0', ,. 71 '" • •• .. .11 15 " "

..,] 10' 1J] 127 , 17 ., '"

, .. " ,.. • 127 ". '" 11 " 11 m 10, ], 1

• 16. lO! ]70 • '" 11. ... ] 120 10' "0 • •• .. m 15 .. ]7 72' 1 '0 .. 71., .. .0 ". , l50 '" ... • 101 122 '0' 11 .0 Jt 1002 " " 1] 51 " ".• 'O. m "0 • 62 Il 712 ,

" 6J m l, 10, 1 1 10] 17 '01 10," 1• •• .0 m • 120 12' ,.. • ,. " 'N ,

" " ••• , •• " '00 0 71 .0 '61

• 100 ,. .., ,. 71 " ... 71 67 Il 610 ] " •• III ] ]0. '01 ... ," 51 lJl

17 ,.'0 m 12 " Il no 17 Il .. '" • .0 '0 9J2 • " 51 112 10,

" 1

" •• ., m 13 ,. 67 m " 67 62 ... • .. " ... , 50 56 712 1 .. 12 '"15 " '0 ... 7. ID, 1 11 " 01 '" • " •• ••• ," 71 ... • " 01 .01

11 JI 22 ,.. 1 " 7] 56' ,. " 11 ,.. 11 " '0 ••• " " " '96 12 ]] ,'0',. .. ]7 ." , 51 ,. 712 " ], 1 12 JI ,.

'01.,

" 110,

" 1

" " 1] " .. 127 0 1.. •• ... I, 11, 1 ,

" Il m .. " m0 lJ1 127 ]., • 117 10. 'DI 1 76 ,. m 0 72 " '"

, 79 " m 10," 1

1 ]0' 127 m • " Il 622 , 111 116 ... 1 51 " ". ] .. 51 'II,

'0 Il 761] '" 171 JI1 , 116 112 '" ] " 76 .ot ] .. 17 '" • .. " '00 10,

" 1• " .. m • 77 71 6J7 • '01 117 ••• • " 22 '", Il 77 m • JI " IJO,

" " 66J 16 JI " 7J7 • •• .. ,.. , .] 92 '" • Il '0 m '0, " ]• ID " '" 7. 11, 1 1 .. ]..., , •• .. '96 • .. " .17 l> " " 712

• '0 32 .,. 0 lJ] '" u. • 61 Il III I, 12, 1 ., " ]ID, ID, 1

• " .. m , 72 76 6JI 10 7] 72 '0' • " " ••• , Il 76 ". .0 16 l2J17 ". ,.. '" ] .. " 10)0 17 ,. .0 m 10 JI 21 m ] 59 .. ... 10, 11, 115 01 .. 922 • 710 m '" 16 " " UO " JI 21 ... • " " ... • "

,'"" " ] • " ]. m " " 1

l, 13, 1 ," .. 712 71, 0, 1

1 " .. ,JO ]0 U " m 0 " 71 ,., 0 " " m • .. " III JI .. 922, 71 71 .., 15 " 2J .0] , 710 10' m , .. " '0', .. '0 m " " m

] 177 li' ] .. 16 JO 2J m ] " " m • " " ••• " " 1 " " ."• 79 " ... 7. 12, 1 • 105 " m l, u. 1 ," " 7IJ 11,

" 1• .. 91 66J 0 67 Il '", 61 .. '" 11 JI 21 m ,

" JI ... , JI , ...• .. " ,.. 1 Il •• 51. , e. " ". I, 15, 1 • Il Il 671 Il, " 1, 71 60 '"

, .. " '" • .. " m JI " 021 10 " .0 021 " " 120

• ID " '" • 10' 125 m 10 .. .0 777 I, II. 1 ., ., 1 U, ], 1

• '0 71 ,.. • " ]. '00 15 " 32 .01 • .. ]] ... 0 71 " m • li Il .9610 .. " m 10 " " '11 17 J7 26 .00 ,

" 2J m , .. .. ... n, ., 1

17 " 51 ..0 7, 13, 1 11 ]] 11 III • .. ]] '11 ," " '51 JO 16 'D<

" " '0 m • " 2J '" " " ]l, II, 1 • " 61 ". Il,

" 111 " " III 1 •• '01 'N , .. 76 ... 0 " ] ... • JO " ... • " " 10],. J] 22 '51 ] .. " 129 • " 32 '" " 0, 1 11 oc ]7 .17 ,

" 16 ...11, " 1 U,

" 112,

" 1]] '0 m "

, ", • JI • "'• JI ] m 11 " " 6J7 • " 21 711 • " 13 711 n, " 1 il, ], 111, " ] 11, 12, 1 .. 21 10 ... n,

" 1,

" • ,.. li .. ."," " ... ] " 16 ... n, " ,

A38

Observed and calculated structure factor of t-Boc-Val-'II[CHz-Oj-Leu-OHColwan. ar_ IOFO lOFe 10000519. tor In.lvnHle.nt..,. rc Il, .,. rc Il, 1 .,. rc 51, 1 u. rc .,. 1 U. rc ." , "'. rc .".,, ., 1 -0, '. , , m 13f,1111 1 '" •• 1207 ,

" .. 1215 0 ,. n UO]

1 .. ;al ll1t 1 " "C 1272 0 ,Ot 112 1195 , .. .. 12~lt 0 ,., '03 1119 • " 55 1237," U lU!! , ,. " un

, ,. " lU2 • ,. 34 141. ," " 1196 0 " Il 1133

0 12 1 161' , .. 71 1220 0 " ft 1351 • " 17 2176 0 " " U" , " U 1131, " 7 lin • " •• 1225 , 10' 113 1195 0 " U lUI , .. " 12'" • " U 1012

0 U 11 1623 • " ]J 1U7 • .. 26 nU , 11 22 III' • ,. ., 1237 • ,. .. "..,.'. 1

," 25 1596 , '0 n 15115 • " " 1323 • .. '0 1149 -4. 12. 1

1 " " 1052 , ,. 36 1233 10 " J!i 1154 -!i, 11, 1 10 " •• ",. 1 .. .. 1157, ,. .0 107' 1 " 21 U'iI1 " " 24 1053 1 " 55 1012 " " .. 1119 ,"

., 1189," 55 1001 • " '0 '0' .', " 1

, '0 20 1679 .', '. 1, .. .. m •

• '0 19 13" .', " , , .. •• 1171 , .. le 1072 1 10' .. 1071 • " " 1711, 11 J1 1051 , " 32 1351 , " " 1742 • ., 61 1065 , .. " 112. • n " 1203

