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SolidphasepeptidesynthesisPartIIApplicationofFmoc/tBustrategyGáborMezőResearchGroupofPeptideChemistryHungarianAcademyofSciencesEötvös

L.UniversityBudapest,HungaryOutline✓Resins;✓Protectinggroups;✓Syntheticprotocol;✓Monitoring;✓Cleavagetechnics;✓Sidereactions;Fmoc/tBu:NHCCH2OCNHCH

CH2COOCOHCCH3CH3CH3NHCHCOOCH2CH2OCH2OOCCH3CH3CH3CRFmoctert-butyl..piperidineTFAWang-resinFmoc-Asp(OtBu)-

Tyr(tBu)-WangresinTypeofresinsforFmoc-chemistryTherearemanydifferentresinsandmostofthemareusedforspecialcasesandinindividuallaboratories.H

ereonlythemostwidelyappliedresinswillbepresented.ResinsarebasedonPS-DVB(1%)copolymer.4-Alkoxybenzylalcohol(

Wang)resin:CH2OPCH2HOAttachmentofthefirstaminoacid:Fmoc-Aaa(X)-OH:DIC:DMAP(2:2:0.2equivtotheresinOHcontent)inDMF,1hatRT.Th

efinalcleavageresultsinpeptideswithCOOHgroupattheC-terminusTheresinisnotavailableforthesynthesisofpeptideswithasequenceontheC-terminalthatissensitiv

efordiketopiperazineformation!SASRIN(SuperAcidSensitiveResIN)(2-methoxy-4-alkoxybenzyl-alcoholresin)CH2O

PCH2HOOCH3Peptideiscleavablewith0.5-1.0%TFAinDCMresultedinprotectedpeptidefragments.CH2OCH2HOCOOH4-Hyd

roxymethylphenoxyaceticacid(HMPA)linker:AttachtoaminomethylPS-DVBresin(CH2)3OCH2HOCOOHOCH34-(4-Hydroxymethyl-3-methoxyp

henoxy)butyricacid(HMPB)linker:RemovalofthepeptidewithTFARemovalofthepeptidewithdilutedTFAAttachtoaminomethylPS-DVBresin2-Chlo

rotritylchloride(ClTrt)resin:ClClPAttachmentofthefirstaminoacid:ThefinalcleavageresultsinpeptideswithCOOHgroupattheC-ter

minusCleavagewith90-95%TFA+scavangersresultsinfreepeptidesCleavagewithAcOH:MeOH(TFE):DCM(1:1:8or2:2:6)results

inprotectedpeptides(availableforfragmentcondensation).ClTrtresinpreventsthediketopiperazineformation!Attachment

ofCysandHisderivativestotheresinisfreefromenantiomerisation!1gClTrt-resin+2mmolFmoc-Aaa(X)-OH+8mmolDIEAi

n3-5mLDCM,for1.5hthen0.8mLMeOHtoblocktheunreactedgroupswashingwithDCM,iPrOH,MeOH,ether(Calculationoftheresincapacity)Det

erminationofloading✓10-20mgofdriedresinareweightedexactlyintoa100mLmeasuringflask(foraloadofca.0.5meq/g20mgissufficient);✓Piperidine/DMF(1:4,V/V)i

saddedtothemark;✓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%piper

idine/DMF30minatRT,thenfiltration30minatRTdilutewithMeOHto25mLdilutewithMeOHto25mLCapacityoftheresin(mmol/g)=1000.

mgly.Aresin301Mgly.mresin.Agly301Mgly=297RinkAmideResin:synthesisofpeptideswithCONH2C-terminusCHNH2PC

H3CHNFmoc-HOCH2-POCH3COH3CleavagewithhighconcentrationofTFAcanleadtothebreakdownofthelinkerbyproducts.UselowTFAc

oncentrationand/ortrialkylsilanesinthecleavagemixture.Peptide-resinbondcanbedetachedwith5%TFA.Removalofprotectinggroupsneeds

aseparatestep.CHNFmoc-HOCH2-CO-Nle-ROCH3COH3RinkAmide-AMandRinkAmide-MBHAresins:CHNH2P2Aminomethyl-PS-DVB4-met

hylbenzhydrylamine-PS-DVBPeptidecleavagewith90-95%TFAsolution.Nleisareferenceforquantitation.Pegylatedresins:c

ompositionofpolyethyleneglycol(Mw:3000-4000)andlow-crosslinkerpolystyrenegel-typeresins.Advantages:excellentpressurestability(continuousflowsynthesis

)excellentswellingproperties(alsoinwater)highdiffusionratesavailablewithmanytypesoffunctionalgroupslowcapacity(0.2-0.6mmol/g),suit

ableforthesynthesisofaggregatingpeptides,foronresincyclisationandfragmentcondansation.ThebasicpolymersupportisaminomethylPEG-PS-DVB

