chemical bioiogy – peptides · occurrence and functions of peptides in nature and every day life...

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Chemical BioIogy – Peptides Prof. Helma Wennemers H 2 N N H O OCH 3 O HO 2 C 1 Prof. H. Wennemers

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Page 1: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

Chemical BioIogy – Peptides

Prof. Helma Wennemers

H2N NH

OOCH3

OHO2C

1 Prof. H. Wennemers

Page 2: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

Oxytocin

NH

OO

HN

CH3CH3

NHO

H2NS S

NH2

O

NH

NH

NO

OO

NH2

O

NH

OHN

H2NO

O

OH

Dendrotoxin

NH

HN

NH

HN CO2

-+H3N

HO

O

O

O

O

Enkephalines

NH

N

O

O OH

NH2

CO2H

Lisinopril H2N NH

OOCH3

OHO2C

Aspartam

2

Prof. H. Wennemers

Page 3: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

The 20 most common proteinogenic amino acids

H2N CO2H H2N CO2H

CH3

H2N CO2H H2N CO2H

H2N CO2H

NH3

H2N CO2H

HN

NH2

NH2

H2N CO2H

N

NH

NH

CO2HH2N CO2H

OH

H2N CO2H

OH

H2N CO2H

CO2

H2N CO2H

CO2

H2N CO2H

CONH2

H2N CO2H

CONH2

H2N CO2H

SH

H2N CO2H

HN

H2N CO2H

OH

Large Diversity

Glycin (Gly, G)

Alanin (Ala, A)

Valin (Val, V)

Isoleucin (Ile, I)

Leucin (Leu, L)

Phenylalanin (Phe, F)

Tyrosin (Tyr, Y)

Tryptophan (Trp, W)

Histidin (His, H)

Lysin (Lys, K)

Arginin (Arg, R)

Aspartat (Asp, D)

Glutamat (Glu, E)

Serin (Ser, S)

Threonin (Thr, T)

Asparagin (Asn, N)

Glutamin (Gln, Q)

Cystein (Cys, C)

Methionin (Met, M)

Prolin (Pro, P)

H2N CO2H

S

H2N CO2H H2N CO2H

Prof. H. Wennemers 3

Page 4: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

Diversity of Peptides – A Number Game

Random combinations of 20 amino acids

3-mer: 203 = 8000 different peptides

5-mer: 205 = 3.2x106 different peptides

10-mer: 2010 = 1.0x1013 different peptides

50-mer: 2050 = 1.1x1065 different peptides

Weight of 1.1x1065 single 50-mer peptide molecules: 1.3x1039 kg

Weight of the earth: 5.9x1024 kg

4 Prof. H. Wennemers

Page 5: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

1. Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide Synthesis a) Aspartam: Properties of amino acids; nomenclature; solution phase synthesis b) Glucagon: Solid phase peptide synthesis c) Fuzeon, an anti HIV drug: Solution and solid phase peptide synthesis 3. Peptide Structures – Nanostructured Materials – Self-assembly a) Fuzeon: α-helices and coiled-coil structures b) Amyloids: β-sheets c) Collagen: PPII helices 4. Applications of Peptides in Chemistry, Biology and Material Sciences a) Peptide-based materials b) Therapeutically active peptides b) Cyclic peptides and cancer imaging c) Asymmetric Catalysis with Peptides – Combinatorial chemistry

Syllabus

5 Prof. H. Wennemers

Page 6: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

1. Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide Synthesis a) Aspartam: Properties of amino acids; nomenclature; solution phase synthesis b) Glucagon: Solid phase peptide synthesis c) Fuzeon, an anti HIV drug: Solution and solid phase peptide synthesis 3. Peptide Structures – Nanostructured Materials – Self-assembly a) Fuzeon: α-helices and coiled-coil structures b) Amyloids: β-sheets c) Collagen: PPII helices 4. Applications of Peptides in Chemistry, Biology and Material Sciences a) Peptide-based materials b) Therapeutically active peptides b) Cyclic peptides and cancer imaging c) Asymmetric Catalysis with Peptides – Combinatorial chemistry

