【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/tBustrategyGáborMezőResearchGroupofPeptideChemistryHungarian

AcademyofSciencesEötvösL.UniversityBudapest,HungaryOutline✓Resins;✓Protectinggroups;✓Syntheticprotocol;✓

Monitoring;✓Cleavagetechnics;✓Sidereactions;Fmoc/tBu:NHCCH2OCNHCHCH2COOCOHCCH3CH3CH3NHCHCOOCH2CH2OCH2OOCCH3CH3CH3CRFmoctert-butyl..piperidine

TFAWang-resinFmoc-Asp(OtBu)-Tyr(tBu)-WangresinTypeofresinsforFmoc-chemistryTherearemanydifferentresinsandmost

ofthemareusedforspecialcasesandinindividuallaboratories.Hereonlythemostwidelyappliedresinswillbepresented.ResinsarebasedonPS-DVB(1%)copolymer.4-Al

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

cleavageresultsinpeptideswithCOOHgroupattheC-terminusTheresinisnotavailableforthesynthesisofpeptideswithasequenceo

ntheC-terminalthatissensitivefordiketopiperazineformation!SASRIN(SuperAcidSensitiveResIN)(2-methoxy-4-alkoxybe

nzyl-alcoholresin)CH2OPCH2HOOCH3Peptideiscleavablewith0.5-1.0%TFAinDCMresultedinprotectedpeptidefragments.CH2OCH2HOCOOH4-Hydroxymethylphenoxya

ceticacid(HMPA)linker:AttachtoaminomethylPS-DVBresin(CH2)3OCH2HOCOOHOCH34-(4-Hydroxymethyl-3-methoxyphenoxy)butyricacid(HMPB)

linker:RemovalofthepeptidewithTFARemovalofthepeptidewithdilutedTFAAttachtoaminomethylPS-DVBresin2-Chlorotritylchloride(ClTrt)resin:ClClPAttachm

entofthefirstaminoacid:ThefinalcleavageresultsinpeptideswithCOOHgroupattheC-terminusCleavagewith90-95%TFA+scav

angersresultsinfreepeptidesCleavagewithAcOH: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(C

alculationoftheresincapacity)Determinationofloading✓10-20mgofdriedresinareweightedexactlyintoa100mLmeasuringflask(foraloadofca.0.5meq/g20m

gissufficient);✓Piperidine/DMF(1:4,V/V)isaddedtothemark;✓Themixtureisshakenthoroughlyandleftfor25-30min;✓Theresinisfilteredoffandtheabsorbance

ofthefiltrateismeasuredat301nm(e=7800).NH2(mmol/g)=[A301.V(ml)/e301.m(mg)].1061.2.ca.4-6mgFmoc-Aaa-resinca.2mgFmoc-Gly-OH+400mL50%piper

idine/DMF+400mL50%piperidine/DMF30minatRT,thenfiltration30minatRTdilutewithMeOHto25mLdilutewithMeOHto25mLCapacityoftheresin(mmol/g)=1000.mgly.Are

sin301Mgly.mresin.Agly301Mgly=297RinkAmideResin:synthesisofpeptideswithCONH2C-terminusCHNH2PCH3CHNFmoc-HOCH2-POC

H3COH3CleavagewithhighconcentrationofTFAcanleadtothebreakdownofthelinkerbyproducts.UselowTFAconcentrationand/

ortrialkylsilanesinthecleavagemixture.Peptide-resinbondcanbedetachedwith5%TFA.Removalofprotectinggroupsneedsaseparatestep.CHNFmoc-HOCH2-CO-N

le-ROCH3COH3RinkAmide-AMandRinkAmide-MBHAresins:CHNH2P2Aminomethyl-PS-DVB4-methylbenzhydrylamine-PS-DVBPeptidecleavagewith90-95%TFAsolution.Nleisar

eferenceforquantitation.Pegylatedresins:compositionofpolyethyleneglycol(Mw:3000-4000)andlow-crosslinkerpoly

styrenegel-typeresins.Advantages:excellentpressurestability(continuousflowsynthesis)excellentswellingproperties(alsoinwater)highdiffus