1 " .. '", .. .. 1274 ,

" " 1212 ," 53 10)2 , '" "0 1081 • .. " lIaI., , " , ,

" 35 12.6 • " :iII 116f, • " 34 10U • '" '" 1123 ," " 1057, " "

.., ," 21 1316 ,

" ]7 1602 ," 13 1419 , 103 '0' 1177 1 ,. .. 1072, " " la" • " J1 1061 • " 32 156] -5, 12. 1 • '" ,,0 1192 -c, 13. 1,

" " 132. .', 0, , ," 75 1233 ,

" 12 1609 , ., ,. 1295 1 " :U UlJl

0 " " ".. ," 5311" • " 61 1150 , " 22 1239 1 n " lJ75 2 " 16 2007,

" 24 1211 ," !il 1179 • " U 1113 • ,. 50 1001 , •• " 1316 , ,. 5' 1125

• U 14 1621 • JI 291381 10 U 9 1720 , ., '0 '" '0 ",. 1227 • ,. 39 1225.,,

'. 1, " 22 16n .'. '. 1 • " .. ••• 11 " " 1061 , 11 23 1649

1 " " 1092 , 21 27 1275 1 2l II 1125 -5, 13, 1 .'. ., l • J2 36 1062, J2 " 1001 ," 17 1412 2 .. 94 1214 l 22 " 1203 1 .. " 1123 ,

" 17 1306, 12 12 Ilt3 • " " ." , 100 " 1199 2 '0 .0 m 2 30 ,. 1405 -4, U. 1

• " " .0' -o. '. l • ., 64 1215 , ,. ,.... , •• " 1175 , 2l 22 1481.,,

" l1 " 57 1177 , 129 133 1211 • .. "

.., ," .0 1211 2 29 25 1217, 30 ,. ..0 2 " J9 1275 • " 29 1666 ,

" " '",

'" ". 1171 , U 10 1180

2 20 " 1317 1 2. 27 1424 ," 22 1912 -5, U, 1 , •• •• 1216 • " ]9 1102, ,. " 1422 • " 40 1172 • 29 21 1279 2 '0 •1775 1 .. 61 1252 , .. " m

• ,. " 1227 ," if 1021 '0 U 13 1411 -o. 0, 1 • U 17 2101 • " " ll6, 21 2. 10SlS 0 " 35 1136 .', " l

l ., ., 1051 10 ,. ]& 1193 -4, 15, 1.,.

" 1,

" 26 1313 1 " " 1255 2 " 71 1016 11 " ,. 9ll l " " 1291, " " '" ·1, •• 1 2 ,." 1231 , .. 90 1037 -'. '. l 2 " "

,.,, 21 25 1055 1 21 25 1246 , 121 121 1197 • ,. 57 1166 1 " J2 1371 , '0 ".,.

0 22 20 1105 2 22 20 UOI ,'" 17& 1112 , m 223 1072 2 .2 80 1199 • .. .. ..., J2 " ... ,

" 21 1305 , Il Il 1237 , •• " 1202, 22 22 1'20 ,

'" ., m·1, " l • " .. 1016 • ., 71 1244 ,

" 100 1205 , ,. 80 11" ." O. l," " '"

," 34 1014 , '0 27 1500 , ,.. 190 1112 ,

'0' 111 1201 1 20' 200 m2 11 10 1625 1 " 14 1675 10 " 25 ION '0 Il U 1273 • .. '0 12C1 2 2.. ". ,.., 2l 2' 1050 ,

" ,.." .', •• l 12 " 21 1003 , .. ,1 1241 , ". "0 Il]

.'. O. 1 .'. " l, ,. 11 2011 .'. 1. l • " II 1177 • ,.. '0'

..,," " 1207 1 J2 30 1101 2 " 23 1510 1 ,. 62 110' • '0 30 1367 ,

" " 1097

2 '0 U 121S 2 2. 21 1212 , .. 91 12]1 2 " 62 1100 10 ,. 31 1010 ," 59 1077, .. es 1113 ,

" 2512" • 92 " 1240,

" 71 1052 11 " 2f 1040 1 '" 195 1071

0 29 21 141" • .. f!I 1067 , "0 105 1201 , 112 171 1030 .'. '. l 1 .. 72 1242

• " !I!I 1242 , ., " '", 11 71 1211 ,

'" 115 10" 1 " !ID 1293 • J2 13 1S1S2, 12 121" 0 " " ." ," ·17 2303 ,

" ID 1201 2 " !II 1305 10 " 17 1970, 19 l.2 1541 -6, 10, 1 • J2 21 lUI ," 56 1274 , 12 71 1223 11 " 54 1201, •• ., m ,. J!I 1000 '0 21 21 1100 • ., 71 1267 1 ., U 1110 12 " 30 1119

• " " '" " 37 1023 -'. '. l • " ID 122' , ., 82 1244 .', 1. l

.'. 1, 1 .. Il .., ," 51 1261 '0 2l 21 1507 • " 51 124' 1 ., •• • '0

1 22 .. ,... 19 15 1113 , 11 10 2)71 11 " 34 1U7 ," 66 1226 2 ,.. '" '0'

2 " " 1215 -l, 11, 1 ," U 1419 12 ,. " '" • .. " lUI

," •• ...,

" " 1241 , 11 13 1715 • .. " 1244 .', 2. l • .. 53 1096 0 '",.. ..,,

" 15 lUI 2 " " ... ," 73 12]1 1 " f!I 1150 10 " " m , ". lU '", ,. U 1411 ,

" " m • 91 tJ 1173 2 '02 104 1011 -0." 1 • lot 111 1033, .. .. 12" .'. 0, 1 1 ,. Il 1110 , .0 71 1062 ,

" 15 UI!I 1 " 35 1311," !II 1122 , 121 114 1165 • " 34 1221 1 ,.