Syllabus

6 Prof. H. Wennemers

Page 7: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

Gastrin = H-Glp-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2

Die Entdeckung von Aspartam - Serendipity in Science

Hormon, das im Magen durch G-Zellen freigesetzt wird und die Bildung von Magensäure anregt. • für physiologische Aktivität nur die vier C-terminalen Aminosäuren notwendig

H-Trp-Met-Asp-Phe-NH2 1965 - James M. Schlatter (G. D. Searle & Company, heute: Monsanto) sucht nach einem Wirkstoff gegen Magengeschwüre und stellt H-Asp-Phe-OCH3 her ....und tut etwas, was man nicht tun sollte...... 7 Prof. H. Wennemers

Page 8: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

BACHEM Holding AG │ Company Presentation Seite 8 │ September 2012

Solution Phase: Calcitonin (salmon)

CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP-NH2

1 - 6 7 - 10 11 - 16 17 - 23 24 - 32

slide by Dr. Thomas Früh, CEO Bachem

Page 9: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

Calcitonin

From: N. Sewald, H.-D. Jakubke, “Peptides: Chemistry and Biology”, 2nd Ed., Wiley-VCH, Weinheim, 2009     9  

„Adversary“ of calcitonin: Parathyroid Hormon (PTH)

Page 10: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

1. Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide Synthesis a) Aspartam: Properties of amino acids; nomenclature; solution phase synthesis b) Glucagon: Solid phase peptide synthesis c) Fuzeon, an anti HIV drug: Solution and solid phase peptide synthesis 3. Peptide Structures – Nanostructured Materials – Self-assembly a) Fuzeon: α-helices and coiled-coil structures b) Amyloids: β-sheets c) Collagen: PPII helices 4. Applications of Peptides in Chemistry, Biology and Material Sciences a) Peptide-based materials b) Therapeutically active peptides b) Cyclic peptides and cancer imaging c) Asymmetric Catalysis with Peptides – Combinatorial chemistry

Syllabus

10 Prof. H. Wennemers

Page 11: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

Solid Phase Peptide Synthesis (SPPS) (Nobelprize for Chemistry 1984: Bruce Merrifield)

Peptidsynthesizer 1966

11 Prof. H. Wennemers

Page 12: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

Solid Phase Peptide Synthesis (SPPS) – Fmoc/tBu Strategie in the example: Wang Linker

O

ClR1

CO2FmocHN Cs LinkerOFmocHNO

R1

LinkerOH2NO

R1

LinkerONH O

R1OFmocHN

R2

DMF

Piperidine:DMF 1:4 (1x 3 min, 1x 7 min)

R2

CO2HFmocHN3 equiv.

3 eq. PyBOP, 3 eq. iPr2NEt

repeat Fmoc deprotection/AA couplings to desired peptide length

LinkerONH O

R1OHN

R2OH2N

RnOHN

H O

R1´OHN

R2´OH3N

Rn´95% TFA

(scavengers)

side-chain deprotected peptide

Wang Linker

12 Prof. H. Wennemers

Page 13: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

Kupplungsreagenzien

N C NR

R

Carbodiimide R = R´ = cyclohexyl: N,N´-Di-Cyclohexyl Carbodiimid (DCC) R = R´ = iso-propyl: N,N´-Di-Isopropyl Carbodiimid (DIC) R = Et, R´= CH2CH2CH2NMe2 : N-Ethyl-N´-(3-Dimethylaminopropyl) Carbodiimid (EDC)

Phosphonium Reagenzien Guanidinium/Uronium Reagenzien

NN

NO P

N

NN

PF6

NN

NO P

NMe2

NMe2NMe2

PF6

NN

N

OO

P OEt

O

OEt

NN

N PF6

NN

ON N

NN PF6

NN

O

NN

N BF4

NN

O

HBTU HATU

TBTU

BOP (Castro`s Reagent)

PyBOP

DEPBT

13 Prof. H. Wennemers

Page 14: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

Commonly used acid sensitive protecting groups for the functional groups of the amino acid side chains

Fmoc: α-NH group Boc: Lysine, Tryptophan, Histidine Pbf: Arginine Trt: Cysteine, Aspartic acid, Glutamic acid Acm: Cysteine tBu: Serine, Threonine, Hydroxyproline, Tyrosine

