多肽合成工藝流程

多肽合成工藝流程2024/3/12多肽合成工藝流程OutlineResins;Protectinggroups;Syntheticprotocol;Monitoring;Cleavagetechnics;Sidereactions;多肽合成工藝流程Fmoc/tBu:NHCCH2OCNHCHCH2COOCOHCCH3CH3CH3NHCHCOOCH2CH2OCH2OOCCH3CH3CH3CRFmoctert-butyl..piperidineTFAWang-resinFmoc-Asp(OtBu)-Tyr(tBu)-Wangresin多肽合成工藝流程TypeofresinsforFmoc-chemistryTherearemanydifferentresinsandmostofthemareusedforspecialcasesandinindividuallaboratories.Hereonlythemostwidelyappliedresinswillbepresented.ResinsarebasedonPS-DVB(1%)copolymer.4-Alkoxybenzylalcohol(Wang)resin:CH2OPCH2HOAttachmentofthefirstaminoacid:Fmoc-Aaa(X)-OH:DIC:DMAP(2:2:0.2equivtotheresinOHcontent)inDMF,1hatRT.ThefinalcleavageresultsinpeptideswithCOOHgroupattheC-terminusTheresinisnotavailableforthesynthesisofpeptideswithasequenceontheC-terminalthatissensitivefordiketopiperazineformation!多肽合成工藝流程SASRIN(SuperAcidSensitiveResIN)(2-methoxy-4-alkoxybenzyl-alcoholresin)CH2OPCH2HOOCH3Peptideiscleavablewith0.5-1.0%TFAinDCMresultedinprotectedpeptidefragments.CH2OCH2HOCOOH4-Hydroxymethylphenoxyaceticacid(HMPA)linker:AttachtoaminomethylPS-DVBresin(CH2)3OCH2HOCOOHOCH34-(4-Hydroxymethyl-3-methoxyphenoxy)butyricacid(HMPB)linker:RemovalofthepeptidewithTFARemovalofthepeptidewithdilutedTFAAttachtoaminomethylPS-DVBresin多肽合成工藝流程2-Chlorotritylchloride(ClTrt)resin:ClClPAttachmentofthefirstaminoacid:ThefinalcleavageresultsinpeptideswithCOOHgroupattheC-terminusCleavagewith90-95%TFA+scavangersresultsinfreepeptidesCleavagewithAcOH:MeOH(TFE):DCM(1:1:8or2:2:6)resultsinprotectedpeptides(availableforfragmentcondensation).ClTrtresinpreventsthediketopiperazineformation!AttachmentofCysandHisderivativestotheresinisfreefromenantiomerisation!1gClTrt-resin+2mmolFmoc-Aaa(X)-OH+8mmolDIEAin3-5mLDCM,for1.5hthen0.8mLMeOHtoblocktheunreactedgroupswashingwithDCM,iPrOH,MeOH,ether多肽合成工藝流程(Calculationoftheresincapacity)Determinationofloading10-20mgofdriedresinareweightedexactlyintoa100mLmeasuringflask(foraloadofca.0.5meq/g20mgissufficient);Piperidine/DMF(1:4,V/V)isaddedtothemark;Themixtureisshakenthoroughlyandleftfor25-30min;Theresinisfilteredoffandtheabsorbanceofthefiltrateismeasuredat301nm(e=7800).

NH2(mmol/g)=[A301.V(ml)/e301.m(mg)].1061.2.ca.4-6mgFmoc-Aaa-resinca.2mgFmoc-Gly-OH+400mL50%piperidine/DMF+400mL50%piperidine/DMF30minatRT,thenfiltration30minatRTdilutewithMeOHto25mLdilutewithMeOHto25mLCapacityoftheresin(mmol/g)=1000.mgly.Aresin301Mgly.mresin.Agly301Mgly=297多肽合成工藝流程RinkAmideResin:synthesisofpeptideswithCONH2C-terminusCHNH2PCH3CHNFmoc-HOCH2-POCH3COH3CleavagewithhighconcentrationofTFAcanleadtothebreakdownofthelinkerbyproducts.UselowTFAconcentrationand/ortrialkylsilanesinthecleavagemixture.Peptide-resinbondcanbedetachedwith5%TFA.Removalofprotectinggroupsneedsaseparatestep.CHNFmoc-HOCH2-CO-Nle-ROCH3COH3RinkAmide-AMandRinkAmide-MBHAresins:CHNH2P2Aminomethyl-PS-DVB4-methylbenzhydrylamine-PS-DVBPeptidecleavagewith90-95%TFAsolution.Nleisareferenceforquantitation.多肽合成工藝流程Pegylatedresins:compositionofpolyethyleneglycol(Mw:3000-4000)andlow-crosslinkerpolystyrenegel-typeresins.Advantages: excellentpressurestability(continuousflowsynthesis) excellentswellingproperties(alsoinwater) highdiffusionrates availablewithmanytypesoffunctionalgroups lowcapacity(0.2-0.6mmol/g),suitableforthesynthesis ofaggregatingpeptides,foronresincyclisationand fragmentcondansation.ThebasicpolymersupportisaminomethylPEG-PS-DVB(NovaSynR