(NovaSynRTG)PEGNH2CH2OCH2HOCOOH4-hydroxymethylphenoxyaceticacidlinkerNovaSynRTGAresinSimilartoWangresinHOCOOH4-carboxytrityllinkerNovaSynRTGTalc

oholresinBeforeusetheresinmustbeconvertedtothechlorideformbyheatingwithAcClorSOCl2intoluene.SimilartoClTrtres

in.CHNH2OCH2OCH3COH32,4-dimethoxy-benzhydryllinkerNovaSynRTGRresinSimilartoRinkAmideMBHAresinCOOHAppliedsidechainprotectinggroupsinFm

oc-chemistry-OH(Ser,Thr,Tyr)Sidechainfunctionalgroupprotectinggroupname(abbreviation)CH3CCH3CH3tert-butyl

(tBu)Trtgroupcanbeusedifon-resinderivatizationisrequired(glycosylation,phosphorylation).Trtcanbecleavedwithdil

utedTFA,whiletBuneeds90%TFAsolutionforeffectiveremoval.-SH(Cys)trityl(Trt)CH2NHCOCH3acetamidomethyl(Acm)ForselectivedeprotectionRacem

isationduringtheattachmentofCysderivativestotheresinsinthepresenceofDMAP:Fmoc-Cys(Trt)-OH>Fmoc-Cys(Acm)-OHHowever,Fmoc-Cys(A

cm)attheC-terminalresultesinsidereaction:CH2OPOCH2CCHNHCH2Acm-SCH2OPOCH2OCCNHCH2OpiperidinepiperidineCCHNHCH2ONH2OC

CHNHCH2OHOCysMcalcDAlaMcalc–34DL-Ala(Pip)Mcalc+41DL-SerMcalc–16Sidechainfunctionalgroupprotectinggroupname(abbreviation)t

ert-butyloxycarbonyl(Boc)eNH2(Lys)OOCCCH3CH3CH3Selectivelyremovableprotectinggroupsforpreparationofmodifiedpe

ptides(labeled,functionalised,branchedorcyclicpeptides):eNH2(Lys)CH34-methytrityl(Mtt)Mttcanberemovedselectiv

elywith1%TFA/DCMsolutioninthepresenceof3-5%TES(triethylsilane)atRTin15-30min.Trtgroupsmaybenotstableeno

ughunderthiscondition.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-ylid

ene)-3-methylbutyl(ivDde)2ivDdeismorestableinbasiccleavagemixtureappliedforFmocremovalthanDde.Bothprotectinggroupscanberemovedwith2%NH2-NH2inDMFeN

H2(Lys)OOCCH2CHCH2allyloxycarbonyl(Aloc)AlocprotectinggroupiscompatiblewithBocaswellasFmoc-chemistry.Itisstable

inacidsandbases.ItcanberemovedinP(Ph)3byPd(0)catalysis.Topreventadditionondoublebondunderothercleavageconditionsapplicationofallylalcoholincleavag

emixturesisrecommended.wCOOH(Asp,Glu)Sidechainfunctionalgroupprotectinggroupname(abbreviation)OCCH3CH3CH3tert-butylester(OtBu)Selectivelyremov

ableprotectinggroupsforpreparationofcyclicpeptides:(pairsofaminoandcarboxylprotectinggroups:Dde-ODmab,Aloc-OAll)CCH3CH3OOCHNHOCH24-{N-[1-(4

,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino}Benzylester(ODmab)SimilarlytoDdeandivDde,theDmabprotectinggroup

canberemovedwith2%hydrazineinDMF.OCH2CHCH2allylester(OAll)ItcanberemovedinP(Ph)3byPd(0)catalysis.Succinimideringformation(Asp):Acidcatalise

dreactionresultsinaorb-Asp-peptides,however,piperidinecatalisedsidereactionunderFmoccleavageprocedureresultsinpiperi

dide:-Asp-Gly-NHCHCONHCH2COCH2COtBuO-Asu-Gly-NHCHNCH2CCOCH2OCONH-tBuOHNHCHCONHCH2COCH2CONHCHCH2CCOONHNCH2COpiperidinepiperidineN

