酶工程作业

AThree-DimensionalPredictiveActiveSiteModelforLipasefrom

Pseudomonascepacia

KarinLemke,MichaelLemke,andFritzTheil*,†

Institutfu¨rAngewandteChemieBerlin-Adlershofe.V.,RudowerChaussee5,D-12484Berlin,Germany

ReceivedMay9,1997X

Athree-dimensionalactivesitemodeloflipasefromPseudomonascepaciasoneofthemostpopularlipasesinorganicsynthesisswasdevelopedonthebasisofthekineticresolutionof3-(aryloxy)pro-pan-2-ols.Sizeandshapeofbothhydrophobicbindingpocketsoftheactivesiteofthislipaseweredeterminedbysubstratemappingincombinationwithmolecularmodelingforsubstratesandnonsubstrates.Thismodelexplainsandpredictswhetheracompoundisacceptedasasubstrateornotandallowstoassesstheenantiomerselectivityofthelipase-catalyzedreaction.

Introduction

Theuseofenzymeshasbeenestablishedasanimportanttoolinorganicsynthesisduringthepastdecade.Duetotheirabilitytodiscriminatebetweenenantiomersandenantiotopicgroups,theyareutilizedinkineticresolutionsofracematesandasymmetrizationsofprostereogenicormesocompoundstoprovideaneasyaccesstoenantiomericallypurebuildingblocks,syntheticandnaturalproducts.1

Amongthebiocatalystsusedinorganicsynthesis,lipases(triacylglycerolhydrolases,EC3.1.1.3)havebeenusedmostfrequentlybecausetheyarecheap,availablefrommanysources,easytohandle,andacceptabroadrangeofsubstrates.2Furthermore,theyareactiveinaqueoussolutionandinpracticallywater-freeorganicsolvents.Particularly,inorganicsolventslipasesremaintheiractivityupto100°C.Lipasescatalyzehydrolysisandformationofcarboxylicestersandformationofamidesuponthereactionconditions.

X-rayanalysesofsomelipases3evidencethattheiractivesitesaresimilartothoseofserineproteasesinwhichtheprimaryhydroxyfunctionofserineofthecatalytictriadeactsasanucleophiletoattackamideor

NewaddressasofSept.1,1997:UniversityofLiverpool,Depart-mentofChemistry,P.O.Box147,LiverpoolL693BX,U.K.

XAbstractpublishedinAdvanceACSAbstracts,August1,1997.(1)(a)Santaniello,E.;Ferraboschi,P.;Grisenti,P.;Manzocchi,A.Chem.Rev.1992,92,1071.(b)Poppe,L;Nova´k,L.SelectiveBioca-talysis;VCH:Weinheim,1992.(c)Roberts,S.M.,Wiggins,K.,Casy,G.,Eds.PreparativeBiotransformations;Wiley:Chichester,1992.(d)Faber,K.BiotransformationsinOrganicChemistry,2nded.;Springer-Verlag:Heidelberg,1995.(e)Drauz,K.,Waldmann,H.,Eds.EnzymeCatalysisinOrganicSynthesis;VCH:Weinheim,1995.(f)Wong,C.-H.;Whitesides,G.M.EnzymesinSyntheticOrganicChem-istry;Pergamon:Oxford,1994.

(2)(a)Chen,C.-S.;Sih,C.J.Angew.Chem.,Int.Ed.Engl.19,28,695.(b)Boland,W.;Fro¨ssl,C.;Lorenz,M.Synthesis1991,1049.(c)Faber,K;Riva,S.Synthesis1992,5.(d)Santaniello,E.;Ferraboschi,P.;Grisenti,P.EnzymeMicrob.Technol.1993,15,367.(e)Theil,F.Catal.Today1994,22,517.(f)Theil,F.Chem.Rev.1995,95,2203.(g)Koskinen,A.M.P.,Klibanov,A.M.,Eds.EnzymaticReactionsinOrganicMedia;BlackieAcademic&Professional:Glas-gow,1996.

