多肽合成工艺流程(英文版)

SolidphasepeptidesynthesisPartIIApplicationofFmoc/tBustrategyGáborMezőResearchGroupofPeptideChemistryHungarianAcademyofSciencesEötvösL.UniversityBudapest,HungaryOutlineResins;Protectinggroups;Syntheticprotocol;Monitoring;Cleavagetechnics;Sidereactions;Fmoc/tBu:NHCCH2OCNHCHCH2COOCOHCCH3CH3CH3NHCHCOOCH2CH2OCH2OOCCH3CH3CH3CRFmoctert-butyl..piperidineTFAWang-resinFmoc-Asp(OtBu)-Tyr(tBu)-WangresinTypeofresinsforFmoc-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!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-DVBresin2-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(Calculationoftheresincapacity)Determinationofloading10-20mgofdriedresinareweightedexactlyintoa100mLmeasuringflask(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=297RinkAmideResin: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.Pegylatedresins:compositionofpolyethyleneglycol(Mw:3000-4000)andlow-crosslinkerpolystyrenegel-typeresins.Advantages:excellentpressurestability(continuousflowsynthesis)excellentswellingproperties(alsoinwater)highdiffusionratesavailablewithmanytypesoffunctionalgroupslowcapacity(0.2-0.6mmol/g),suitableforthesynthesisofaggregatingpeptides,foronresincyclisationandfragmentcondansation.ThebasicpolymersupportisaminomethylPEG-PS-DVB(NovaSynRTG)PEGNH2CH2OCH2HOCOOH4-hydroxymethylphenoxyaceticacidlinkerNovaSynRTGAresinSimilartoWangresinHOCOOH4-carboxytrityllinkerNovaSynRTGTalcoholresinBeforeusetheresinmustbeconvertedtothechlorideformbyheatingwithAcClorSOCl2intoluene.SimilartoClTrtresin.CHNH2OCH2OCH3COH32,4-dimethoxy-benzhydryllinkerNovaSynRTGRresinSimilartoRinkAmideMBHAresinCOOHAppliedsidechainprotectinggroupsinFmoc-chemistry-OH(Ser,Thr,Tyr)Sidechainfunctionalgroupprotectinggroupname(abbreviation)CH3CCH3CH3tert-butyl(tBu)Trtgroupcanbeusedifon-resinderivatizationisrequired(glycosylation,phosphorylation).TrtcanbecleavedwithdilutedTFA,whiletBuneeds90%TFAsolutionforeffectiveremoval.-SH(Cys)trityl(Trt)CH2NHCOCH3acetamidomethyl(Acm)ForselectivedeprotectionRacemisationduringtheattachmentofCysderivativestotheresinsinthepresenceofDMAP:Fmoc-Cys(Trt)-OHFmoc-Cys(Acm)-OHHowever,Fmoc-Cys(Acm)attheC-terminalresultesinsidereaction:CH2OPOCH2CCHNHCH2Acm-SCH2OPOCH2OCCNHCH2OpiperidinepiperidineCCHNHCH2ONH2OCCHNHCH2OHOCysMcalcDAlaMcalc–34DL-Ala(Pip)Mcalc+41DL-SerMcalc–16Sidechainfunctionalgroupprotectinggroupname(abbreviation)tert-butyloxycarbonyl(Boc)eNH2(Lys)OOCCCH3CH3CH3Selectivelyremovableprotectinggroupsforpreparationofmodifiedpeptides(labeled,functionalised,branchedorcyclicpeptides):eNH2(Lys)CH34-methytrityl(Mtt)Mttcanberemovedselectivelywith1%TFA/DCMsolutioninthepresenceof3-5%TES(triethylsilane)atRTin15-30min.Trtgroupsmaybenotstableenoughunderthiscondition.Sidechainfunctionalgroupprotectinggroupname(abbreviation)eNH2(Lys)OCCH3CH3ORR=metil1,isopropyl21-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl(Dde)11-(4,4-dimeth