Dinuclear nickel complexes modeling the structureand function of the acetyl CoA synthase active siteMikinao Ito, Mai Kotera, Tsuyoshi Matsumoto1, and Kazuyuki Tatsumi1

Department of Chemistry, Graduate School of Science, and Research Center for Materials Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya464-8602, Japan

Edited by Robert Huber, Max Planck Institute for Biochemistry, Martinsried, Germany, and approved May 29, 2009 (received for review January 15, 2009)

A dinuclear nickel complex with methyl and thiolate ligands,Ni(dadtEt)Ni(Me)(SDmp) (2), has been synthesized as a dinuclearNid–Nip-site model of acetyl-CoA synthase (ACS) (dadtEt is N,N�-diethyl-3,7-diazanonane-1,9-dithiolate; Dmp is 2,6-dimesitylphe-nyl). Complex 2 was prepared via 2 methods: (i) ligand substitutionof a dinuclear Ni(II)–Ni(II) cation complex [Ni(dadtEt) Ni(tmtu)2] (OTf)2(1) with MeMgBr and KSDmp (tmtu is tetramethylthiourea), (ii)methyl transfer from methylcobaloxime Co(dmgBF2)2(Me)(Py) (5)to a Ni(II)–Ni(0) complex such as [Ni(dadtEt)Ni(cod)] (3), generatedin situ from Ni(dadtEt) and Ni(cod)2, followed by addition of KSDmp(cod is 1,5-cyclooctadiene; dmgBF2 is difluoroboryl-dimethylglyoxi-mate). Method ii models the formation of Nip–Me species proposedas a plausible intermediate in ACS catalysis. The reaction of 2 withexcess CO affords the acetylthioester CH3C(O)SDmp (8) with con-comitant formation of Ni(dadtEt)Ni(CO)2 (9) and Ni(CO)4 plusNi(dadtEt). When complex 2 is treated with 1 equiv of CO in thepresence of excess 1,5-cyclooctadiene, the formation of 9 andNi(CO)4 is considerably suppressed, and instead the dinuclearNi(II)–Ni(0) complex is generated in situ, which further affords 2upon successive treatment with Co(dmgBF2)2(Me)(Py) (5) and KS-Dmp. These results suggest that (i) ACS catalysis could include theNid(II)–Nip(0) state as the active species, (ii) The Nid(II)–Nip(0) speciescould first react with methylcobalamin to afford Nid(II)–Nip(II)–Me,and (iii) CO insertion into the Nip–Me bond and the successivereductive elimination of acetyl-CoA occurs immediately when CoAis coordinated to the Nip site to form the active Nid(II)–Nip(0)species.

acetylthioester formation � dinuclear nickel-site model � N2S2ligand � nickel acyl complex

Acetyl-CoA synthase/CO dehydrogenase (ACS/CODH), abifunctional metalloenzyme found in acetogenic, methano-

genic, and sulfate-reducing bacteria, has the important functionof fixing CO and CO2 in the global carbon cycle (1–3). WhereasCODH catalyzes the reversible conversion of CO2 to CO, ACSassembles acetyl-CoA from CO, CoA, and a methyl moiety thatis derived from the methylcobalamin of the corrinoid iron–sulfurprotein (CFeSP). Recently, the crystal structures of the ACS/CODH system from the bacteria Moorella thermoacetica andCarboxydothermus hydrogenoformans have been elucidated (4–6); the ACS active site denoted as the A-cluster contains a[Fe4S4] cubane cluster and a Nid–Nip dinuclear site as shown inFig. 1, where the 2 nickels designated as Nid and Nip occupy distaland proximal positions, respectively, to the [Fe4S4] cluster. Thegeometry around Nid is square planar, composed of 2 cysteinesulfurs and 2 carboxyamide nitrogens of the tripeptide Cys-Gly-Cys from the protein backbone. Nip carries an unidentifiedligand X and 3 bridging cysteine sulfurs, 2 from the aforemen-tioned tripeptide, and 1 from the [Fe4S4] cluster.