(3)(a)Winkler,F.K.;D’Arcy,A.;Hunzicker,W.Nature1990,343,771.(b)Brady,L.;Brzozowski,A.M.;Derewenda,Z.S.;Dodson,E.;Dodson,G.;Tolley,S.;Turkenburg,J.P.;Christiansen,L.;Huge-Jensen,B.;Norskov,L.;Thim,L.;Menge,U.Nature1990,343,767.(c)Grochulski,P.;Li,Y.;Schrag,J.D.;Bouthillier,F.;Smith,P.;Harrison,D.;Rubin,B.;Cygler,M.J.Biol.Chem.1993,268,12843.(d)Noble,M.E.M.;Cleasby,A.;Johnson,L.N.;Egmond,M.R.;Frenken,L.G.J.FEBSLett.1993,331,123.(e)Uppenberg,J.;Hansen,M.T.;Patkar,S.;Jones,T.A.Structure1994,2,293.(f)Cygler,M.;Grochulski,P.;Kazlauskas,R.J.;Schrag,J.D.;Bouthillier,F.;Rubin,B.;Serreqi,A.N.;Gupta,A.K.J.Am.Chem.Soc.1994,116,3180.

†

estercarbonylgroups.Furthermore,thesestructuredeterminationsshowacommoncatalyticmachineryforalllipases.Despitethisfact,substrateacceptanceandthedegreeofenantiodifferentiationisverydifferentdependinguponthenaturalsourceofthelipase.Itisacceptedingeneralthatsubstraterecognition,stabiliza-tion,andenantioselectivetransformationisdeterminedbytwohydrophobicbindingregionsorpocketswhicharenotseparatedfromthecatalyticsite.3f

OneofthemostpopularlipasesusedinorganicsynthesisislipasefromPseudomonascepaciafromAmanoPharmaceuticalCo.,Ltd.(Nagoya,Japan),calledlipaseAmanoPS.BeforereidentificationofthebacterialsourceitwascalledlipasefromP.fluorescens(lipaseP).DuetotheflexibilityofitsbindingsiteslipasePSacceptsabroadrangeofsubstrates.Thislipasehasbeenusedforregio-andstereoselectivehydrolysis4andalcoholysis5ofcarboxylicestersandanhydrides6andfortheregio-andstereoselectivetransesterificationofalcohols.4a,b,d,7ThereseemstobealmostnorestrictionregardingthestructureofcompoundswhichareacceptedassubstratebylipasePS.

Inordertorationalizeandtopredictreactivityandselectivityoflipase-catalyzedbiotransformations,adeeper

(4)(a)Klempier,N.;Faber,K.;Griengl,H.Synthesis19,933.(b)Foelsche,E.;Hickel,A.;Ho¨nig,H.;Seufer-Wasserthal,P.J.Org.Chem.1990,55,1749.(c)Goergens,U.;Schneider,M.P.J.Chem.Soc.,Chem.Commun.1991,10.(d)Goergens,U.;Schneider,M.P.J.Chem.Soc.,Chem.Commun.1991,1066.(e)Chenevert,R.;Gagnon,R.J.Org.Chem.1993,58,1054.(f)Hirose,Y.;Kariya,K.;Nakanishi,Y.;Kurono,Y.;Achiwa,K.TetrahedronLett.1995,36,1063.(g)Tanaka,K.;Shogase,Y.;Osuga,H.;Suzuki,H.;Nakamura,K.TetrahedronLett.1995,36,1675.

(5)(a)Bianchi,D.;Cesti,P.J.Org.Chem.1990,55,5657.(b)Bianchi,D.;Bosetti,A.;Cesti,P.;Golini,P.TetrahedronLett.1992,33,3231.(c)Koichi,Y.;Suginaka,K.;Yamamoto,Y.J.Chem.Soc.,PerkinTrans.11995,15.

(6)(a)Yamamoto,K.;Nishioka,T.;Oda,J.;Yamamoto,Y.Tetra-hedronLett.1988,29,1717.(b)Ozegowski,R.;Kunath,A.;Schick,H.LiebigsAnn.Chem.1993,805.