NM=Mcalc+57Applicationofothercleavagereagents(DBU,TBAF,DEA,morpholine)eliminatethepiperidideformation,butnotthesuccinimid

eformation.AdditionofHOBttothecleavagemixturecanreducethesuccinimideringclosure.ButthebestresultsmaygetwiththeuseofFmoc-(Hmb)-aminoacidderi

vatives:Hmb:2-hydroxy-4-methoxybenzyl(removablewithTFA)NHCHCONCH2COCH2COtBuO(Hmb)aminoacidderivativesaresecundaryami

nes:RemovalofFmocgroupandtheattachementofthenextAspderivativeisdifficult,needslongertime.Ninhydrintestcan’tdetecttheefficacyofthecoupling.Fmoc-(Fmo

c-Hmb)Gly-OH1g=370EUR(NovaBiochem)Theincreasingofthesolubilityofprotectedpeptidefragmentsaswellaspreventingofaggre

gationof”difficult”sequencescanbereachbytheapplicationofHmbgroups.Sidechainfunctionalgroupprotectinggroupname(abbreviation)wCONH2(Asn,Gl

n)trityl(Trt)ThesolubilityofFmoc-Asn-OHandFmoc-Gln-OHisextremelybad.TheTrtprotectinggroupincreasesthesolubilityandpreventsthede

hydratationaswellasringclosuresidereactionsduringthesynthesis.N-terminalGlnorAsn-Gly(Arg,Ser,Ala,Asn)sequencemaycauseproblemsafterthecl

eavageoftheprotectinggroup.NNH(His)ptimidazolgrouptert-butyloxymethyl(Bum)(p)CH2OCCH3CH3CH3Thesameproblemasincase

ofBominBocstartegy.Don’tuseitforthesynthesisofpeptidescontainingCysattheN-terminal!Sidechainfunctionalgroupprotectinggroupname(abbreviation)NNH(Hi

s)ptimidazolgrouptrityl(Trt)(t)TrtgroupprotectsthetN.However,itsapplicationpreventsbothepimerisaton(notincaseofattachmenttoresins)andalkylati

on.-NH-C-NH2NHguanidinogroupCH3SOOCH3CH3CH3O4-methoxy-2,3,6-trimethylbenzene-sulfonyl(Mtr)(Arg)MtristoostableinT

FA.Elevatedtemperature(30oC)and/orincreasedtime(4-6hrs)isnecessaryforeffectivecleavage.1MTMSOBr-thioaniso

l/TFAmixtureisanalternativecleavagemixturethatcanremoveMtrmoreeffectively.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)SOOCH3CH3CH3OC

H3CH3SOOCH3CH3CH3OCH3CH3PmccanbecleavedwithTFAin2-3hrs,butPbfprotectinggroupcanberemoved1.5-2timesfasterthanPmc.Pbfalsogivesrise

tolesssulfonatedTrpbyproductthanPmcorMtr.UseFmoc-Arg(Pbf)-OHforthesynthesisofoligo-Argasacellpenetratingpeptide!Sidechainfunctionalgroupprotectingg

roupname(abbreviation)indoleNH(Trp)tert-butyloxycarbonyl(Boc)OOCCCH3CH3CH3TheprotectionofindolesidechainofTrpi

snotnecessary,buttheapplicationofBocgroupisrecommended.UnderTFAcleavagetheappearanceofinN-carboxyindoleprotectsTrpvs

alkylationandsulfonation.inN-carboxygroupisremovedunderaqueousconditioninworkingupprocedure.ProtectionofthesidechainofMeti

snotneededinFmoc-strategy.Fmoc/Bzl(benzyltypeprotectinggroupsforblockingofsidechains)strategyisappliedforthesynthesisofprotectedp

eptidefragments,becauseofthebettersolubilityofbenzylprotectedfragmentsovertert-butylandtritylprotectedfragments.1)Washtheresin3xwithDMF;0.5-1.

0mineach2)CleavageofFmocprotectionwith3)2%piperidine+2%DBU/DMF;2+2+5+10min*3)Washtheresin8xwithDMF;0.5-1.0mineach**4)Cou

pling:Fmoc-aminoacidderivative-DIC-HOBtinDMF***(3equiveachcalculatedtotheresincapacity);60min5)Washtheresin2xwit

hDMF;0.5-1.0mineach6)Washtheresin2xwithDCM;0.5-1.0mineach7)NinhydrinmonitoringSyntheticprotocolofFmoc-strategy(-)yellow(+)blue*DBUisthe

cleavagereagent,piperidineisforthecaptureofdibenzofulvene20%or50%piperidineinDMF,50%morpholineorDEAinDMFan

d20mMTBAFinDMFarealsousedascleavagemixture.**After4DMFwashing,IPAwashingmaybeappliedforshrinkingthere

sin.AnunefficientremovalofbasefromtheresinmaycauseFmoccleavageinthenextcouplingstep.***DICisusedinsteadofDCCinthismethod,becauseofthelimit

edsolubilityofDCUintheappliedsolvents.N,N’-diisopropylcarbodiimide(DIC,DIPCDI))CouplingagentsNNCN,N’-dicyclohexylcarbodiimide(DC