Biological investigations suggest that the oxidized state of thecluster Aox should be formulated as {Nid2�–Nip2�–[Fe4S4]2�}(7–9). However, the electronic configuration of the active re-duced state, probably reduced by 2 electrons from Aox, which isproposed to be {Nid2�–Nip1�–[Fe4S4]1�}, has not yet been firmlyestablished (10–11). The order of the addition of the substrates,

Me, CO, and CoA, to the active site is also controversial, andseveral mechanisms have been proposed on the basis of biolog-ical and computational studies (12–14). On the other hand,synthetic studies that probe the reaction mechanisms are limitedin number, although various thiolato-bridged dinuclear nickelcomplexes have been reported as A-cluster models (15–23).

Proposed ACS mechanisms commonly involve the followingsteps: (i) methyl transfer from methylcobalamin to the reducedNip(0) [or Nip(I)] site, (ii) CO insertion into the Nip–Me bond,and (iii) formation of acetyl-CoA via the reductive eliminationat the Nip site (1–3, 7–14). These reactions had been studied byusing mononuclear nickel complexes. CO insertion into theNi–Me bonds of Ni(NS3

R)Me [NS3R is N(CH2CH2SR)3; R is i-Pr,

t-Bu], investigated by Stavropoulos et al. (24) , resulted in theformation of acetylthioesters CH3C(O)SR and Ni(0) species inhigh yield upon treatment with CO. Likewise, the reaction of[Ni(bpy)(SR)(Me)] with �3 equiv of CO was found to yield[Ni(bpy)(CO)2] and acetylthioesters (25). Before these reports,Kim et al. (26) had shown that the reaction of Ni(dppe-)(SAr)(Me) with CO generated CH3C(O)SAr. Recently, Ramp-ersad et al. (27) demonstrated the acetylthioester formation inthe reaction of Ni–Pd dinuclear complex, Ni(bme-daco)Pd(CO-)(COMe), with NaSMe. Eckert et al. (28) reported the methyl-transfer reaction from methylcobaloxime to (triphos)Ni0(PPh3)to gave [(triphos)NiMe]2�. They have also reported that thedinuclear nickel methyl complex [Ni(phma)Ni(Me)(dppe)]� re-acts with CO to give [Ni(phma-CO-Me)]� and (dppe)Ni(CO)2,in which acetylation occurs at the thiolato sulfur of the phmaligand (29).

We report herein ACS model studies using a series of thiolato-bridged dinuclear nickel complexes in the Ni(II)–Ni(II) andNi(II)–Ni(0) states, including a key Ni(II)–Ni(II) dinuclearcomplex carrying a methyl and a thiolate, Ni(dadtEt)Ni(Me)(S-

Author contributions: T.M. and K.T. designed research; M.I. and M.K. performed research;M.I. and M.K. contributed new reagents/analytic tools; M.I., M.K., and T.M. analyzed data;and T.M. and K.T. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates have been deposited with the Cambridge Crys-tallographic Data Centre, Cambridge CB2 1EK, U.K. (CSD accession nos. 716127–716134).

1To whom correspondence may be addressed. E-mail: i45100a@nucc.cc.nagoya-u.ac.jp ortmatsu@chem.nagoya-u.ac.jp.

This article contains supporting information online at www.pnas.org/cgi/content/full/0900433106/DCSupplemental.

SN

SNNid

Nip

XS

CysS

Fe

S

FeFe

SFe

S S

S

S Cys

Cys

Cys

O

O

O Cys

Cys

Gly

Fig. 1. Schematic view of the ACS active site.

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Dmp) (2). Although the diaminedithiolate dadtEt ligand appliedto this model study would have different electronic propertiesfrom the diamidodithiolate Cys-Gly-Cys ligand of A-cluster inACS, the results herein would provide a clue to the reactionmechanism for the ACS catalysis.