(7)(a)Kim,M.-J.;Choi,Y.K.J.Org.Chem.1992,57,1605.(b)Moris,F.;Gotor,V.J.Org.Chem.1992,57,2490.(c)Takano,S.;Yamane,T.;Takahashi,M.;Ogasawara,K.Synlett1992,410.(d)Bovara,R.;Carrea,G.;Ottolina,G.;Riva,S.Biotechnol.Lett.1993,15,169.(e)Bovara,R.;Carrea,G.;Ferrara,L.;Riva,S.Tetrahedron:Asymmetry1991,2,931.(f)Lambusta,D.;Nicolosi,G.;Patti,A.;Piattelli,M.Tetrahedron:Asymmetry1993,4,919.(g)Sato,M.;Hirokawa,T.;Hattori,H.;Toyota,A.;Kaneko,C.Tetrahedron:Asymmetry1994,5,975.(h)Nakano,H.;Okuyama,Y.;Iwasa,K.;Hongo,H.Tetrahedron:Asymmetry1994,5,1155.(i)Takano,S.;Yamada,O.;Iida,H.;Ogasawara,K.Synthesis1994,592.(j)Sibi,M.-P.;Lu,J.L.TetrahedronLett.1994,35,4915.(k)Boaz,N.W.;Zimmerman,R.L.Tetrahedron:Asymmetry1994,5,153.(l)Chadha,A.;Manohar,M.Tetrahedron:Asymmetry1995,6,651.(m)Ema,T.;Maeno,S.;Takaya,Y.;Sakai,T.;Utaka,M.Tetrahedron:Asymmetry1996,7,625.(n)Theil,F.;Ballschuh,S.Tetrahedron:Asymmetry1996,7,3565.

S0022-3263(97)00838-4CCC:$14.00©1997AmericanChemicalSociety

ActiveSiteModelforLipasefromP.cepaciainsightintotheactivesitesoflipasesregardingthecatalyticmechanismaswellasshapeandsizeofthebindingpocketsisnecessary.Therearetwoapproachestocontributetothesolutionofthisproblemwhicharecomplementaryoneanother:X-raystructuredetermi-nationofcrystallinelipasesandsubstratemapping.Veryrecently,thestructureofcovalentcomplexesofCandidarugosalipasewithtransitionstateanalogsforthehydrolysisofmenthylesterswasusedtoexplainthechiralpreferenceoflipasesingeneral.3fHowever,lipasesaretypicalinduced-fitenzymesandthereforeX-raystructureasafrozenconformationcannotbeusedtoexplainandpredictwhichcompoundisacceptedasasubstratebythelipase.Inordertodeterminespatialandconstitutionalrequirementstoidentifysubstratesandnonsubstratessubstratemappingincombinationwithmolecularmodelingseemstobeverypromising.Inordertorationalizesubstratepropertiesandtopredictsubstratepropertiesforenzyme-catalyzedreac-tions,itisofgreatimportancetodevelopactivesitemodels.

Cubic-spaceactivesitemodelshavebeendevelopedforporcineliveresterase,8cyclohexanonemonooxygenaseforBaeyer-Villigeroxidation9orsulfoxidation,10anit-rilase,11andaverycrudemodelforlipaseYSfromAmano.12

InthecaseoflipasefromP.cepacia,therearesomeattemptstoexplaintheenantiomerselectivitybydiffer-entsizesofthehydrophobicpocketsbasedonsubstratemappingofthislipasewithoutdeterminingtheshapesandsizesofthebindingsites.13Kazlauskas’rules14explainandpredictthestereochemicaloutcomeforthekineticresolutionofestersbyhydrolysisandtranses-terificationofalcoholsonthebasisofdifferentsizesofthesubstituentsadjacentwiththestereogeniccenter.ApreliminaryX-rayanalysisofP.cepacialipase15givesduetoalowresolutionnoinformationontheactivesiteofthisenzyme.

ResultsandDiscussion

Itwasouraimtodevelopathree-dimensionalactivesitemodeloflipasefromP.cepaciabycombinationofsubstratemappingandmolecularmodeling.

SubstrateMapping.Veryrecentlywefoundthatreactivityandenantiomerselectivityinthekinetic

(8)(a)Toone,E.J.;Werth,M.J.;Jones,J.B.J.Am.Chem.Soc.1990,112,4946.(b)Toone,E.J.;Jones,J.B.Tetrahedron:Asymmetry1991,2,1041.(c)Provencher,L.;Wynn,H.;Jones,J.B.;Krawczyk,A.R.Tetrahedron:Asymmetry1993,4,2025.(9)Ottolina,G.;Carrea,G.;Colonna,S.;Ru¨ckmann,A.Tetrahe-dron:Asymmetry1996,7,1123.