" 1171 2 " 5' 1277 • " 36 1390

• " 31 lue 2 " 40 1331 • " J3 1022 , m 151 lOI, , ,.. 150 1177 • m 132 1195

• ,. " m , ., ID 1201 .'. •• l , .. 10 11'1 , 92 12 1211 10 30 35 142'

.', 2. 1 ," 41 1310 1 " 21 1417 , '" 52 1210 • lU lU 1202 11 " 44 1247

1 " 7J 1201 ," 1] 12. 2 .0 71 UII • " " lU'

, .. 4!1 1270 12 " JI 1154

2 " 12 12U ," 30 1415 , ,. 91 1171 • " 51 1301 1 2. 32 1465 .'. 2. l,

" !II 1270 1 " 21 22,. 1 " 52 1315 '0 " " 1224, ,. 17 17U 1 10> '02 .",

" 45 1271 • ,. 21 1163 , Il 77 1111 " .. 45 1072 • 30 35 ION 2 " " 991," 15 123e 10 " .. ,.. 1 30 3!1 1402 12 " " '" 10 " 11 1297 , " flO 107., .. CI 12,. -'. 1, 1 ,

" J3 1225 .,.'. 1

-fi, 10, 1 • ., ., tl1, .. .. 1175 1 21 J1 1121 • " 2J 12", .. 60 1061 1 92 ., 1201 , 20 20 1702

• " 51 1021 2 '01 110 1174 • " " ,., 2 " JI 1311 2 " " 1212 , 01 !II 1105

• 22 II 1122 , ft H 1111 .'. " l, m 106 10fll ,

" " 1237 ,'" 1101111

.', '. l 1 m III 11N 1 " JJ lue • '" 255 1053 , .. 76 1116 • 121 130 1111

1 .. 53 1273 ; 11 12 2155 2 '0 51 1294 ," U 1320 , 1JO UO 1165 ,. .. 91 1205

2 " 77 1191 , 102 107 1213 , '0 fil 1225 1 " I!I 1200 • ., " 1211 11 .. 23 lU', 11 71 UH ," U 144' • " " 1271

, '0 69 1257 , ,. 42 1276 12 21 27 13U," li 1210 • lB 11 1115 , Il fl2 un • ,. 74 1111 , 19 l' 1332 -" ,. 1, .. 70 1112 '0 " U 10U 1 " 12 12.7 10 " J2 1301 • " 21 1224 1 201 2.0 '", .. 1] 1110 11 •• .. "0 , 2t n lll2 12 JI " BI] -', 11, 1 2 '''' ". III, .. fil 1071 .'. 2. l • " J5 1044 .'. '. l 1 " !lfI U17 , 1]0 110 ..., .0 3. 1041 ,. 10 1214 , •• " '"

, Il 42 1230 2 .. 61 1211 1 "' '" ...• 21 11 1010 11 13 2520 -5, 10. 1 2 "' 170 1032 ,

" 21 1557 ,'" ... ."

A39

• Col=••r. 10ro lOFe 10000519 •• '0' Inalc;nl11C11ntkTo re ", 1 kTo re '" 1 "'0 re '" • "'0 re al, 1 "'0 re al, "'0 ro ",-', " 1 • 54 66 1132 , 106 106 '" • U II 1365 • .. 21 1265 -l,

" 1• " " 153. 10 " " '" , 190 '" .10 , ., flO 11.3 -2, 15, 1 1 '" '" 'li7 .. ., 1128 Il " " '" • '" m m • '01 150 1016 1 " 511nO , .. 17 "0• lJI ..0 1115 ·3, ID, 1 , ,.. Uo '" 7 " 11 lt24 , .. " 1136,

'" '" ."• ... ... 1194 1 106 10' 1169 • " 51 1002 • '" 2' 1101 , Il 10 1115 • " " 11110 " .. ..00 ,

'" '" 1145 7 77 7. 1052 • '0 n 152. • .. 691116 , 100 101 '"11 11 " Ulll ," " 1:117 • m 152 ua' 10 " '1 1111 , 01 ... lUI • .. 52 10'.

12 " " 1079 • .. Il no' • m 1'73 1171 11 " oU 1166 • " .0 ... 7 " li lU,-', " 1

, 10. le' 1204 10 17 Il 1231 -2," 1

7 .. 11 Ual • " Ji l'52• 151 ... m • " U 1J9O 11 " 54 12&0 • " 50 1032 -2. II, 1 • lJO 1231201, lJ 11 19112 7 " 71 1212 " 11 9 :Z01' , m ". ... 1 " 60 1099 10 " n 12'3,'" ,.. ... • .. 70 1169 -" " 1

, 10' 10. ... , U fil 1163 11 " U 127,

• Il .. 000 • " 45 11&5 1 ,,,'" '" • .. 107 1035 ,

" 36 1210 " lJ 15 lU.• " " 1018 10 .. .. '" , 'O, '0' ... • " 63 lU4 • '0 23 ail -l, ., ,• 54 " 1126 -J. 11, l , m '" ... • .. 22 lin • " :n 1132 1 .. " "'7 11 76 1192 1 .. 17 1196 • '" J30 m 7 ". 157 1176 • .. 20 1165 , m 150 ."• " 89 1202 , .0 18 1204 • .0 n ..0 • " Il un ·2, 17, 1 , lJO ,.. '"• " 51 1330 , 106 lU 1190 • .. .. 951 • " 5' lUi 1 '0 " lUl • m lJl '"10 U 25 18.551 • '" U9 Ul7 7 '" UO Ion 10 " JI 1267 ,

'0 " uo. , m 171 ."11 54 61 1139 • lJI 131 1196 • .. 79 1150 11 27 JO 1325 ," H 1011 • " 7'S 1025

" " U 1073 7 JI 32 lUI • 110 120 1191 -2," 1 • " 21 1105 7 '0 n 1017-', 5, 1 • 20 .6 12.0 10 27 U 1710 1 m '" ... • 10 .S 119] • " U n.5

1 '" '" '" • " ., 1211 11 " 14 .031 2 01 37 1220 ~2, li, 1 • " ID nu2 .. " ... -3, 12, 1 Il " 141U3 1 111 111 1047 1 .. ]0 m 10 11 67 USD1 100 95 '" 1 " " U19 '2, ], 1 • " '76 1101 2 " " ... 11 11 16 171'• .. 17 1001 2 .. 01 "00 1 "0 '" '" • " 31 1211 -l, 0, 1 12 " 4J 10n• .. BB 1011 ] " 61 122' 2 122 l2l '" • 12 ID 1200 1 11 12 lUI -l, " 1• lJ7 "0 10.8 • 11 2. 2209 ,

'" 111 717 7 110 125 1202 2 ]0' ]20 112 1 111 !li 70'7 " 95 1175 • " 42 U24 • '" 102 112 • 11 75 1250 1 10' 10. ... 2 211 20' "t• .. 90 1218 7 " .6 118. • 20] ,.. m • 21 31 1501 , m m ... , 2ll 200 17.t 21 .7 1'54 • " 50 1010 • " 57 1011 10 " 19 1152 , n. '" m • ". '" ".10 " 6. 1196 • 11 11 .01 7 .. 27 1325 11 " 3. 1030 • 170 171 III , 112 lU ."11 72 7J 1011 -J, U, 1 • 101 105 1143 -2, 10, 1 7 172 171 .71 • 17 76 10U

