【文档说明】多肽合成工艺流程(英文版).pptx，共(29)页，206.350 KB，由精品优选上传

转载请保留链接：https://www.ichengzhen.cn/view-331811.html

以下为本文档部分文字说明：

SolidphasepeptidesynthesisPartIIApplicationofFmoc/tBustrategyGáborMezőResearchGroupofPeptideChemistryHungarianAcademyofSciencesEötvösL.Universit

yBudapest,HungaryOutline✓Resins;✓Protectinggroups;✓Syntheticprotocol;✓Monitoring;✓Cleavagetechnics;✓

Sidereactions;Fmoc/tBu:NHCCH2OCNHCHCH2COOCOHCCH3CH3CH3NHCHCOOCH2CH2OCH2OOCCH3CH3CH3CRFmoctert-butyl..piperidineTFAWang-resinFmoc-Asp(OtBu)

-Tyr(tBu)-WangresinTypeofresinsforFmoc-chemistryTherearemanydifferentresinsandmostofthemareusedforspecialcasesandinind

ividuallaboratories.Hereonlythemostwidelyappliedresinswillbepresented.ResinsarebasedonPS-DVB(1%)copo

lymer.4-Alkoxybenzylalcohol(Wang)resin:CH2OPCH2HOAttachmentofthefirstaminoacid:Fmoc-Aaa(X)-OH:DIC:DMAP(2:2:0.2equivtotheresinOHcontent)inDM

F,1hatRT.ThefinalcleavageresultsinpeptideswithCOOHgroupattheC-terminusTheresinisnotavailableforthesynthesisofpeptideswithasequenceon

theC-terminalthatissensitivefordiketopiperazineformation!SASRIN(SuperAcidSensitiveResIN)(2-methoxy-4-alkoxybenz

yl-alcoholresin)CH2OPCH2HOOCH3Peptideiscleavablewith0.5-1.0%TFAinDCMresultedinprotectedpeptidefragments.CH2OCH2HOCOOH4-Hydro

xymethylphenoxyaceticacid(HMPA)linker:AttachtoaminomethylPS-DVBresin(CH2)3OCH2HOCOOHOCH34-(4-Hydroxymethyl-3-methoxyphenoxy)butyrica

cid(HMPB)linker:RemovalofthepeptidewithTFARemovalofthepeptidewithdilutedTFAAttachtoaminomethylPS-DVBresin2-Chlorotritylchloride(ClTr

t)resin:ClClPAttachmentofthefirstaminoacid:ThefinalcleavageresultsinpeptideswithCOOHgroupattheC-terminusCleav

agewith90-95%TFA+scavangersresultsinfreepeptidesCleavagewithAcOH:MeOH(TFE):DCM(1:1:8or2:2:6)resultsinprotectedpeptides(availabl

eforfragmentcondensation).ClTrtresinpreventsthediketopiperazineformation!AttachmentofCysandHisderivativestotheresin

isfreefromenantiomerisation!1gClTrt-resin+2mmolFmoc-Aaa(X)-OH+8mmolDIEAin3-5mLDCM,for1.5hthen0.8mLMeOHtoblocktheunreactedgroupswashi

ngwithDCM,iPrOH,MeOH,ether(Calculationoftheresincapacity)Determinationofloading✓10-20mgofdriedresinareweightede

xactlyintoa100mLmeasuringflask(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+400

mL50%piperidine/DMF30minatRT,thenfiltration30minatRTdilutewithMeOHto25mLdilutewithMeOHto25mLCapacityoftheresin(mmol/g)=1000.mgly.Aresin301Mgly.

mresin.Agly301Mgly=297RinkAmideResin:synthesisofpeptideswithCONH2C-terminusCHNH2PCH3CHNFmoc-HOCH2-POCH3COH3CleavagewithhighconcentrationofTFAcanl

eadtothebreakdownofthelinkerbyproducts.UselowTFAconcentrationand/ortrialkylsilanesinthecleavagemixture.Peptide-resinbondcanbedetache

dwith5%TFA.Removalofprotectinggroupsneedsaseparatestep.CHNFmoc-HOCH2-CO-Nle-ROCH3COH3RinkAmide-AMandRinkAm

ide-MBHAresins:CHNH2P2Aminomethyl-PS-DVB4-methylbenzhydrylamine-PS-DVBPeptidecleavagewith90-95%TFAso

lution.Nleisareferenceforquantitation.Pegylatedresins:compositionofpolyethyleneglycol(Mw:3000-4000)andlow-crosslinkerpolystyrenegel-typeres

ins.Advantages:excellentpressurestability(continuousflowsynthesis)excellentswellingproperties(alsoinwater)highdiffusionratesav