Results and DiscussionDinuclear Nickel Complex Having Methyl and Thiolato Ligands.We have recently reported the synthesis of a series of thiolato-bridged dinuclear nickel complexes such as Ni(dadtEt)Ni(SAr)2and [Ni(dadtEt)Ni(L)2]2� (L is tmtu, tBuNC; tmtu is tetramethy-thiourea) (30, 31). During these studies, we found that thetmtu-substituted dinuclear complex [Ni(dadtEt)Ni(tmtu)2]-(OTf)2 (1) reacted with MeMgCl and KSDmp stepwise inTHF at �60 °C to afford the dinuclear nickel complex[Ni(dadtEt)Ni(Me)(SDmp)] (2) carrying a methyl and a thiolatein 76% yield as a reddish-purple powder (Scheme 1). Complex2 is an intriguing entity modeling the dinuclear nickel site ofthe A-cluster. The Me and SDmp moieties closely resemble thesubstrates of the ACS mediating reaction, a methyl group andCoA, respectively. The 1H NMR spectrum of 2 shows a singletsignal at �0.66 ppm that is attributable to the Ni–Me group(25, 26, 29).

The molecular structure of 2 was confirmed by X-ray crystal-lography. Complex 2 crystallized with 2 crystallographicallyindependent molecules, 2A and 2B, and Fig. 2 shows theirstructures. The metric parameters of 2A and 2B are mostlysimilar. One of the structural differences between 2A and 2Bderives from the geometry of dadtEt ligand; the conformation ofthe Ni(1)-N(2)-C(6)-C(7)-S(2) ring and the orientation of 1 ethylgroup differ between 2A and 2B. The other intriguing structuraldisparity is the folding of the 2 Ni-coordination planes along the�S–�S vector, where the dihedral angle defined by the 2Ni(�-S)2 planes is 119.4° for 2A and 103.5° for 2B. As a result, theNi–Ni distance of 2A [2.9029(3) Å] is markedly longer than thatof 2B [2.6935(3) Å]. This observation points to a soft nature ofthe potential energy curve for the folding motion. The Ni–Mebond lengths of 2A and 2B are comparable to that of [Ni(ph-ma)Ni(Me)(dppe)]� [1.966(2) Å] (28). Because of the strongtrans influence of the methyl group, the Ni(2)–S(1) distances areelongated by 0.10 and 0.12 Å compared with the Ni(2)–S(2)distances.

The bond lengths and angles of 2A and 2B also compared wellwith the relevant structural parameters reported for the A-clus-ters of ACSMt (from Moorella thermoacetica) and ACSCh (fromCarboxydothermus hydrogenformans) (5, 6), as shown schemati-cally in Fig. 3. Although the Cys-Gly-Cys tripeptide of theA-cluster must have different geometrical/electronic propertiesfrom the dadtEt model ligand, the core structures of 2A (and 2B)and the A-cluster are very much alike. Like 2A, B, the Ni(2)-�Sbonds are longer than the Ni(1)–�S bonds, except for Ni(2)–S(2)of ACSMt,, which is trans to the Ni(2)–S(3) bond. The dihedralangle between the Ni(�-S)2 planes is somewhat larger for theA-clusters. Of note are the elongated Ni(2)–S(1) distances

observed for both 2A, B, and the A-clusters, compared with theNi(2)–S(2) lengths. This elongation indicates that the unidenti-fied ligand X of ACS has a strong trans influence, which may beas strong as a Me group.

Synthesis of 2 via a Nid(II)–Nip(0) Complex. A plausible mechanismof ACS catalysis includes the Nid(II)–Nip(0) state of the A-clus-ter as an active, reduced form (1, 3, 9). The formation of‘‘[Ni(S2N2�)Ni(cod)]2�’’ in situ from [Ni(S2N2�)]2� and Ni(cod)2[S2N2� is N,N�-ethylenebis(3-mercaptopropionamide)] has beenproposed by Rauchfuss and coworkers (16), and here we reportthe synthesis of a similar Nid(II)-Nip(0) model by the reaction ofNi(dadtEt) and Ni(cod)2. When an acetonitrile solution ofNi(dadtEt) was added to a toluene solution of Ni(cod)2 at�20 °C, the solution immediately turned dark purple. Althoughwe have not yet isolated this species because of its thermalinstability, the formation of the Ni(II)–Ni(0) complex such asNi(dadtEt)Ni(cod) (3) was strongly indicated by a trappingreaction with fumaronitrile, affording Ni(dadtEt)Ni(�2-(E)-NCCH�CHCN) (4) in 77% yield (Scheme 2). The molecularstructure of 4 was determined by X-ray crystallography [seesupporting information (SI) Text]. To get further insights intothe Ni(II)–Ni(0) complex formed in situ, we examined thereaction at low temperature and monitored by 1H-NMR. Be-cause the dissociation of 1,5-cyclooctadiene and the consump-tion of Ni(dadtEt) and Ni(cod)2 were confirmed, the formationof the Ni(II)–Ni(0) complex is indicated. However, the signalsexcept for those of the dissociated 1,5-cyclooctadiene are con-siderably broad, and therefore the Ni(II)–Ni(0) complex has not

21

tmtutmtu

NiSNSN

Ni1) MeMgCl2) KSDmp

SDmpMe

NiSNSN

Ni

[OTf]2

Dmp =

Scheme 1.