(10)Ottolina,G.;Pasta,P.;Carrea,G.Colonna,S.;Dallavalle,H.;Holland,L.Tetrahedron:Asymmetry1995,6,1375.

(11)Deigner,H.P.;Blencowe,C.;Freyberg,C.E.J.Mol.Catal.B1996,1,61.

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(13)(a)Laumen,K.Ph.D.Thesis,Universita¨t-Gesamthochschule,Wuppertal,1987.Kloosterman,M.;Kierkels,J.G.T.;Guit,R.P.M.;Vleugels,L.F.W.;Gelade´,E.T.F.;vandenTweel,W.J.J.;Elferink;V.H.M.;Hulshof,L.A.;Kamphuis,J.InLipases:Structure,MechanismandGeneticEngineering;Alberghina,L.,Schmid,R.D.,Verger,R.,Eds.;VCH:Weinheim,1991;p187.Xie,Z.-F.Tetrahe-dron:Asymmetry1991,2,733.Exl,C.;Ho¨nig,H.;Renner,G.;Rogi-Kohlenprath,R.;Seebauer,V.;Seufer-Wasserthal,P.Tetrahedron:Asymmetry1992,3,1391.

(14)(a)Kazlauskas,R.J.;Weissfloch,A.N.E.;Rappaport,A.T.;Cuccia,L.A.J.Org.Chem.1991,56,2656.(b)Weissfloch,A.N.E.;Kazlauskas,R.J.J.Org.Chem.1995,60,6959.

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J.Org.Chem.,Vol.62,No.18,19976269

Scheme1

resolutionof3-(aryloxy)propan-2-olderivativesbytrans-esterificationwithvinylacetateinorganicsolventsinthepresenceoflipasefromP.cepaciasignificantlydependonthesubstituentsatthearomaticringandinthe1-positionofthepropylskeleton16(Scheme1).Inourconceptprobingthethree-dimensionalstructureoftheactivesiteoflipasePSbysubstratemapping,itwasveryimportanttoidentifynonsubstratesforthislipase.Non-substratesarecompoundswhichdifferfromsubstratesinthesizeofthesubstituentsatthestereogeniccenterandarenottransformedinthepresenceoflipasefromP.cepacia.Thesetof613-(aryloxy)propan-2-olsscreenedassubstratescanbesubdividedregardingtheirchemicalreactivityintosubstrates(Group1)whichareacylatedwithadifferentdegreeofenantiomerselectivityex-pressedastheEvalue17andnonsubstrates(Group2)whichresistlipase-PS-catalyzedtransesterificationasshowninFigure1.Withregardtotheirsubstitutionpattern,compoundsofGroup1canbesubdividedintotheSubgroups1.1-1.5andthoseofGroup2canbesubdividedintotheSubgroups2.1and2.2(Figure1).SummarizingthefactsdepictedinFigure1,itcanbeconcludedthatreactivityandenantiomerselectivityofthelipase-catalyzedtransesterificationstronglydependonthesubstituentsR1andR2.Ingeneral,goodsub-stratesarecharacterizedbyR1representinghydrogenorparasubstituentsatthearylring(compounds1-8,Subgroup1.1withE>50)andbyR2beinganun-branchedacyloxyresidue(compounds14-21,Subgroup1.2withE>50).Eithershortacyloxygroupssuchasacetateorextremelylongacyloxygroupssuchasn-hexadecanoateareconvertedsmoothlywithhighenan-tiomerselectivity.Branchedresiduessuchasisobutyrate22,3-phenylpropanoate23and6-phenylhexanoate24(Subgroup1.2withE>50)aregoodsubstratesaswell.SubstratesinwhichR1isasmallorthosubstituentatthearylring(compounds3-13,Subgroup1.1withE<50)andinwhichR2isabranchedacyloxy(compounds30-34,Subgroup1.2withE<50),analkyloxy(com-pounds35-38,Subgroup1.3),orazido(compound39,Subgroup1.4)residueareconvertedwithasignificantlylowerenantiomerselectivity.Theamines40and41(Subgroup1.4)aresubstratesforlipasePS,butinthiscasetheenzymeisnotabletodistinguishbetweentheenantiomers.However,chemicalreactioncouldbeex-cludedbecauseintheabsenceoflipasePSthesubstrates40and41arenotacylatedbyvinylacetate.Withtheexceptionofthecompound48substratesofSubgroup1.5areacylatedbylipasePSwithpoorenantiomerselectiv-ity.