ailablewithmanytypesoffunctionalgroupslowcapacity(0.2-0.6mmol/g),suitableforthesynthesisofaggregatingpeptides,foronresincyclisationandfragmen

tcondansation.ThebasicpolymersupportisaminomethylPEG-PS-DVB(NovaSynRTG)PEGNH2CH2OCH2HOCOOH4-hydroxymethylphenoxyaceticacidlink

erNovaSynRTGAresinSimilartoWangresinHOCOOH4-carboxytrityllinkerNovaSynRTGTalcoholresinBeforeusetheres

inmustbeconvertedtothechlorideformbyheatingwithAcClorSOCl2intoluene.SimilartoClTrtresin.CHNH2OCH2OCH3COH

32,4-dimethoxy-benzhydryllinkerNovaSynRTGRresinSimilartoRinkAmideMBHAresinCOOHAppliedsidechainprotectinggroupsinFmoc-chemistry-OH(Ser,Thr,Tyr)Sidec

hainfunctionalgroupprotectinggroupname(abbreviation)CH3CCH3CH3tert-butyl(tBu)Trtgroupcanbeusedifon-resinderivatizati

onisrequired(glycosylation,phosphorylation).TrtcanbecleavedwithdilutedTFA,whiletBuneeds90%TFAsolutionforeffectiveremoval.-SH(Cys)trity

l(Trt)CH2NHCOCH3acetamidomethyl(Acm)ForselectivedeprotectionRacemisationduringtheattachmentofCysderivativestotheresinsintheprese

nceofDMAP:Fmoc-Cys(Trt)-OH>Fmoc-Cys(Acm)-OHHowever,Fmoc-Cys(Acm)attheC-terminalresultesinsidereaction:CH2OPOCH2CCHNHCH2Acm-SCH2OPOCH2OCCNHCH

2OpiperidinepiperidineCCHNHCH2ONH2OCCHNHCH2OHOCysMcalcDAlaMcalc–34DL-Ala(Pip)Mcalc+41DL-SerMcalc–16Sid

echainfunctionalgroupprotectinggroupname(abbreviation)tert-butyloxycarbonyl(Boc)eNH2(Lys)OOCCCH3CH3CH3Selectivelyremovableprotecti

nggroupsforpreparationofmodifiedpeptides(labeled,functionalised,branchedorcyclicpeptides):eNH2(Lys)CH34-methytrityl(Mtt)Mttca

nberemovedselectivelywith1%TFA/DCMsolutioninthepresenceof3-5%TES(triethylsilane)atRTin15-30min.Trtgroupsmaybenotstableenoughunderthiscondition.S

idechainfunctionalgroupprotectinggroupname(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)2ivDdeism

orestableinbasiccleavagemixtureappliedforFmocremovalthanDde.Bothprotectinggroupscanberemovedwith2%NH2-NH2inD

MFeNH2(Lys)OOCCH2CHCH2allyloxycarbonyl(Aloc)AlocprotectinggroupiscompatiblewithBocaswellasFmoc-chemi

stry.Itisstableinacidsandbases.ItcanberemovedinP(Ph)3byPd(0)catalysis.Topreventadditionondoublebondunderothercleavageconditionsapplicationofa

llylalcoholincleavagemixturesisrecommended.wCOOH(Asp,Glu)Sidechainfunctionalgroupprotectinggroupname

(abbreviation)OCCH3CH3CH3tert-butylester(OtBu)Selectivelyremovableprotectinggroupsforpreparationofcyclicpeptides:(pairsofaminoandcarb

oxylprotectinggroups:Dde-ODmab,Aloc-OAll)CCH3CH3OOCHNHOCH24-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbuty

l]-amino}Benzylester(ODmab)SimilarlytoDdeandivDde,theDmabprotectinggroupcanberemovedwith2%hydrazineinDMF.OCH2CHCH2allyl

ester(OAll)ItcanberemovedinP(Ph)3byPd(0)catalysis.Succinimideringformation(Asp):Acidcatalisedreactionresults

inaorb-Asp-peptides,however,piperidinecatalisedsidereactionunderFmoccleavageprocedureresultsinpiperidide:-Asp-Gly-NHCHCONHCH2COCH2COtBuO-Asu-Gl

y-NHCHNCH2CCOCH2OCONH-tBuOHNHCHCONHCH2COCH2CONHCHCH2CCOONHNCH2COpiperidinepiperidineNNM=Mcalc+57Applicationofothercleavagereagents(DBU,