12 " 14 1425 1 lU 1.0 1169 • 72 70 1225 1 10 12 1096 • ,"0 234 1079 7 Il Il 110t.],

" 1, 107 106 1177 .0 00 99 1210 2 .. 6. 1144 • " 56 1256 • .. '2 1191

1 127 120 950 ] " 36 1565 11 .0 19 1242 ] " 59 1192 10 " 66 122' • " li 11052 .. " 1025 • .0 15 11a1 12 27 JO lil9 • " 57 nu 11 " 50 1231 10 Il 72 1251] " Il 10U • .0 40 12., -2,

" 1, 11 40 1362 12 20 2t 1261 11 10 JO 1271

• 21 " 155' 7 " 17 1417 1 171 ," ... • lU 116 1115 11 17 l' 1312 12 '" 25 lUI

• m 114 107. • 11 " ... 2 201 200 n. 7 100 106 1217 -l, l, 1 -l, 7, 1

• " 41 1409 -3, 14, 1 1 1.. 11. m • " 41 U07 1 lli7 '" 16.4 1 10] .0. li'7 " 7' 1224 1 .. 64 1171 • '0] ,.. .11 • " 13 12.2 ,

'" m ... 2 ... 110 111

• 100 106 12J2 2 01 17 1242 ,'" 111 .90 10 .. 24 1226 1 172 112 '" ] m 27t l1J

• JI Jl UU 1 .. 22 2210 • .12 '" ." 11 11 11 1617 • 111 li> ... • '" III '"'0 " 31 1327 • " 37 1215 7 " 16 1091 -2, 11, 1 • 217 '" m , 12 U 111.11 11 31 1152 • ]0 27 1261 • 12 19 2103 1 '" 117 111. • 17 .. ... B " 37 11"12 11 18 1121 • .. 201419 • 2Z 22 1611 2 207 205 1112 7 111 III ... 7 .. 50 1251

.], 7, 1 7 01 01 '" 10 " 61 1229 ," 54 1337 • " 40 1191 • " 3. U42

1 151 151 ... -3, U, 1 11 .. 61 1150 ," ID 1232 • 77 75 1237 • " li 145., 121 122 ... 1 ]2 JO 1295 12 11 11 1163 7 2Z 22 1920 10 10. 106 1236 10 11 19 l'51

] " 91 1011 2 10 21 1619 -2," 1 • " 75 1144 11 " 52 1261 11 " 16 1415

• 101 103 1013 ] 21 11 1361 1 m m 7" • Il 16 1042 12 11 11 1655 12 11 6 1511

• .. 67 1110 ," JO 1186 2 m l2l m 10 01 " '" lJ 11 2. 1075 -l, " ,• .. Il 1113 • J2 J5 1167 1 77 " 121 -2, 12, 1 '1, 2, 1 1 202 100 '"7 .. 105 1205 • 21 24 1174 • 100 .. '" 1 " 52 121' 1 ". m no 2 U7 lU '"• " 51 1211 -J, li, 1 , 127 l2l '" 2 101 201 lUI 2 201 "7 '" ] 101 117 lt7

• " 23 1619 1 01 U 1071 • 01 " 102. 1 200 191 1170 1 110 "' .01 • .. 62 1001'0 21 19 1629 2 70 70 ... 7 .. 96 lU1 , m 114 1111 • 177 li> '"

," 26 lU'

11 " " '" ] Il 45 1019 • 1J] 136 1115 , 110 131110 • 172 17J 710 • .0 31 130J-],

" 1 • n 11 1291 • Il 47 1211 • .. t3 1212 • 72 " ". 7 " 57 12111 .. 13 1011 -3, 17, 1 10 " 57 1211 • " 21 lUS 7 " 5. 1042 • " 76 12222 111 114 1073 1 " " III 11 •• 31 1217 t .. 47 lDœ • ,.. 111 1095 t Il 71 1252] .. 69 11.' 2 " " OU 12 " 33 1153 10 2Z 21 1006 • Il ID 1212 10 .. U 11la

• m 125 1111 ] 21 25 1052 -2," 1

-2, 13, 1 10 110 111 1215 -l, ., 1, 111 121 11U -2, 0, 1 1 20. lOi m 1 10 li 1225 11 " " 1211 1 1lI m 107

• 100 115 UN 1 .. '0 702 2 " " '" 2 " 59 1237 Il li 71 1017 2 171 17J '"7 " 50 1345 2 Il " ... ] 70 72 ... • n 73 1230 11 20 16 1011 1 " ID 10"

• " .5 U5. 1 172 171 ". • JI 2Z 10" , .0 52 1224 -l, ], 1 • tl !Il 1163

• " .0 1362 • '151 '" '" • " 42 1162 • " ]0 lU' 1 .0' ... '",

" 11 Ul'10 11 JS 1213 ,

~OI 111 nt • ]0 U 1310 7 " 53 UU 2 ". ". '" • 72 72 111411 " 32 114. " ~7 J4 119~ 7 11. n, 1121 • " 26 12]. 1 ... m '21 7 11 JI 1351

-l, t, 1 7 77 7. 1051 • 117 121 1115 • " .. .11 • '" '" 707 • 107 10) 12011 .. '6 1123 • " 32 1211 • ut 116 1201 -2, 14, 1 ,

" .. '" t .. 12 122t, UO n9 1102 • " J4 U22 10 11 35 1343 1 Il Il 1222 • .. 72 .., 10 " JI lUI1 l1l 11" 1112 10 .. 2] 1613 11 .. 1 1121 2 150 Ut nu 7 111 185 1010 11 lO, 16 U7t

• Il Il 1111 11 " II UI2 12 J2 J3 112. ] 111111 "" • '" 156 1111 -1, 10, 1, 111 ln 1112 12 15 • 190] -2, 7, 1 • " ~2 1217 t m lU 11tS 1 lJ1 '" tI2

• '" lU 1112 1J ]0 21 171 • 201 ,.. ... • li 25 nu 10 11 .0 150) ,'" la' 10U

7 .. 100 1200 -2, l, 1 2 li' m 17. • 27 J1 13J. 11 " 51 1204 1 171 n'Ion• " 4t 1211 1 ". ". .OS 1 17. '" ... 7 " Il ... 12 11 11 1100 , .. .. uu

ColLCJ81. are 10Fo lOFe 1000051';1. for In.l;n1tlcant1 ,",0 ,. '" 1 ,",0 ,. al, 1 "'0 r. al, 1 "'0 r. SI. 1 ,",0 r. al, 1 "'0 r. al,

-l, ID, 1 10 '0 n 1294 • .. 17 1222 , '0 " 1125 0 ,.. ,.. '" 1 119 121 ".