Whentheisobutanoate22(Subgroup1.2)andthepivaloate56(Subgroup2.2)distinguishedbyamethylgroupattheacylresidueinthe1-positionarecompared,

(16)(a)Theil,F.;Weidner,J.;Ballschuh,S.;Kunath,A.;Schick,H.J.Org.Chem.1994,59,388.(b)Theil,F.;Lemke,K.,Ballschuh,S.;Kunath,A.;Schick,H.Tetrahedron:Asymmetry1995,6,1323.(c)Lemke,K.;Theil,F.Kunath,A.;Schick,H.Tetrahedron:Asymmetry1996,7,971.

(17)Chen,C.-S.;Fujimoto,Y.;Girdaukas,G.;Sih,C.J.J.Am.Chem.Soc.1982,104,7294.

6270J.Org.Chem.,Vol.62,No.18,1997Figure1.Surveyof3-(aryloxy)propan-2-olderivativesscreenedassubstrates.

asignificantlydifferentbehaviorofbothcompoundswasobserved.Whiletheisobutyrate22wassmoothlycon-

Lemkeetal.

Figure2.Structuralrequirementsofnonsubstrates.

Scheme2

Table1.KineticResolutionoftheEthanediol

Derivatives(RS)-62-69substrate

R3

R2

E62n-C8H17OAc463n-C10H21OAc10n-C12H25OAc865n-C14H29

OAc866cyclo-C6H11OOAc>10067PhOAc

7468PhOCO-n-C9H194769

Ph

OCO-n-C15H31

>100

vertedbylipasePSwithhighenantiomerselectivity(E)78),itshomologuethepivaloate56completelyresistslipasePS-catalyzedacylation.Basedonthisfinding,itwaspredictedandfoundthatcomparablebulkysubstit-uentsinthe1-positionleadtononsubstratessuchas57-61.ThenonsubstratesdonotcompetewithsubstratesinaccommodatingtheactivesiteoflipasefromP.cepacia.Forexample,iflipasePSispreincubatedwiththenonsubstrates56-61andthenincubatedwiththeisobu-tanoate22,thelatterisacylatedwiththesamerateandselectivityasintheabsenceofnonsubstrates.Ingeneral,thenonsubstrates50-61(Figure2)arecharacterizedeitherbyabulkysubstituentintheorthopositionofthearomaticring(Subgroup2.1)orsubstituentsinthe1-positioninwhichthegroupsAandBareatomswithfourligands(allhydrogenreplaced)(Subgroup2.2).Inordertoinvestigatetheinfluenceofthe(aryloxy)m-ethylresidueingeneralonreactivityandenantiomerselectivity,itwasreplacedbyothersubstituents.Scheme2andTable1demonstratetheresultsforthekineticresolutionofethanediolderivatives(RS)-62-69inwhichR3representsphenyl,n-alkyl,andcyclohexyloxyresidues.Table1demonstratesthatthearyloxysubstituentcanbereplacedbyalkyl,phenyl,orthecyclohexyloxyresidue.However,ifR3isann-alkylchainasforthesubstrates62-65,theenantiomerselectivitydropsdownashasalreadybeenfoundforshorteralkylgroups.16aButveryhighselectivitywasobservedforthecyclohexyloxyderivative66whichisevidencethatthearomaticchar-acterofR3isnotnecessaryforanefficientkineticresolution.Thephenylethanediolderivatives67-69showpropertiescomparabletothosefoundforthecorresponding(aryloxy)methylcompounds14,19,and21.Thepoorenantiomerselectivityofthealkylderivatives62-65comparedwiththosecompounds66,67,andthearyloxyderivativesofGroup1showthatalongalkylsidechaincannotmimicacyclicresiduebysizeandshape.IngeneraltheenantiomerselectivityofthesubstratesacetylatedbylipasePSisinaccordancewithKazlauskas’rule.However,forsubstrateswithlongacyloraromatic

ActiveSiteModelforLipasefromP.cepaciaJ.Org.Chem.,Vol.62,No.18,19976271

Figure3.Superpositionof20low-energyconformersofsubstrate21.