TBAF,DEA,morpholine)eliminatethepiperidideformation,butnotthesuccinimideformation.AdditionofHOBttothecleavagemixturecanreducethesuccinimideringc

losure.ButthebestresultsmaygetwiththeuseofFmoc-(Hmb)-aminoacidderivatives:Hmb:2-hydroxy-4-methoxybenzyl(removablewithTFA)NHCHCON

CH2COCH2COtBuO(Hmb)aminoacidderivativesaresecundaryamines:RemovalofFmocgroupandtheattachementofthenextAspderivativeisdi

fficult,needslongertime.Ninhydrintestcan’tdetecttheefficacyofthecoupling.Fmoc-(Fmoc-Hmb)Gly-OH1g=370EUR(NovaBiochem)Theincreasing

ofthesolubilityofprotectedpeptidefragmentsaswellaspreventingofaggregationof”difficult”sequencescanbereachbytheapplicationofHmbgroups.Si

dechainfunctionalgroupprotectinggroupname(abbreviation)wCONH2(Asn,Gln)trityl(Trt)ThesolubilityofFmoc-

Asn-OHandFmoc-Gln-OHisextremelybad.TheTrtprotectinggroupincreasesthesolubilityandpreventsthedehydratationaswellasringclosur

esidereactionsduringthesynthesis.N-terminalGlnorAsn-Gly(Arg,Ser,Ala,Asn)sequencemaycauseproblemsafterthecleavageoftheprot

ectinggroup.NNH(His)ptimidazolgrouptert-butyloxymethyl(Bum)(p)CH2OCCH3CH3CH3ThesameproblemasincaseofBominBocstartegy.Do

n’tuseitforthesynthesisofpeptidescontainingCysattheN-terminal!Sidechainfunctionalgroupprotectinggroupnam

e(abbreviation)NNH(His)ptimidazolgrouptrityl(Trt)(t)TrtgroupprotectsthetN.However,itsapplicationpreventsbothepimerisaton(notincaseofattachmenttore

sins)andalkylation.-NH-C-NH2NHguanidinogroupCH3SOOCH3CH3CH3O4-methoxy-2,3,6-trimethylbenzene-sulfonyl(M

tr)(Arg)MtristoostableinTFA.Elevatedtemperature(30oC)and/orincreasedtime(4-6hrs)isnecessaryforeffectivecleavage.1MTMSOB

r-thioanisol/TFAmixtureisanalternativecleavagemixturethatcanremoveMtrmoreeffectively.Sidechainfunctionalgroupprotectinggroupname

(abbreviation)-NH-C-NH2NHguanidinogroup(Arg)2,2,5,7,8-pentamethyl-chroman-5-sulfonyl(Pmc)2,2,4,6,7-pentamethyl-dihydrobenzofuran

e-6-sulfonyl(Pbf)SOOCH3CH3CH3OCH3CH3SOOCH3CH3CH3OCH3CH3PmccanbecleavedwithTFAin2-3hrs,butPbfprotectinggroupcanberemoved1.5-2timesfastertha

nPmc.PbfalsogivesrisetolesssulfonatedTrpbyproductthanPmcorMtr.UseFmoc-Arg(Pbf)-OHforthesynthesisofoligo-Argasacellpenetratingpept

ide!Sidechainfunctionalgroupprotectinggroupname(abbreviation)indoleNH(Trp)tert-butyloxycarbonyl(Boc)OOCCCH3CH3CH3Theprotectiono

findolesidechainofTrpisnotnecessary,buttheapplicationofBocgroupisrecommended.UnderTFAcleavagetheappearanceofinN-carboxyindol

eprotectsTrpvsalkylationandsulfonation.inN-carboxygroupisremovedunderaqueousconditioninworkingupprocedure.ProtectionofthesidechainofMetisnotneededi

nFmoc-strategy.Fmoc/Bzl(benzyltypeprotectinggroupsforblockingofsidechains)strategyisappliedforthesynthesisofprotectedpeptidefragments,becauseof

thebettersolubilityofbenzylprotectedfragmentsovertert-butylandtritylprotectedfragments.1)Washtheresin3xwithDMF;0.5-1.0mineach2)Cl

eavageofFmocprotectionwith3)2%piperidine+2%DBU/DMF;2+2+5+10min*3)Washtheresin8xwithDMF;0.5-1.0mineach**4)Coup

ling:Fmoc-aminoacidderivative-DIC-HOBtinDMF***(3equiveachcalculatedtotheresincapacity);60min5)Washtheresin2xwithDMF;0.5-1.0min

each6)Washtheresin2xwithDCM;0.5-1.0mineach7)NinhydrinmonitoringSyntheticprotocolofFmoc-strategy(-)yellow(+)blue*DBUisthecleavagereagent,piperid

ineisforthecaptureofdibenzofulvene20%or50%piperidineinDMF,50%morpholineorDEAinDMFand20mMTBAFinDMFarealsousedascleavagemixture.**After4DMFwashing