C)NNCCHHCCH3CH3H3CH3CNNHCOCOCHX-NHRX:Boc,FmocO-acyl-isoureaderivativesNNHCOCOCHX-NHRN-acyl-ureaderivativesO-NacylshiftNHNHCOureaderiva

tives:DCU,DIUCOCHX-NHROBtHOBtinsituactiveester+NNNOH1-hydroxybenzotriazole(HOBt)NNNOHN1-hydroxy-7-aza-benzotriazole(HOAt)NN

NOP+(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’tneedDCCorDICforpreparationo

finsituactiveesterHexamethylphosphoramide(carcinogen)!>AOPPyAOP2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluroniumhexafluorophosphateHBTUNN

NOC+(CH3)2NN(CH3)2PF6-NNNOC+(CH3)2NN(CH3)2BF4-2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluroniumtetraf

luoroborateTBTUNNNOC+N(CH3)2PF6-N(CH3)2+-N-[(1H-benzotriazol-1-yl)(dimethylamino)-methylene]-N-methanaminiumhexafluorophosphateN-oxideAcco

rdingtoNMRandröntgendiffractionstudiesanewstructurewassuggested:HATU,TATU,HBPyU,HAPyU,etc.Guanylationwithuroniumtypeco

uplingreagentsNNNOC+(CH3)2NN(CH3)2PF6-COCHHNHRNH-PEPTIDE+COCHNHRNH-PEPTIDEC(CH3)2NN+(CH3)2+HOBtPF6-Don’tuseexcessofcouplingagen

t(cyclisation,fragmentcondensation);Makepreactivationoftheincomingaminoacid;Apply:X-Aaa-OH:HBTU:DIEA=3:2.9:3(equiv)totheresincap

acity.FmoccleavageflowchartDoesthepeptidecontainN-terminalFmocgroup?yesnoRemoveFmocDoesthepeptidecontainArg,Met,TrporTrt?yesnoDoesthepeptidecontainAr

g,Met?UsecleavagemixtureAyesnoUsecleavagemixtureBDoesthepeptidecontainTrporTrt?noyesUsecleavagemixtureCA:0.5mLd.i.water9.5mL

TFAC:0.25mLEDT0.25mLd.i.Water9.50mLTFAB:0.75gcryst.phenol0.25mLEDT0.50mLthioanisole0.50mLd.i.water10mLTFABoc/BzlorFmoc/tBustrat

egyAminoacidderivativesandresinsforBoc-strategyisstillcheaper:Boc-Ala-OH(Mw:189)5g11EUR,1mmol0.416EURFmoc-Ala-OH(Mw:311)5g11EUR,

1mmol0.684EURBoc-Arg(Tos)-OH(Mw:429)5g32EUR,1mmol2.746EURFmoc-Arg(Pbf)-OH(Mw:649)5g90EUR,1mmol11.682EURMBHAresin(0.4-1

.2mmol/g)5g49EURRinkAmideMBHAresin(0.4-0.8mmol/g)5g168EURCleavageofprotectinggroups(decapeptide):15EUR(Boc),5EUR(Fmoc)DCM(forpeptides

ynthesis)49EUR/LDMF(forpeptidesynthesis)111EUR/LHowever,applicationofBoc-strategyneedsaspecialHFcleavageapparatus!ManysynthesizersaredesignedforFmo

cchemistry.TheyareTFAsensitive.Orderingofpiperidinemightneedallowance,becauseitisthestartingmaterialinthesynth

esisofmorphine.BocFmoc➢ItisbetterforavoidingDKPformation;➢ThereisnoproblemwiththeBoccleavage,soitisbetterincaseofpeptidest

hataggregateeasily.AggregatesaredestroyedineveryTFAcleavagestep;➢Becauseoftheextraneutralisationstep,thesyntheticcycletakeslongertime;➢Resinsfo

rBoc-strategyareavailableforFmoc-chemistry,too.Twostepscleavageproceduremayresultsinbettercrudeproduct.Firs

tstepTFAcleavage(sidechainprotectinggroups)thenHF(peptide-resinbond).Moresuitableforpreparationofbranchedpeptides.➢

ClTrtresinmustbeusedtopreventDKPformation;➢IncompleteFmocdeprotectionincaseofaggregatingpeptides;➢Itisbetterforacidsensitivepep

tides(Trp,Met),oxidation,alkylationcanbeavoided.Asp-Probondishighlyacidsensitive.➢especiallyrecommendedforO-

glycosylatedorsulfatedpeptides;➢BecauseoftheorthogonalityofNaandsidechainprotectinggroupsfullyprotectedsequencesca

nbeprepared.