ionratesavailablewithmanytypesoffunctionalgroupslowcapacity(0.2-0.6mmol/g),suitableforthesynthesisofaggregati

ngpeptides,foronresincyclisationandfragmentcondansation.ThebasicpolymersupportisaminomethylPEG-PS-DVB(No

vaSynRTG)PEGNH2CH2OCH2HOCOOH4-hydroxymethylphenoxyaceticacidlinkerNovaSynRTGAresinSimilartoWangresinHOCOOH4-carboxytrityllinkerNovaSynRTGTalc

oholresinBeforeusetheresinmustbeconvertedtothechlorideformbyheatingwithAcClorSOCl2intoluene.SimilartoClTrtre

sin.CHNH2OCH2OCH3COH32,4-dimethoxy-benzhydryllinkerNovaSynRTGRresinSimilartoRinkAmideMBHAresinCOOHAppliedsi

dechainprotectinggroupsinFmoc-chemistry-OH(Ser,Thr,Tyr)Sidechainfunctionalgroupprotectinggroupname(abbreviation)CH3CCH3CH

3tert-butyl(tBu)Trtgroupcanbeusedifon-resinderivatizationisrequired(glycosylation,phosphorylation).TrtcanbecleavedwithdilutedTFA,

whiletBuneeds90%TFAsolutionforeffectiveremoval.-SH(Cys)trityl(Trt)CH2NHCOCH3acetamidomethyl(Acm)ForselectivedeprotectionRacemisationduringt

heattachmentofCysderivativestotheresinsinthepresenceofDMAP:Fmoc-Cys(Trt)-OH>Fmoc-Cys(Acm)-OHHowever,Fmoc-Cys(Acm)attheC-terminalresultesinside

reaction:CH2OPOCH2CCHNHCH2Acm-SCH2OPOCH2OCCNHCH2OpiperidinepiperidineCCHNHCH2ONH2OCCHNHCH2OHOCysMcalcDAlaMcalc–34DL-Ala(Pip)Mcalc+4

1DL-SerMcalc–16Sidechainfunctionalgroupprotectinggroupname(abbreviation)tert-butyloxycarbonyl(Boc)eNH2(Lys)OOCCCH3CH3CH3Selectivelyremovab

leprotectinggroupsforpreparationofmodifiedpeptides(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-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl(

ivDde)2ivDdeismorestableinbasiccleavagemixtureappliedforFmocremovalthanDde.Bothprotectinggroupscanberemove

dwith2%NH2-NH2inDMFeNH2(Lys)OOCCH2CHCH2allyloxycarbonyl(Aloc)AlocprotectinggroupiscompatiblewithBocaswellasFmoc-chemistry.Itisstableinacidsand

bases.ItcanberemovedinP(Ph)3byPd(0)catalysis.Topreventadditionondoublebondunderothercleavageconditionsa

pplicationofallylalcoholincleavagemixturesisrecommended.wCOOH(Asp,Glu)Sidechainfunctionalgroupprotectinggroupname(abbreviation)OCCH3CH3CH3tert-butyl

ester(OtBu)Selectivelyremovableprotectinggroupsforpreparationofcyclicpeptides:(pairsofaminoandcarboxylprotectinggro

ups:Dde-ODmab,Aloc-OAll)CCH3CH3OOCHNHOCH24-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino}Benz

ylester(ODmab)SimilarlytoDdeandivDde,theDmabprotectinggroupcanberemovedwith2%hydrazineinDMF.OCH2CHCH2allylester

(OAll)ItcanberemovedinP(Ph)3byPd(0)catalysis.Succinimideringformation(Asp):Acidcatalisedreactionresultsinaorb-As

p-peptides,however,piperidinecatalisedsidereactionunderFmoccleavageprocedureresultsinpiperidide:-Asp-