Ni(1)

S(2)

S(1)

Ni(2)

S(3)

C(12)

Ni(1) S(1)

S(2)

S(3)

Ni(2)

C(12)

2A

2B

N(1)

N(2)

N(2)

N(1)

C(6)

C(7)

C(6)

C(7)

Fig. 2. ORTEP drawing of Ni(dadtEt)Ni(Me)(SDmp) (2A, 2B). Thermal ellip-soids are shown at 50% probability.

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yet completely characterized. The broadness of the signals maysuggest the fractional cod coordination to the Ni(0) site.

Modeling the methyl-transfer step from methylcobalamin tothe Nip site, proposed for the ACS catalytic mechanism, thereaction of the Ni(II)–Ni(0) complex with methylcobaloximeCo(dmgBF2)2(Me)(Py) (5) was examined (28). Because of thethermal instability, the reaction of the Ni(II)–Ni(0) complex and5 was conducted at �30 °C, although the reaction was rathersluggish at this temperature. After stirring the solution contain-ing the Ni(II)–Ni(0) complex and 5 for 37 h, addition of KSDmpand workup gave 2 in 60% yield (Scheme 2). This result indicatesthat ACS catalysis could include the Nid(II)–Nip(0) state as anactive state, reacting with methylcobalamin to afford the Nid(I-I)–Nip(II)–Me species.

Methylation of Ni(0) can also be achieved by oxidative addi-tion of MeI to the Ni(II)–Ni(0) complex, affording complex 2upon successive treatment with KSDmp (Scheme 3). However,this route produces an inferior yield of �20%, probably becausethe MeI addition to the Ni(0) site competes with electrophilicaddition of MeI to the �-thiolates of the dadtEt ligand asobserved for analogous reactions of Ni(N2S2) complexes (32).The selective methylation of the Nip site in ACS could be due tothe steric bulk of the methylcobalamin, which may hinder thenucleophilic methylation of the cystein sulfurs bridging the 2nickel centers. Although the putative MeI adduct of the Ni(II)–Ni(0) complex, [Ni(dadtEt)Ni(Me)I], was not isolated, a similarreaction of the Ni(II)–Ni(0) complex with iodopentafluoroben-zene confirms the oxidative addition process, giving the analo-gous adduct Ni(dadtEt)Ni(C6F5)I (6) in 24% yield. Complex 6was further converted to Ni(dadtEt)Ni(C6F5)(SDmp) (7) viasubsequent addition of KSDmp (Scheme 3). The molecular

structures of 6 and 7 were analyzed by X-ray crystallography (seeSI Text).

Acetylthioester Formation. To model the formation of acetyl-CoAat the ACS active site, we examined the reaction of 2 with CO.Treatment of 2 with an atmospheric pressure of CO in THF atroom temperature resulted in an immediate color change fromreddish purple to reddish brown. After removal of volatiles, thematerials soluble to ether were subjected to silica gel columnchromatography to afford the acetylthioester CH3C(O)SDmp(8) in 89% yield. This result proves that the dinuclear nickelcomplex 2 can carry out a reaction similar to that of ACS. Theresidual reddish brown solid, insoluble to ether, was identified asNi(dadtEt), which was recovered in quantitative yield.