TG)PEGNH2CH2OCH2HOCOOH4-hydroxymethylphenoxyaceticacidlinkerNovaSynR

TGAresinSimilartoWangresin多肽合成工藝流程HOCOOH4-carboxytrityllinkerNovaSynR

TGTalcoholresinBeforeusetheresinmustbeconvertedtothechlorideformbyheatingwithAcClorSOCl2intoluene.SimilartoClTrtresin.CHNH2OCH2OCH3COH32,4-dimethoxy-benzhydryllinkerNovaSynR

TGRresinSimilartoRinkAmideMBHAresinCOOH多肽合成工藝流程AppliedsidechainprotectinggroupsinFmoc-chemistry-OH(Ser,Thr,Tyr)Sidechainfunctionalgroupprotectinggroupname(abbreviation)CH3CCH3CH3tert-butyl(tBu)Trtgroupcanbeusedifon-resinderivatizationisrequired(glycosylation,phosphorylation).TrtcanbecleavedwithdilutedTFA,whiletBuneeds90%TFAsolutionforeffectiveremoval.-SH(Cys)trityl(Trt)CH2NHCOCH3acetamidomethyl(Acm)Forselectivedeprotection多肽合成工藝流程RacemisationduringtheattachmentofCysderivativestotheresinsinthepresenceofDMAP:Fmoc-Cys(Trt)-OH>Fmoc-Cys(Acm)-OHHowever,Fmoc-Cys(Acm)attheC-terminalresultesinsidereaction:CH2OPOCH2CCHNHCH2Acm-SCH2OPOCH2OCCNHCH2OpiperidinepiperidineCCHNHCH2ONH2OCCHNHCH2OHOCys McalcDAla Mcalc–34DL-Ala(Pip)Mcalc+41DL-Ser Mcalc–16多肽合成工藝流程Sidechainfunctionalgroupprotectinggroupname(abbreviation)tert-butyloxycarbonyl(Boc)eNH2(Lys)OOCCCH3CH3CH3Selectivelyremovableprotectinggroupsforpreparationofmodifiedpeptides(labeled,functionalised,branchedorcyclicpeptides):eNH2(Lys)CH34-methytrityl(Mtt)Mttcanberemovedselectivelywith1%TFA/DCMsolutioninthepresenceof3-5%TES(triethylsilane)atRTin15-30min.Trtgroupsmaybenotstableenoughunderthiscondition.多肽合成工藝流程Sidechainfunctionalgroupprotectinggroupname(abbreviation)eNH2(Lys)OCCH3CH3ORR=metil1,isopropyl21-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl(Dde)11-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl(ivDde)2ivDdeismorestableinbasiccleavagemixtureappliedforFmocremovalthanDde.Bothprotectinggroupscanberemovedwith2%NH2-NH2inDMFeNH2(Lys)OOCCH2CHCH2allyloxycarbonyl(Aloc)AlocprotectinggroupiscompatiblewithBocaswellasFmoc-chemistry.Itisstableinacidsandbases.ItcanberemovedinP(Ph)3byPd(0)catalysis.Topreventadditionondoublebondunderothercleavageconditionsapplicationofallylalcoholincleavagemixturesisrecommended.多肽合成工藝流程wCOOH(Asp,Glu)Sidechainfunctionalgroupprotectinggroupname(abbreviation)OCCH3CH3CH3tert-butylester(OtBu)Selectivelyremovableprotectinggroupsforpreparationofcyclicpeptides:(pairsofaminoandcarboxylprotectinggroups:Dde-ODmab,Aloc-OAll)CCH3CH3OOCH3CH3CHNHOCH24-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino}Benzylester(ODmab)SimilarlytoDdeandivDde,theDmabprotectinggroupcanberemovedwith2%hydrazineinDMF.OCH2CHCH2allylester(OAll)ItcanberemovedinP(Ph)3byPd(0)catalysis.多肽合成工藝流程Succinimideringformation(Asp):Acidcatalisedreactionresultsinaorb-Asp-peptides,however,piperidinecatalisedsidereactionunderFmoccleavageprocedureresultsinpiperidide:-Asp-Gly-NHCHCONHCH2COCH2COtBuO-Asu-Gly-NHCHNCH2CCOCH2OCONH-tBuOHNHCHCONHCH2COCH2CONHCHCH2CCOONHNCH2COpiperidinepiperidineNNM=Mcalc+57多肽合成工藝流程Applicationofothercleavagereagents(DBU,TBAF,DEA,morpholine)eliminatethepiperidideformation,butnotthesuccinimideformation.AdditionofHOBttothecleavagemixturecanreducethesuccinimideringclosure.ButthebestresultsmaygetwiththeuseofFmoc-(Hmb)-aminoacidderivatives:Hmb:2-hydroxy-4-methoxybenzyl(removablewithTFA)NHCHCONCH2COCH2COtBuOCH2CH3OHO(Hmb)aminoacidderivativesaresecundaryamines:RemovalofFmocgroupandtheattachementofthenextAspderivativeisdifficult,needslongertime.Ninhydrintestcan’tdetecttheefficacyofthecoupling.Fmoc-(Fmoc-Hmb)Gly-OH1g=370EUR(NovaBiochem)Theincreasingofthesolubilityofprotectedpeptidefragmentsaswellaspreventingofaggregationof”difficult”sequencescanbereachbytheapplicationofHmbgroups.多肽合成工藝流程Sidechainfunctionalgroupprotectinggroupname(abbreviation)wCONH2(Asn,Gln)trityl(Trt)ThesolubilityofFmoc-Asn-OHandFmoc-Gln-OHisextremelybad.TheTrtprotectinggroupincreasesthesolubilityandpreventsthedehydratationaswellasringclosuresidereactionsduringthesynthesis.N-terminalGlnorAsn-Gly(Arg,Ser,Ala,Asn)sequencemaycauseproblemsafterthecleavageoftheprotectinggroup.