,IPAwashingmaybeappliedforshrinkingtheresin.AnunefficientremovalofbasefromtheresinmaycauseFmoccleavagein

thenextcouplingstep.***DICisusedinsteadofDCCinthismethod,becauseofthelimitedsolubilityofDCUintheappliedsolvents.N,N’-diisoprop

ylcarbodiimide(DIC,DIPCDI))CouplingagentsNNCN,N’-dicyclohexylcarbodiimide(DCC)NNCCHHCCH3CH3H3CH3CNNHCOCOCHX-NH

RX:Boc,FmocO-acyl-isoureaderivativesNNHCOCOCHX-NHRN-acyl-ureaderivativesO-NacylshiftNHNHCOureaderivatives:DCU,DIUCOCHX-NHROBtHO

Btinsituactiveester+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)phosphoniumhexafluoro

phosphate(PyBOP)Theydon’tneedDCCorDICforpreparationofinsituactiveesterHexamethylphosphoramide(carcinogen)!>AOPPyAOP2-(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-metha

naminiumhexafluorophosphateN-oxideAccordingtoNMRandröntgendiffractionstudiesanewstructurewassuggested:HATU,TATU,HBPyU,HAPyU,etc.

GuanylationwithuroniumtypecouplingreagentsNNNOC+(CH3)2NN(CH3)2PF6-COCHHNHRNH-PEPTIDE+COCHNHRNH-PEPTIDEC(CH3)2NN+(CH3)2+HOBtPF6-Don’tuseexcesso

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

)totheresincapacity.FmoccleavageflowchartDoesthepeptidecontainN-terminalFmocgroup?yesnoRemoveFmocDoesthepeptidecontainArg,Met,TrporTrt?

yesnoDoesthepeptidecontainArg,Met?UsecleavagemixtureAyesnoUsecleavagemixtureBDoesthepeptidecontainTrporTrt?noyesUsecleavag

emixtureCA:0.5mLd.i.water9.5mLTFAC:0.25mLEDT0.25mLd.i.Water9.50mLTFAB:0.75gcryst.phenol0.25mLEDT0.50mLthioan

isole0.50mLd.i.water10mLTFABoc/BzlorFmoc/tBustrategyAminoacidderivativesandresinsforBoc-strategyisstillcheaper:Boc-Ala-OH(Mw:189)5g11EUR,1mmol0.416

EURFmoc-Ala-OH(Mw:311)5g11EUR,1mmol0.684EURBoc-Arg(Tos)-OH(Mw:429)5g32EUR,1mmol2.746EURFmoc-Arg(Pbf)-OH(Mw:649)5g90EUR,1mmol11.682EURMBHAresi

n(0.4-1.2mmol/g)5g49EURRinkAmideMBHAresin(0.4-0.8mmol/g)5g168EURCleavageofprotectinggroups(decapeptide):15EUR(Boc),5

EUR(Fmoc)DCM(forpeptidesynthesis)49EUR/LDMF(forpeptidesynthesis)111EUR/LHowever,applicationofBoc-strategyneedsas

pecialHFcleavageapparatus!ManysynthesizersaredesignedforFmocchemistry.TheyareTFAsensitive.Orderingofpiperidinemightneedallowance,becauseitisthestar

tingmaterialinthesynthesisofmorphine.BocFmoc➢ItisbetterforavoidingDKPformation;➢ThereisnoproblemwiththeBoccleavage,soitisbetterincaseofpepti

desthataggregateeasily.AggregatesaredestroyedineveryTFAcleavagestep;➢Becauseoftheextraneutralisationstep,thesynt

heticcycletakeslongertime;➢ResinsforBoc-strategyareavailableforFmoc-chemistry,too.Twostepscleavageproceduremayresultsinbettercrudeproduct.Fir

ststepTFAcleavage(sidechainprotectinggroups)thenHF(peptide-resinbond).Moresuitableforpreparationofbranchedpeptides.➢ClTrtres

inmustbeusedtopreventDKPformation;➢IncompleteFmocdeprotectionincaseofaggregatingpeptides;➢Itisbetterforacidsensitivepeptides(Tr

p,Met),oxidation,alkylationcanbeavoided.Asp-Probondishighlyacidsensitive.➢especiallyrecommendedforO-glycosylatedorsulfatedpeptides;➢Becauseoftheo

rthogonalityofNaandsidechainprotectinggroupsfullyprotectedsequencescanbeprepared.