Gly-NHCHCONHCH2COCH2COtBuO-Asu-Gly-NHCHNCH2CCOCH2OCONH-tBuOHNHCHCONHCH2COCH2CONHCHCH2CCOONHNCH2COpiperidinepiperidineNNM=Mcalc+57Applicationof

othercleavagereagents(DBU,TBAF,DEA,morpholine)eliminatethepiperidideformation,butnotthesuccinimidefo

rmation.AdditionofHOBttothecleavagemixturecanreducethesuccinimideringclosure.Butthebestresultsmaygetwiththeuse

ofFmoc-(Hmb)-aminoacidderivatives:Hmb:2-hydroxy-4-methoxybenzyl(removablewithTFA)NHCHCONCH2COCH2COtBuO(Hmb)aminoacidderivativ

esaresecundaryamines:RemovalofFmocgroupandtheattachementofthenextAspderivativeisdifficult,needslongertim

e.Ninhydrintestcan’tdetecttheefficacyofthecoupling.Fmoc-(Fmoc-Hmb)Gly-OH1g=370EUR(NovaBiochem)Theincreasingofthesol

ubilityofprotectedpeptidefragmentsaswellaspreventingofaggregationof”difficult”sequencescanbereachbytheapplicationof

Hmbgroups.Sidechainfunctionalgroupprotectinggroupname(abbreviation)wCONH2(Asn,Gln)trityl(Trt)ThesolubilityofFmoc-Asn-OHandFmoc-Gln-OHisex

tremelybad.TheTrtprotectinggroupincreasesthesolubilityandpreventsthedehydratationaswellasringclosuresidereactionsduringthesynthesis.N-termi

nalGlnorAsn-Gly(Arg,Ser,Ala,Asn)sequencemaycauseproblemsafterthecleavageoftheprotectinggroup.NNH(His

)ptimidazolgrouptert-butyloxymethyl(Bum)(p)CH2OCCH3CH3CH3ThesameproblemasincaseofBominBocstartegy.Don’tusei

tforthesynthesisofpeptidescontainingCysattheN-terminal!Sidechainfunctionalgroupprotectinggroupname(abbreviation)NNH(

His)ptimidazolgrouptrityl(Trt)(t)TrtgroupprotectsthetN.However,itsapplicationpreventsbothepimerisaton(notincaseofattachmenttoresins)anda

lkylation.-NH-C-NH2NHguanidinogroupCH3SOOCH3CH3CH3O4-methoxy-2,3,6-trimethylbenzene-sulfonyl(Mtr)(Arg)MtristoostableinTFA.Elevatedtemp

erature(30oC)and/orincreasedtime(4-6hrs)isnecessaryforeffectivecleavage.1MTMSOBr-thioanisol/TFAmixturei

sanalternativecleavagemixturethatcanremoveMtrmoreeffectively.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)SOOCH3

CH3CH3OCH3CH3SOOCH3CH3CH3OCH3CH3PmccanbecleavedwithTFAin2-3hrs,butPbfprotectinggroupcanberemoved1.5-2timesfasterthanPmc.Pbfalsogive

srisetolesssulfonatedTrpbyproductthanPmcorMtr.UseFmoc-Arg(Pbf)-OHforthesynthesisofoligo-Argasacellpenetratingpeptide!Sidechainfunctionalgrouppr

otectinggroupname(abbreviation)indoleNH(Trp)tert-butyloxycarbonyl(Boc)OOCCCH3CH3CH3TheprotectionofindolesidechainofTrpisnotnecessary,buttheapplicati

onofBocgroupisrecommended.UnderTFAcleavagetheappearanceofinN-carboxyindoleprotectsTrpvsalkylationandsulfonation.

inN-carboxygroupisremovedunderaqueousconditioninworkingupprocedure.ProtectionofthesidechainofMetisno

tneededinFmoc-strategy.Fmoc/Bzl(benzyltypeprotectinggroupsforblockingofsidechains)strategyisappliedforthesynthesisofprotectedpeptidefr

agments,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)Coupling:Fmoc-aminoacidderivative-DIC-HOBtinDMF***(3equiveachcalculatedtotheresincapacity);60min5)Washtheresin2xwithDMF;0.5-1

.0mineach6)Washtheresin2xwithDCM;0.5-1.0mineach7)NinhydrinmonitoringSyntheticprotocolofFmoc-strategy(-)yellow(+)blue*DBUisthecleavagereage

nt,piperidineisforthecaptureofdibenzofulvene20%or50%piperidineinDMF,50%morpholineorDEAinDMFand20mMTBAFinDMFarealsousedascleavagemixtu

re.**After4DMFwashing,IPAwashingmaybeappliedforshrinkingtheresin.AnunefficientremovalofbasefromtheresinmaycauseFmoccl

eavageinthenextcouplingstep.***DICisusedinsteadofDCCinthismethod,becauseofthelimitedsolubilityofDCUintheappliedsolvents.N