NNH(His)ptimidazolgrouptert-butyloxymethyl(Bum)(p)CH2OCCH3CH3CH3ThesameproblemasincaseofBominBocstartegy.Don’tuseitforthesynthesisofpeptidescontainingCysattheN-terminal!多肽合成工藝流程Sidechainfunctionalgroupprotectinggroupname(abbreviation)NNH(His)ptimidazolgrouptrityl(Trt)(t)TrtgroupprotectsthetN.However,itsapplicationpreventsbothepimerisaton(notincaseofattachmenttoresins)andalkylation.-NH-C-NH2NHguanidinogroupCH3SOOCH3CH3CH3O4-methoxy-2,3,6-trimethylbenzene-sulfonyl(Mtr)(Arg)MtristoostableinTFA.Elevatedtemperature(30oC)and/orincreasedtime(4-6hrs)isnecessaryforeffectivecleavage.1MTMSOBr-thioanisol/TFAmixtureisanalternativecleavagemixturethatcanremoveMtrmoreeffectively.多肽合成工藝流程Sidechainfunctionalgroupprotectinggroupname(abbreviation)-NH-C-NH2NHguanidinogroup(Arg)2,2,5,7,8-pentamethyl-chroman-5-sulfonyl(Pmc)2,2,4,6,7-pentamethyl-dihydrobenzofurane-6-sulfonyl(Pbf)SOOCH3CH3CH3OCH3CH3SOOCH3CH3CH3OCH3CH3PmccanbecleavedwithTFAin2-3hrs,butPbfprotectinggroupcanberemoved1.5-2timesfasterthanPmc.PbfalsogivesrisetolesssulfonatedTrpbyproductthanPmcorMtr.UseFmoc-Arg(Pbf)-OHforthesynthesisofoligo-Argasacellpenetratingpeptide!多肽合成工藝流程Sidechainfunctionalgroupprotectinggroupname(abbreviation)indoleNH(Trp)tert-butyloxycarbonyl(Boc)OOCCCH3CH3CH3TheprotectionofindolesidechainofTrpisnotnecessary,buttheapplicationofBocgroupisrecommended.UnderTFAcleavagetheappearanceofinN-carboxyindoleprotectsTrpvsalkylationandsulfonation.inN-carboxygroupisremovedunderaqueousconditioninworkingupprocedure.ProtectionofthesidechainofMetisnotneededinFmoc-strategy.Fmoc/Bzl(benzyltypeprotectinggroupsforblockingofsidechains)strategyisappliedforthesynthesisofprotectedpeptidefragments,becauseofthebettersolubilityofbenzylprotectedfragmentsovertert-butylandtritylprotectedfragments.多肽合成工藝流程Washtheresin3xwithDMF;0.5-1.0mineachCleavageofFmocprotectionwith2%piperidine+2%DBU/DMF;2+2+5+10min*Washtheresin8xwithDMF;0.5-1.0mineach**Coupling:Fmoc-aminoacidderivative-DIC-HOBtinDMF***(3equiveachcalculatedtotheresincapacity);60minWashtheresin2xwithDMF;0.5-1.0mineachWashtheresin2xwithDCM;0.5-1.0mineachNinhydrinmonitoringSyntheticprotocolofFmoc-strategy(-)yellow(+)blue*DBUisthecleavagereagent,piperidineisforthecaptureofdibenzofulvene20%or50%piperidineinDMF,50%morpholineorDEAinDMFand20mMTBAFinDMFarealsousedascleavagemixture.**After4DMFwashing,IPAwashingmaybeappliedforshrinkingtheresin.AnunefficientremovalofbasefromtheresinmaycauseFmoccleavageinthenextcouplingstep.***DICisusedinsteadofDCCinthismethod,becauseofthelimitedsolubilityofDCUintheappliedsolvents.