• JO " UIO 11 " lZ 1376 10 JO J7 1336 • " " lO'~ 1 II 20 10Z0 2 m '" '"," " nu 12 102 Il 1017 11 " 11 1143 • " " ... 2 .. ., ... , '00 94 1019

• " Il un 0, " , 12 " l' 119. 0, 14, 1 , 111 110 m • '09 109 1013

• tI tI UU , m '" uo.. 0," 1

0 20' 205 1173 • 211 '",., , .. 51 UID

10 '0 2t 1142 2 121 121 ... 1 240 m m 1 '" ut 1112 , 119 124 '" • " 5. 1252

-l, 11. 1 ,"" '" m 2 m 121 '" 2 " 29 11:17 • 41 Ji 1010 ,

" 23 1542

1 m 111 1052 • m m ... , 121 120 m , " 12 1]20 , 51 52 1177 • " 77 121.

2 Il ., 1115 ,'" '" '" • !JI '" ... • JI U 12" • J2 21 U.O, • 51 56 1206, .. 100 IU6 • 12 Il .01 , 12 SI 2036 ,

" ;U 1513 • .. 30 l'" 10 41 J7 1044

• '''' 10' 114' ,'" 160 1003 • 22 19 1592 • JJ JJ 1239 10 22 17 1743 l, ID, 1, " al 1200 • ., 61 1162 , JI JI 1434 , " 7. lO;Z! 11 .. 41 1172 0 " " 220

• " 10. 1201 • " n 1313 • 110 115 120fj • " .. '" 12 " .. 1046 1 .2 " 1016,"' III 117' 10 .. .. 131' • 92 94 1253 0, 15. 1 " " 1

2 JJ JI 1300

• .0 tI 1159 11 63 65 1211 10 " 32 1321 1 66 .. 12" 0 m no '0', 10' 105 ..016

• Il 51 10.5 12 11 17 155. 11 " 111 1510 2 20 19 1611 1 26J ... '" • "0 130 111&

10 " 20 1414 0, 2, 1 0," 1

," 13 2131 2 2U 212 '"

," 102 1177

-l, 12, 1 0 '" '" '" 0 110 119 120 , 26 25 131. , 121 112 7Jl • 11. 120 1217

1 '" l' lU' ,'" '" .01 1 lJ2 m 122 , 16 14 1330 • 1J1 !JI '0'

, " 19 1751, 119 120 lU. , 611 426 117. , '" m "'0 0, 16, 1 , 51 62 91' • JJ 30 1426, ., '1 12JI , nt 226 '",

" " 92. 0 101 110 1113 • " " 1011. • Il 1S UU

• 61 51 122. • '" '" ... • 10' 10. 910 1 " 76 1156 , 161 161 110] 10 " 20 1397, 61 116 1231 , 141 U. '", 119 116 10•• , U 61 1267 • .. " 12.5 l, 11, 1

• 21 20 lUO • .. " '" • 12 7J 1132 , JJ H l:2U • " .. 1100 0 6J 63 '", 10' 112 11112 ," SI 1139 , 16 9 1977 • .. 1019" 10 " " 1537 1 " 77 1085

• 61 70 1121 • .. fi 1132 • " 51 1270 , 11 13 1511 Il " 13 11105 , 60 60 1171

• '" 63 102. • 66 U 12•• • 63 60 1221 • JJ 3D 1029 12 " .. ua3 ," ]7 U3.

10 21 21 ... 10 " 75 1250 10 JI 2' 1361 0, 17, 1 " " 1 • 61 8S 1189

·1, 13, 1 Il '" 37 13U Il Il 5 1953 1 .. .2 1171 0 J11 J20 ... ," 77 1226, .. " 1H7 12 6J 6) 1096 O. " 1 2 .. 59 1056 1 '" '" '" • " 53 1299, m 1J9 1179 0, '. 1 1 112 163 "'

, .. .S 1073 , '00 201 '0', .. 27 IS38, JI J2 U97 , 10' 120 1109 , '" '" 921 • J2 :u lOM , lJ2 12' ". • 29 28 1320

• " " 1216 , lJ6 lJ1 ,.. , 126 120 ... , 20 23 112' • 121 119 '" • U 37 1130," " 1271 , ,., 260 ... • '66 lU 1020 O. li, 1 , .. " '" l, 12, 1

• 61 1) U.1 • lU lU 712 ," 61 lUO 0 " 2. 1211 • '" 151 1035 0 121 119 '", •• fol 119. , 121 129 '" • .. .6 1327 1 50 .. .'" , 51 59 1257 1 " 36 1373

• 29 2. 1222 • 120 l2J '", JO 38 1UO , 21 20 114. • " 96 1232 , .. 60 1223

• •• u 'U , 111 110 10.0 • ,. 20 2U2 " 0, 1 • JI .0 1401 , " 70 1215

·1, 1., 1 • " 76 1170 • 51 !l3 1191 0 '" 26. '''' 10 11 15 l!i1!i13 • ,. 2. 1539

1 102 91 121' • JO 31 1U9 Il 21 27 1011 1 m 369 nl3 Il 29 22 l20t ," 5& 1283,

" 70 1Z75 10 " 56 1317 0, 10, 1 , '01 '00 '" l, " , • JJ 37 130t, .. ta 1321 Il 61 57 1213 " '" 1t1 ". , 152 '" m 0 m m 69J , 61 !li 116.