When CO gas was charged to a THF solution of 2 at �60 °C,it was noticed that the color first changed from reddish purpleto a dark green before becoming reddish brown. After stirringthe solution for 3 min at �60 °C, a dark-green solid wasprecipitated from the reaction mixture upon addition of hexane.The IR spectrum of the nearly colorless THF/hexane solutionshowed �CO bands at 2,044 and 1,710 cm�1, which are attribut-able to Ni(CO)4 and 8, respectively. The dark-green precipitatewas crystallized from acetonitrile/ether, and the resulting dark-green needles were subjected to X-ray structural analysis. Thecrystals were found to contain the dinuclear nickel dicarbonylcomplex Ni(dadtEt)Ni(CO)2 (9) and Ni(dadtEt) in a 1:1 ratio(Scheme 4).

The formation of the acetylthioester, CH3C(O)SDmp (8),probably proceeds via insertion of CO into the Ni–Me bond of2 with successive reductive elimination. The latter step, whereNi(2) in Fig. 2 is reduced from NiII to Ni0, would be facilitated

Fig. 3. Core structures of Ni(dadtEt)Ni(Me)(SDmp) (2A) and A-cluster in ACSMt. In square, a view of structure from the S1–S2 vector.

Ni(cod)SN

SNNi

SN

SNNi

NC

CN

NiSN

SNNi

Ni(cod)2

3

4

NC CN

Co(dmgBF2)2(Me)(Py)2) KSDmp

SDmpMe

NiSN

SNNi

2

NNNN

O BF2

OOOF2B Co

Me

Py

1)

"Ni(dadtEt)Ni(0)"

Scheme 2.

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by coordination of CO at Ni(2), to generate 9 and Ni(CO)4. Inan attempt to detect the possible nickel(II) acetyl intermediate,we examined the reaction of 2 with 1 equiv of CO at �60 °C.However, the detectable products were the same as thoseobtained from the reaction of 2 with atmospheric CO, i.e.,CH3C(O)SDmp (8), 9, and Ni(dadtEt), except for the presenceof unreacted starting complex 2. The result indicates that thereductive elimination of the acetylthioester facilitated by COcoordination is faster than the preceding CO insertion into theNi–Me bond.

An ORTEP drawing of 9 is given in Fig. 4. The coordinationgeometries of Ni(1) and Ni(2) are square planar and tetrahe-dral, typical for Ni(II) and Ni(0) oxidation states, respectively.The long Ni(2)–�S bonds of 2.3847(8)–2.3577(8) Å corrobo-rate the Ni(0) state for Ni(2), and the Ni(1)–�S bond lengthsof 2.1866(7)–2.1956(6) Å are similar to those of the Ni(II)–Ni(II) dinuclear nickel models (15, 21, 30, 31). Interestingly,the dihedral angle of 97.6° between the 2 Ni(�-S)2 planes isnotably smaller than those of Ni(II)–Ni(II) dinuclear nickelmodels (10, 18–23). It is also smaller than that (109.7°) of theclosely related complex, [Et4N]2[{Ni(S2N2�)}{Ni(CO)2}], re-ported by Linck et al. (16). The IR spectrum of the dark-greenneedles shows 2 �CO bands at 1,971 and 1,890 cm�1, which arereasonable for a Ni(0) dicarbonyl such as 9; the �CO bands of[Et4N]2[{Ni(S2N2�)}{Ni(CO)2}] were reported to appear at1,948 and 1,866 cm�1. This shift of �CO to lower wave numbersindicates greater back donation to CO for the negativelycharged dinuclear complex.

To obtain further insight into the CO insertion into the Ni–Mebond, the dinuclear complex carrying an electron-withdrawingbenzenethiolate, Ni(dadtEt)Ni(Me)(SC6Cl5) (10), was preparedin a similar fashion from 1, MeMgCl, and KSC6Cl5 (25). Whena dichloromethane solution of 10 was treated with atmosphericCO at room temperature, MeC(O)SC6Cl5 was produced in 36%yield. However, when the same reaction was performed at�40 °C, the IR spectrum exhibits an intense absorption at 1,629cm�1 attributable to an acyl CO stretch. X-ray structural analysisof brown crystals obtained at �40 °C under a CO atmosphere

confirms the formation of the acyl complexNi(dadtEt)Ni(COMe)(SC6Cl5) (11) (see SI Text). Notably COinsertion is reversible in this particular case. The �(CO) band inthe IR spectra weakened and then disappeared upon evacuationof the solution at �40 °C, from which complex 10 was recoveredquantitatively (Scheme 5). In ACS catalysis, CO insertion intothe Nip–Me species would be reversible, and the reductiveelimination of acetyl-CoA would immediately occur when theCoA is coordinated to Nip site.