,N’-diisopropylcarbodiimide(DIC,DIPCDI))CouplingagentsNNCN,N’-dicyclohexylcarbodiimide(DCC)NNCCHHCCH3CH3H3CH3CNNHCOCOCH

X-NHRX:Boc,FmocO-acyl-isoureaderivativesNNHCOCOCHX-NHRN-acyl-ureaderivativesO-NacylshiftNHNHCOureaderivatives:DCU,DIUCOCHX-NHROBtHOB

tinsituactiveester+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(PyBO

P)Theydon’tneedDCCorDICforpreparationofinsituactiveesterHexamethylphosphoramide(carcinogen)!>AOPPyAO

P2-(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-meth

anaminiumhexafluorophosphateN-oxideAccordingtoNMRandröntgendiffractionstudiesanewstructurewassuggested:HATU,TATU,HBPyU,

HAPyU,etc.GuanylationwithuroniumtypecouplingreagentsNNNOC+(CH3)2NN(CH3)2PF6-COCHHNHRNH-PEPTIDE+COCHNHRNH-PEPTIDEC(CH3)2NN+(CH3)

2+HOBtPF6-Don’tuseexcessofcouplingagent(cyclisation,fragmentcondensation);Makepreactivationoftheincom

ingaminoacid;Apply:X-Aaa-OH:HBTU:DIEA=3:2.9:3(equiv)totheresincapacity.FmoccleavageflowchartDoesthepeptidecontainN-terminalFmocgroup?yesnoR

emoveFmocDoesthepeptidecontainArg,Met,TrporTrt?yesnoDoesthepeptidecontainArg,Met?UsecleavagemixtureAyesnoUsecleavagemixtureBDoesthepeptidec

ontainTrporTrt?noyesUsecleavagemixtureCA: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,1mmol0.416EU

RFmoc-Ala-OH(Mw:311)5g11EUR,1mmol0.684EURBoc-Arg(Tos)-OH(Mw:429)5g32EUR,1mmol2.746EURFmoc-Arg(Pbf)-OH(Mw:649)5g90EUR,1mmol11.6

82EURMBHAresin(0.4-1.2mmol/g)5g49EURRinkAmideMBHAresin(0.4-0.8mmol/g)5g168EURCleavageofprotectinggrou

ps(decapeptide):15EUR(Boc),5EUR(Fmoc)DCM(forpeptidesynthesis)49EUR/LDMF(forpeptidesynthesis)111EUR/LHowever,applicationofBoc-str

ategyneedsaspecialHFcleavageapparatus!ManysynthesizersaredesignedforFmocchemistry.TheyareTFAsensitive.Orderingofpiperidinemight

needallowance,becauseitisthestartingmaterialinthesynthesisofmorphine.BocFmoc➢ItisbetterforavoidingDKPformation

;➢ThereisnoproblemwiththeBoccleavage,soitisbetterincaseofpeptidesthataggregateeasily.AggregatesaredestroyedineveryTFAcleavagestep;➢Be

causeoftheextraneutralisationstep,thesyntheticcycletakeslongertime;➢ResinsforBoc-strategyareavailabl

eforFmoc-chemistry,too.Twostepscleavageproceduremayresultsinbettercrudeproduct.FirststepTFAcleavage(sidechainprotectinggroups)thenHF(peptide

-resinbond).Moresuitableforpreparationofbranchedpeptides.➢ClTrtresinmustbeusedtopreventDKPformation;➢IncompleteFmocdepro

tectionincaseofaggregatingpeptides;➢Itisbetterforacidsensitivepeptides(Trp,Met),oxidation,alkylationcanbeavoided.Asp-Probondishighlyacid

sensitive.➢especiallyrecommendedforO-glycosylatedorsulfatedpeptides;➢BecauseoftheorthogonalityofNaandsidechainprote

ctinggroupsfullyprotectedsequencescanbeprepared.