• " 56 125' 12 " 13 13•• 1 220 211 ... • m m '", 196 lU 716 • 21 22 1229,

" S7 1263 0," 1

,'" 120 1002 , ••• m 122 , m ". m l, 13, 1

• 6J ... 1190 0 520 !l37 2002 , 121 121 1027 • 121 111 ... 1 lJ6 1J7 ". 0 " " .", 12 73 1027 1 '" ". m • Il 11 111' , '" 153 1055 • .. .. ... 1 " 95 1199

• ,. JO ... , ". m '",

" 53 1220 • .. !li 1195 , 219 J11 '",

" 70 12"·1, 15, 1 ,

" " '" • " 5. 1210 • .. .) 1320 • '" " 109S , 2J 23 1'30

1 .. .. 12.' • 221 217 '", 120 120 1209 10 " 37 1.75 , 41 .0 1291 • Il 78 1214, 2t 27 1503 ,

'" '" 160 • " U 1221 11 " 22 1511 • .. 61 1251 ," U 1214, 62 .. lUS • '09 100 ... • 51 51 1110 12 .. 61 1011 • " Il 122. • JI 31 1309

• Il Il lU' , JI 37 123' 10 " 51 10.2 l, l, 1 Il 12 5 1690 ," 211 1329

• " li "'. • '" 173 1144 0, 11, 1 0 120 126 m " " 1 • .. 20 1109

• " 14 lU. • " 15 1250 1 .. '2 1101 , ... 676 1120 0 m .., ". l, U, 1, 2t 26 1060 10 .. Il lU) ," 13 1071 ,

'" 62J 21U 1 '" '" 710 0 126 12J '"·1, 16, 1 11 " 2' lU. , '00 95 1101 ,'" m UJ , ... 221 III 1 lOI 110 1194

1 Il 61 1171 12 J7 31 1119 • 17 22 2161 • m '" '" ," " ." , J7 13 1315,

" " 1207 0," 1

," 2' 1319 , 110 11' '" • " 56 1000 , ., ., 1295, '0 29 1253 1 '01 202 101 • 61 " 12.2 • " " ". ,

'" 93 Ion • 26 J1 1527

• J2 ]] 1250 , '0 26 '", .. U 1399 , 26 291477 • 12 " UU

, .. 60 1110, Il 11 Ul' ,'li 211 '09 • 2t 11 1." • " 25 U71 ,

" .. 1204 • " 55 1099

-l, 17, 1 • Il Il '" • J2 11 1216 • JJ JI 1521 • ., fol un ," 5!i1 1012

1 " .. 1011 , Il te ... " .. 11 150) 10 21 H 1611 • 15 12 19.5 • " 20 1191, 61 12 1001 • "' Il 1000 0, 12, 1 11 2J 2t 1123 10 16 13 1551 1, 15, 1

• li " 1009 , 51 62 111' 0 210 20. 10'1 12 .. 9 l'50 11 " 21 1260 • " 25 1097

• 11 16 1'13 • .. 13 1272 1 lU lU 1102 l," 1 " " 1

, 7J 75 121'

·1, II, 1 • 121 111 1231 ," 11 1152 0 916 1020 120 0 112 111 ''''

, 10' 10) 1172

1 11 li 127t 10 Il 5013" ," 70 1203 1 '" 412 1"" , m '" .n , .. 8' 1153, 12 , 1305 11 " 16 lUI • .. n UOt 2 261 :11:11 "0 , 51 ,. ". • JO 35 1207, 11 12 1"2 12 21 2t lU2 ," 52 123' , 192 1t7 m , .. .. 1013 ,

" 22 133.

0, 0, 1 0, " 1 • .. 51 13]0 • '" '" m • " 5. 1111 • .. .. 1011

1 ,.. 171 n'l • '" m ... , '0 )0 1505 , 220 211 '",

" 51 1111 l, 16, 1, 16 .. .'" , '01 " ... • " .2 1102 • " 15 1501 • " 90 1171 0 .. 12 .09,'01 m ... ,

'" 112 711 O. 13. 1 ," :J5 12" , 122 119 119. 1 50 5fi 1201

• 11 Z4 127. , >U 210 '" 1 " 103 1110 • " 51 12~ • " 11 1266 , 2J 22 1"7, •• " IJI • m 212 ." , ., ,. 121• • JI 3' 1314 • " 6. 1210 , 1J 11 ano• .. J7 tI2 ,

" .. 960 , Il Il 120' 10 2t 26 UU 10 20 19 US) • Il 11 1722, 161 161 ... • u la 1031 • 12 " 12.. 21 " 21 1324 21 " 23 11" • J7 JI l7J

• 26 li 1631 ,'" 124 1101 , .. .1 1271 12 n .5 1051 " " 1

l, 17, 1

• ., •• 125. • " 32 1.5. • 112 1111151 " " 10 nt m 66' 0 Jt 41 m

A40

A41

ColulIlfla a:r. IOFo lOFe: 1OO00S1;. fc.r Inalgn1!Jcanl

"0 re ." 1 "0 re BI, 1 '" Fe ." "0 Fe .,. 1 "0 re '" 1 "0 Fe '"l, 17. 1 0 ... '" ,.. , 71 17 1167 " " 1, .. l6 1332 , 51 50 un,

" SC 1011 1 ,. Il '" • " 21 l1SlI 0 m m .09 ," Cl 112. • " " litS,

" 31 1155 , ll1 122 '",

" 19 1600 1 ln l70 ... • " u un , 11 " 1112, J1 JO ,.. ," " '" • 12 U US, , .. " '"

," J& IU2 1 " " nu.

l, II, 1 • 111 117 10U , J1 10 1111 , 136 lU 1000 • " 26 U1l • " " m• .. .. ... , 6J 71 1159 1 n " '" • " " 1134 ," :Z2 1257 " " 11 JI JI Olt • .. 14 1115 2, U. 1 ,

" 21 1!190 1 16 11 1234 0 101 ,., '"," '0 '"

, ., 93 1223 0 60 " 161 • .. 19 1207 J, 12. 1 1 116 m 1015," " 961 1 JJ 36 1411 1 " " 1352 ,

" 21 1625 0 " Il m ," " 1110

" 0, 1 • " 24 1365 ," " un 1 20 21 lU!! 1 19 3D 1111 ,

" " un0 J1 " 610 10 12 11 1160 , .. n 1265 • " <II 1286 ,

" 70 1253 • 112 lU 11111 " " 1023 U 19 11 1170 • " II 1799 10 " 13 1325 , .. " 11U , Il 9J 12U, 360 '56 '" " " 1

, 20 ;n 1441 " " 1 • " " lUI • " " U4S• " 61 '" 0 'Ol 'Ol 602 • " 39 1005 0 " 19 '",

" " lftS!! ," " 1311, .. 102 .,. 1 128 122 ... 2, 15, 1 1 " " "' • 20 16 1579 1 " 20 \&03• •• 4e 1199 , lU 10. 9JO 1 " 34 125. ,

" 56 1076 , 21 " 1115 • 20 22 11117 " 40 142. ,

" 71 IOU , .. 46 1163 , .. 99 1045 J, 13, 1 " " 1• " :a 1470 • 107 lOI 1052 ," 17 1611 • 165 1H la" 0 .. .. m 0 15 1 15!111• " 53 1305 ,