Because complex 9 obtained after treatment of 2 with excessCO does not react with methyl cobaloxime, the acetylthioesterformation reported here is stoichiometric. However, the activeNi(II)–Ni(0) complex such as 3 can be regenerated when com-plex 2 is treated with 1 equiv of CO in the presence of 20 equivof 1,5-cyclooctadiene. This significantly suppresses the forma-tion of 9 and Ni(CO)4, and complex 2 and CH3C(O)SDmp (8)were obtained in 46% and 93% yields, respectively, upon treat-ment with methylcobalamin 5 and KSDmp (Scheme 6). Accord-ing to these results, CO addition to the Nip site in ACS catalysismust be strictly regulated to prevent the formation of inactiveNi(0)-carbonyl species, such as 9, and therefore CO transfer mayperhaps be controlled by opening and closing the tunnel con-necting CODH and ACS (5, 6). The cystein thiolate of the[4Fe-4S] cluster located at the Nip site may be important for theregulation of CO coordination and promotion of reductiveelimination of acetyl-CoA, a role played by 1,5-cyclooctadiene inour model reaction.

Concluding Remarks. We have synthesized the dinuclear nickelcomplex Ni(dadtEt)Ni(Me)(SDmp) (2), having a methyl groupand a thiolate, which is a good mimic of the structure of thedinuclear nickel site of the A-cluster, although the diamidodi-thiolate Cys-Gly-Cys ligand of the A-cluster is different from thediaminedithiolate dadtEt ligand. Complex 2 has been synthesizedfrom the Ni(II)–Ni(0) species such as [Ni(dadtEt)Ni(cod)] (3) viamethyl transfer from Co(dmgBF2)2(Me)Py (5), which models thereaction of the active state of the A-cluster with methylcobal-

Fig. 4. ORTEP drawing of Ni(dadtEt)Ni(CO)2 (9). Thermal ellipsoids are shownat 50% probability.

2

CO (excess)

SDmpMe

NiSNSN

Ni

8

CO

OC

NiSNSN

Ni +

Ni(CO)4

Me SDmp

O9

+

SNSN

Ni

Scheme 4.

11

SC6Cl5

Me

NiSNSN

Ni

O

10

SC6Cl5Me

NiSNSN

Ni

CO

– CO

Scheme 5.

Scheme 3.

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amin. Notably an ACS model reaction was found to take placeon the Nip site of 2, featuring CO insertion into the Ni–Me bond,successive reductive elimination of the acetylthioester,CH3C(O)SDmp (8), and generation of such Nid(II)–Nip(0)species. These results are consistent with an ACS mechanism, inwhich the Nip site in the A-cluster first takes up a Me moiety andCO insertion follows (12). However, the alternative mechanism,

which involves initial CO coordination at Nip(I) and subsequentreaction with Me, remains to be examined (13). In either case,Nid is not the catalytic site but plays a structural role to hold the2 bridging cysteinyl sulfurs of the Cys-Gly-Cys backbone in afavorable and perhaps flexible position for coordination to Nip(33–35).

Materials and MethodsAll reactions and the manipulations were performed under nitrogen or argonatmospheres by using standard Schlenk techniques. Solvents were dried,degassed, and distilled from CaH2 (CH2Cl2), or from Mg turnings (methanol)under nitrogen. Hexane, ether, toluene, THF, and CH3CN were purified bycolumns of activated alumina and a supported copper catalyst supplied byHansen.

All experimental procedures and details of characterization of complexesare given in SI Text. Molecular structures of 4, 6, 7, 10, and 11 are given in Figs.S1–S5, and selected bond distances and angles for 2, 4, 6-7, 9, 10-11 are listedin Tables S1–S5, respectively. Crystal data are summarized in Table S6.

ACKNOWLEDGMENTS. We are grateful to Prof. Roger E. Cramer for discus-sions and careful reading of the manuscript. This work was supported byGrants-in-Aid for Scientific Research 18GS0207 and 18065013 from the Min-istry of Education, Culture, Sports, Science, and Technology, Japan.

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Scheme 6.

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