Prof. H. Wennemers 14

Page 15: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

Linker für die Festphasensynthese

O

OR

O

O

OR

O OMe

O

Ph

R

O

Cl

O

HN

R

OMeO

MeO

NH

OOR

O

NO2

O

O

R

O

HN

O

S NH

O

NH

R

O OO

Wang SASRIN® Chlorotrityl

Rink Amide Hycram

o-Nitrobenzyl Kenner Safety Catch

15 Prof. H. Wennemers

Page 16: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

Peptidsynthese an fester Phase (Nobelpreis für Chemie 1984 für Bruce Merrifield)

Peptidsynthesizer 1966 heute

16 Prof. H. Wennemers

Page 17: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

BACHEM Holding AG │ Company Presentation Seite 17 │ September 2012

Solid Phase: Glucagon (1-29, human)

HSQGTFTSDYSKYLDSRRAQDFVQWLMNT

slide by Dr. Thomas Früh, CEO Bachem

Page 18: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

BACHEM Holding AG │ Company Presentation Seite 18 │ September 2012

Insulin    (Insulin  Superfamily)  inac+ve  hexameric  form  –  bound  to  Zn2+:  

Insulin and Glucagon

Glucagon    (Secre3n  Family)  

18  

Page 19: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

BACHEM Holding AG │ Company Presentation Seite 19 │ September 2012

Comparison: Solution vs. Solid Phase

Calcitonin Glucagon -  Produced since 1986 1996 -  Synthetic steps 104 56 -  Purification CCD

two-dim. HPLC three-dim. HPLC -  Derivatives needed 158 kg 29 kg

for the production of 1kg -  Time for completion 2.5 years 0.6 years

Page 20: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

Human  Parathyroid Hormone (1-84)

Parathyroid Hormone (PTH)    

T.  Michels,  R.  Dölling,  U.  Haberkorn,  W.  Mier,  Org.  Le(.,  2012  ,  14,  5218-­‐5221.  R.  Balling,  R.  G.  Erben,  Nature  Medicine,  2000,  6,  860-­‐861.  

PTH  fragment  (46-­‐84):    AGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ-­‐NH2  

addition of 5% formic acid to the piperidine

20  

Page 21: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

Aspartimid Formation

D.  Housset,  C.  Habersetzer-­‐Rochat,  J.-­‐P.  As3er,  J.  C.  Fontecilla-­‐Camps,  J.  Mol.  Biol.,  1994,  238,  88–103.M.  Mergler,  F.  Dick,  B.  Sax,  P.  Weiler,  T.  Vorherr,  J.  Pep>de  Sci.  2003,  9,  36–46.    T.  Michels,  R.  Dölling,  U.  Haberkorn,  W.  Mier,  Org.  Le(.,  2012  ,  14,  5218-­‐5221.      

R NH

HN

NH

O

O

O

O

R1

R2

OtBu

O

R NH

NNH

O

O

OO

R1

R2

O

R NH

HN

NH

O

O

O

O

R1

R2

OH

O

R NH

HN

NH

O

O

O

O

R1

R2

NO

HN

NHO

O

O

R1

R2

HN

O

R

OHO

HN

NHO

O

O

R1

R2

HN

O

R

NO

Acid or Base(epimerisation)

Hydrolysis(epimerisation)

20% Piperidine/DMF(epimerisation)

α-PeptidePiperidide of α-Peptide

21  

Page 22: Chemical BioIogy – Peptides · Occurrence and Functions of Peptides in Nature and Every Day Life …hormones, neurotransmitters, therapeutics, artificial sweetener, … 2. Peptide

Side-reactions of Met

Y. S. Kim, J. A. Moss, K. D. Janda, J. Org. Chem. 2004, 69, 7776-7778.D. Picone et al, ChemBioChem 2006, 7, 257 – 267.T. Lührs, C. Ritter, M. Adrian, D. Riek-Loher, B. Bohrmann, H. Döbeli, D. Schubert, R. Riek, PNAS 2005, 102, 48, 17342–17347.  

Aqueous Solution Structure of the Alzheimer's Disease Aβ Peptide 3D Structure of Alzheimer's Aβ(1-42) fibrils

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