" Sc 11116 • JO 29 1175 ," 91 1203 1 )J " 1369 1 " .. 1111

10 " 23 alS • ..0 135 1114 , JI 39 1042 • .. '7 126t ," 51 un ,

" " 11112, l, 1 , 71 70 1238 • " "

.,. , .. 67 1237 ," Il lu5 , ,., 10' 1112

0 lJJ 131 ..0 1 " :ZZ 1592 2, 16, 1 • " 21 1..2 • " 117 1230 • '" 120 12001 126 126 ." , J9 31 1211 0 ID ,. 77J 10 " U 1111 , J5 J6 11U ,

" " uu2 265 m '" 2, " , 1 " 70 1069 " " 1 • ,. " on • " " un, 119 112 77J 0 " 101 ... ,

" \9 1561 0 120 m 6l' 3, U, 1 ," " 1412• m m .5O 1 " 53 1013 ,

" 3~ 1103 1 " 55 1071 0 JJ JJ 967 1 " .. 1071, ., " 11113 2 " U 1415 • 50 " ,U 2 " 711 10601 1 " .0 1171 " " 1• .. .. 1202 ," 75 1062 , 51 50 m , .. 3. 13701 2 " 71 109. 0 .. Il ..,

• J6 21 13.' • ltl l!U 10" 2, 17, 1 • '" 231 11U , 51 t9 lUl 1 " .. 1~6]

10 " 19 1711 , .., 15. 1156 0 11 19 1005 , ,.. 171 1179 , 11 19 1271 ," 36 1319

21 " " 1353 • " 56 1261 1 " 33 1013 • 11 11 2061 3, 15, 1 • 12 10 23662, 2, 1 , JI .5 1577 2 " 25 1130 , 60 36 119' 0 20 " 103& ,

" 22 17170 210 '01 .., • 11 19110 , 12 17 16U • ,. 21 1575 1 " " lU5 • 12 U 2323,

'" J50 657 • 2J 20 1299 2, li, 1 10 19 19 115. 2 .. 22 U50 7 " 21 11172 "' 123 '" 10 21 23 1201 0 12 12 .., " " 1

, U " 1036 • 15 11 lnl, m m '" " " 11 " " '" 0 17 .. HO • " " 1171 " " 1• '"

,., 167 0 ,. .. 7Jl " 0, l, 1 21 22 U6' 3, 16, 1 0 " 11 1197, 192 ,.. '" 1 ., .. 1021 0 60 " ,oa , Jl 31 1326 0 60 .. nt 1 " " 111.

• al 51 1120 2 22 2& 1524 1 111 111 m ," 25 lU7 1 .. 30 1100 ,

" 67 12437 110 114 1151 ,

" 35 1312 2 lJJ 12' 1:32 • JI 27 U51 2 19 21 1110 ," 5& 1333

• 2. 25 1647 • .. 95 1137 , m 126 ou , ,.. 153 11U 3, 17, 1 • al 65 124910 " 23 1310 , lU 141 1110 ,

" 69 1151 • .. 90 1239 0 " " m ," 56 125.

11 21 19 12&& • .. 62 1241 • " 37 1461 ," 31 1307 1 2J 20 ... • " U 1207

2. " , , 21 25 1174 ," &5 1269 1 " 21 1311 " 0, 1 ,

" n 11620 ". m .65 • ID 31 12.4 ,

" U 1110 • " " ".. 0 lU III '06 • ,. 25 10561 ,.,

'".., 10 ,. 19 1176 10 " 21 1069 " " , 1 .. J9 1193 " " 12 '" m '" 2, 10, 1 " l, 1 0 " " '"

," ., un 0 " " ••0, 136 131 119 0 " .. 110 0 111 115 '" 1 " 55 1171 ," 75 1135 1 " " 135,J

• 2Jl ". III 1 ., 15 1157 1 109 112 .,. , .. 46 1201 • II &0 un ," " 1511, ., •• 1001 , 60 60 116' , 111 12' ... , •• U 1219 , 11 23 2313 ," 24 lin

• ,., 160 1077 ," 37 Ul2 , 120 111 ." • 60 51 12,J3 • 112 112 1117 • 96 98 1200, •• 66 1217 • .. 36 1331 • " 69 1073 ,

" 96 1220 , .. U 1330 , 15 ')3 n64

• 50 &1 1322 • " 72 12.. ," 90 1111 • 21 16 1715 , JO 29 1065 • " 12 1127,

" 33 13.3 , .. 31 1225 • " &5 1333 ," Uo 1725 " l, , , .. U 10n

11 " 29 1126 1 J9 36 1229 ," 21 1591 • 31 29 1231 0 " " '" • " 25 1000

2, " , ," 21 1211 • " 35 U11 ,

" " 1)" 1 " 70 104' " ., 1

• ID .,"0 2, 11, 1 • " 21 1516 " " 1

, .. 31 1211 0 II 35 10711 '" ... m 0 .. .. III " " 1

0 " " ..' ," 52 1111 1 100 105 1202

2 15 Il .Ol 1 " " 1140 0 " " .11 1 ., 66 1116 • .. 13 1110 : •• 73 125., lJO m ." , 120 111 1146 1 15 22 1631 ," 56 1244 ,

" 7012117 ," 52 1266

• .. .. .u , 111 123 1169 , 160 15' ,.. , 101 107 1160 • " 301U7 • ,. 61 1205, lU lU 1009 • .. 104 1225 ," .. ,.. • 50 57 1214 ,

" 23 15M ," 36 1216

• ,. 37 1322 , .. Il 1210 • lOI 10& 10n , .. 31 13" • 20 21 U30 • Il ,JI 1115, ,. 76 1205 • 11 11 1.21 ," 11 1512 • 21 29 1&12 • JO 3'J 1051 ,

" u un

• 26 31 1557 , .. 30 1401 • 60 62 1249 ," 22 1311 " " , 4, 10, 1

11 11 14 U21 • U 39 10M , 59 57 1302 • 16 l' 1531 0 '" 119 m 0 " JO 10152,

" 1 • " 20 1102 1 •• U 1375 • 10 10 1787 1 " 72 1017 1 " " 13700 ". 250 m 2, 12, 1 ,