Figure4.SuperpositionofsubstrateswithE>50.

residuesinthe1-positionsuchas18-21,23,24,27-29,and33,theenantiomerselectivitycannotbepredictedandexplainedbyKazlauskas’rule.14aThereasonforthisbehaviorishydrophobicpocketswhichdiffernotbyvolumebutbyshape.Furthermore,thepresenceofanacyloxygroupinthe1-positionhasanadditionalstabiliz-ingeffectonthesubstratefitintotheactivesite,becausethecorrespondingethers35-38areacetylatedwithasignificantlowerenantiomerselectivitythanthecorre-spondingacyloxyderivatives.Theamines40and41areconvertedbylipasePSwithoutanyenantiomerselectiv-ity.Therefore,thebeneficialeffectoftheacyloxyresidueinthe1-positionmaybeattributedtothecarbonylgroupeitherasanelectrondonatorforahydrogenbridgebondtoasuitableaminoacidinthebindingregionortodipole-dipoleinteractions.

MolecularModeling.Formolecular-modelingin-vestigationsallsubstrateswithE>50andnonsubstrateswereselected.ThegeometryofthesemoleculeswasdeterminedwiththeaidofthemodelbuildprogramHyperchemRelease4.018providingstandardbondlengthandangels.Furtheroptimizationtofindalocalmini-mumwascarriedoutwiththesemiempiricalmethodAM1.19Onthebasisofthelocalminimumfound,conformationalanalysiswascarriedoutusingtheCon-formationalSearchprogramofChemplusRelease1.0.18Comparisonofthelow-energyconformationsfoundbyconformationalanalysiswiththestartingconformerswhichwerefoundafterthefirstgeometryoptimization

(18)Hypercube,Inc.,Waterloo,Ontario,Canada,1994.

(19)Dewar,M.J.S.;Zoebisch,E.G.;Healy,E.F.;Stewart,J.P.J.Am.Chem.Soc.1985,107,3902-3909;1993,115,5348.

6272J.Org.Chem.,Vol.62,No.18,1997Lemkeetal.

Figure5.Superpositionofsubstrates(black)withE>50andnon-substrates(red).

withAM1revealedthatthelong-stretchedconformersusedasstartingconformersareenergeticallymorefavoredthancoiledconformers.Figure3shows20ofthelow-energyconformersofsubstrate21superimposedinthestereogeniccenter.AsimilarconsiderationbetweentheconformersofallothersubstrateswithE>50revealedthatinallcasesthelong-stretchedconformerswerethemoststableandbysuperimposinginthestereogeniccentertheyaretheonlyconformerswithcommonstructuralregions(Figure4).Geometryopti-mizationforthenonsubstratesshowsthesamebehavior.Figure5showsthesuperpositionofthesubstrates1-8,14-24,48,66,67,and69(black)andnonsubstrates50-61(red).Thestereogeniccenterscoincidewiththeoriginofthecoordinatesystem,theC-HbondwiththeX-axisandtheC-OHbondwiththey/zplane(behindthedrawingplane).Figure5showsthatthenonsubstratesdonotfittothesuperimposedsubstratesintheregionsdepictedinFigure2whichwererecognizedasprobableforacompoundtobeasubstrateornotforthelipasefromP.cepacia.

Thecorrespondenceoftheresultsobtainedbysub-stratemappingandmolecularmodelingclearlyindicatesthatsubstratesdonotfitthehydrophobicbindingregionsoftheactivesiteoflipasefromP.cepaciainacoiledconformation.Ifthisisthecase,thereshouldbenoreasonthatthemoleculesidentifiedasnonsubstratesareexcludedfromthelipase-catalyzedtransformation.Fur-thermore,substrateconformationsinvacuumdeterminedbymolecularmodelingandofthesubstratesintheactivesiteshouldbeverysimilar.ThisisinaccordancewithM.J.S.Dewars’desolvationmodel20whichpostulatessimilaritiesbetweenenzymaticreactionsinsolutionandgasphasereactions.