多肽合成工藝流程N,N’-diisopropylcarbodiimide(DIC,DIPCDI))CouplingagentsNNCN,N’-dicyclohexylcarbodiimide(DCC)NNCCHHCCH3CH3H3CH3CNNHCOCOCHX-NHRX:Boc,FmocO-acyl-isoureaderivativesNNHCOCOCHX-NHRN-acyl-ureaderivativesO-NacylshiftNHNHCOureaderivatives:DCU,DIUCOCHX-NHROBtHOBtinsituactiveester+多肽合成工藝流程NNNOH1-hydroxybenzotriazole(HOBt)NNNOHN1-hydroxy-7-aza-benzotriazole(HOAt)NNNOP+(CH3)2NN(CH3)2N(CH3)2PF6-benzotriazol-1-yl-oxy-tris(dimethylamino)-phosphoniumhexafluorophosphate(BOP)NNNOP+PF6-NNNbenzotriazol-1-yl-oxy-tris(pyrrolidino)phosphoniumhexafluorophosphate(PyBOP)Theydon’tneedDCCorDICforpreparationofinsituactiveesterHexamethylphosphoramide(carcinogen)!>AOPPyAOP多肽合成工藝流程2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluroniumhexafluorophosphateHBTUNNNOC+(CH3)2NN(CH3)2PF6-NNNOC+(CH3)2NN(CH3)2BF4-2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluroniumtetrafluoroborateTBTUNNNOC+N(CH3)2PF6-N(CH3)2+-N-[(1H-benzotriazol-1-yl)(dimethylamino)-methylene]-N-methanaminiumhexafluorophosphateN-oxideAccordingtoNMRandr?ntgendiffractionstudiesanewstructurewassuggested:HATU,TATU,HBPyU,HAPyU,etc.多肽合成工藝流程GuanylationwithuroniumtypecouplingreagentsNNNOC+(CH3)2NN(CH3)2PF6-COCHHNHRNH-PEPTIDE+COCHNHRNH-PEPTIDEC(CH3)2NN+(CH3)2+HOBtPF6-Don’tuseexcessofcouplingagent(cyclisation,fragmentcondensation);Makepreactivationoftheincom