" 23 141& 3, 10, 1 ," 61 1251 , Il 15 "01,

" 31 1151 0 U " 0J3 10 11 11 1"4 0 161 '" .., ," 11 1\12 , 56 " 1244

2 .., '" m 1 " " 11'5 " " , 1 Il 63 1245 • •• Il UN • .. .. 11'], •• .. '", 62 6t 1279 0 m 112 ,.. , ., 51 1220 , JI 37 13.1 , U •• 1158

• ID .. ,.. i " 12 12&2 1 " " ... , 59 51 12.)4 • " n 1421 • " 2' 1115, 115 120 1053 • 37 .0 1321 2 97 .. '" • .. 66 1231 , 12 15 2310 , n 15 112&

• " 76 1153 , 11 19 2001 ," .. 1000 , l6 19 2199 • 11 14 1754 '. 11, 1,

" 57 1276 , 50 46 1101 • '" 17' 10&2 • .. 15 2031 • " 11 1155 0 " l& 1054

• " 27 1421 • 10 1 1760 ," U 11&' ,

" 51 1015 " " 11 51 !li2 12.3

• 11 13 1917 2, U, 1 • " 77 1199 • 21 25 lOS! 0 21 " ... , .. &1 1325,. 11 13 1475 0 " " on ," 31 1410 J, 11, 1 1 .. U 12:10 ,

" •• lut11 19 17 1230 1 51 55 1279 • " 37 1&70 0 " " 915 2 li 16 2113 • " 4" 12"

'. " , , .2 '0 1225 10 11 1& lUI 1 12 Il 1201 ," " 1171

," 32 1217

A42

C'c lulllt\. Ar. lOFo lOFe lOOOOSlg, • 'or III.l;nUlcant'''0 re "0 1 ,",0 re "0 1 "0 re "0 1 "0 '< "0 1 ", re "0 1 "0 re SIOC, n. • 17 20 U29 0 .. .. '" 1 •• 10 1092 0 Il Il '" 0 .. " '"• 32 " Ion 1 107 lOi 1110 1 " J7 132. • " t6 1060 1 19 57 n9~ 1 " " 10U4, 12. 1 , 61 60 127] 2 7J 7J 1230 5 ,. ]] 1065 2 " " lU6 2 " 17 12&)

0 •• ., 119 • .. 56 lUi! ," 59 1246 5. 10. 1 ,

" " 1100 , 19 " 9511 51 51 UJ! 5 50 '" 1119 • " J) 1336 0 " " 0J7 • " " nu " " 12 " " 11.. • " U lU6 • ,. 2. 1164 1 " fol lUI '. '. 1 0 " " 1021, 32 " UO" 7 Il 11 un J " " m J 19 39 10~J 0 " " "" 1 " " 1201

• Il " ".. 5. 2. 1 5, '. 1 • " 41 10U 1 lO 32 1400 2 ",. un

" 13. 1 0 10' 10' 1143 • " " ... 5. 11. 1 2 JJ JO UC. 1 17 15 1195

• JJ " '" 1 " 57 1434 1 .. .. 1329 0 " " '",

" 57 lU" '. •• 11 19 " 1203 2 " " 1350 2 " 57 1502 1 " 37 1:Il7 • " 10 1311 • ,. ,. 7912 11 15 1895 ,

" " llfot • ,. ,. lllt 2 " U Ul1 5 20 11 1096 1 ,. Il 1'~", 5. " 1029 5 JJ " 1290 5 " U 1167 ," 39 1009 '. " 1 2 " 10 lUt

• 20 " '290 • " " 1582 • 10 1020n • " " ." 0 7J 71 0J7 6, 10, 1". 101. 1 7 12 17 1971 5. 7. 1 5, 12. 1 1 lO JJ 1345 0 JO " lU

0 65 .. 722 5. '. 1 0 76 75 m 0 32 32 m 2 52 " 1162 1 ,. • 16'71 "

,. 1185 0 59 •• "' 1 .. .. 1241 1 ,. 29 1127 , ,. " 11" 7. 0, 12 " " 1013 1 " 35 UO, 2 " " 12'0 2 21 25 1020 • " JI 912 0 " 52 ..,, 20 " 1259 2 JI 37 1461 ,

" .. 1192 5, n • .1 5 " 19 1011 1 " 10 110't. 15, 1 , 57 41 1420 • " 57 1265 0 lO JO 71. '. 5. 1 7. 1. 1

0 20 " III • " 52 1265 5 11 17 1713 1 " " '" 0 77 70 10. 0 " 22 on1 " .. .00 5 22 21 1576 • " JI 1013 '. O. 1 1 " " 1113 7. 2. 12 " " '" • " 28 1453 5. 8, J 0 1:1 1) 1920 2 10 17 16St 0 59 " ..,

5, O. 1 5. '. 1 0 li 12 1756 2 16 11 1853 , 21 ,. 125' 1 " 57 •••0 6J 61 .., 0 " .. ... 1 " " 10159 , li 15 2102 • 12 11 ~!l6. 7. ,. 1

1 " li 1630 1 " JO 1706 2 67 " 1185 • " 26 1238 '. '. 1 0 .. " 0", 10 19 1964 , GO 59 1271 1 Il JI 12!i14 '. 1. 1 0 " li 105. 1 61 " 126

3 16 11 2063 , li 18 1563 5 22 21 1218 0 15 3J 10ClO 1 " " 1170 7. '. 1• 10 11 1&92 • 15 12 1921 • 10 • 1645 1 .. 6) 11(,4 2 .. .7 1045 • " " 7595 11 4 2230 • 11 8 21119 5. 0, 1 3 " 15 2039 ,

" " ... 1 31 '0 9177 Il 27 1070 7 11 12 1724 0 29 J2 1020 • '0 17 1512 • " 12 1331 7. 5. 1

5. '. 1 5. 5. 1 , .. .. 1149 O• 2. 1 " 7. 1 0 " .. '"

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A44

•IH 300 MHz 2D NOE speetra of t-Boc-Val-Leu-OH at 296K in [2HslDMSO.

OV3UH23. S"lC D.TE~ 5-9-0

Q tOI 0 f" '.0

• 0 1.5~ '"• D 2.0

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ILO "'-,5 1.0 6.5 6.0 5.5 S.O 4.S '.0 3.5 3.0 2.5 2.D 1.5 1.0pp,

A4S

•IH 300 MHz 2D NOE spectra of t-Boc-Val-'II[CH2-Oj-Leu-OH at 296K in[2H6 jDMSO

A46

8§JOaINOI'lS.SJ'lXAU PROC:

NOESY • .lUDATE 4~9-D

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1

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HIDC VAL PSI CH2D LEV OH

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