Finally,tovisualizetheboundariesoftheactivesitemodeloflipasePS,amodelingprogram21wasusedtowrapthesuperimposedsubstratesincludingtheirvan

(20)Dewar,M.J.S.Enzyme1986,36,8.

(21)Molwrap:OverlayingofMoleculesintheirStereogenicCenterandWrappingoftheSuperstructure,developedbyLemke,M.,Berlin,Germany,1996,accessviae-mail:mtlemke@aol.com.

Figure6.Superpositionofwrappedsubstrateswithun-wrappednonsubstrates.

Figure7.Simplifiedcubic-spacemodelofthewrappedsubstrates.

derWaalsradii.Figure6showsthepatchrepresentationofthewrappedthree-dimensionalactivesitemodelsuperimposedwiththenonsubstratesandFigure7thesimplifiedcubic-spacerepresentation.

SummaryandConclusion

Apredictivethree-dimensionalactivesitemodelforlipasefromP.cepaciawasdevelopedbysubstratemap-pingandmolecularmodeling.Thismodelclearlyindi-catesthatsubstratebindingandorientationiscausedbytwohydrophobicpocketswhichareverydifferentbyshapesatube-likestretchedwithaverylimiteddiameter

ActiveSiteModelforLipasefromP.cepacianeartheactiveserineandasecondwithasphericalshapeneartheactiveserine.Highenantiomerselectivityistheresultofanoptimalorientationofthesecondaryhydroxygroupofthefasterreactingenantiomerinthedirectionofthecatalytictriadeofthisenzymewhichiscausedbyanoptimalstabilizationofthesubstituentsadjacentatthestereogeniccenter.Themorespherically-shapedpocketpreferentiallyaccommodatessubstituentssuchasphenyl,phenoxymethyl,para-substitutedphe-noxymethyl,(trityloxy)methyl.7a(tert-butyloxy)methyl,4c(phenylthio)methyl,4c((tert-butyldimethylsilyl)oxy)methyl,4dor(p-tosyloxy)methyl7kwhichactasanchoringgroupswhereasthetube-likepockethostsstretchedsubstituentssuchasacetoxymethyluptoatleast(n-hexadecanoy-loxy)methyl,methyl,4c,d,7aethyl,4c,d,7aorvinyl.4d,7kOth-erwise,twocarbonchainsofquitedifferentlengthdonotallowanefficientstereodifferentiationbetweenbothenantiomersbecausealong-stretchedsubstituentisnotabletoanchorthesubstrateinthemoreshericalpocket.Nonsubstratesarecharacterizedbytwostericallyde-mandingsubstituentsatthestereogeniccenter.There-fore,thesecompoundsdonotfitintotheactivesite.

J.Org.Chem.,Vol.62,No.18,19976273

HighenantiomerselectivityinlipasePS-catalyzedreactionscanbepredictedifsecondaryalcoholsortheiresterspossesssubstituentsatthestereogeniccenterwhichareverydifferentbyshapeandnotbyvolume.

Acknowledgment.ThisworkwassupportedbytheMinistryofEducation,Science,Research,andTechnol-ogyoftheFederalRepublicofGermanyandtheBerlinSenateDepartmentforScience,Research,andCulture(ProjectNo03C005).FinancialsupportbytheDeutscheForschungsgemeinschaft,theDeutscheAkademiederNaturforscherLeopoldina,andtheFondsderChemis-chenIndustriearealsogratefullyacknowledged.LipasePSwasagiftfromAmanoEnzymeEuropeLtd.

SupportingInformationAvailable:Experimentalandspectroscopicdataforthekineticresolutionofthecompounds62-69(8pages).Thismaterialiscontainedinlibrariesonmicrofiche,immediatelyfollowsthisarticleinthemicrofilmversionofthejournal,andcanbeorderedfromtheACS;seeanycurrentmastheadpagefororderinginformation.JO970838D