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SolidphasepeptidesynthesisPartIIApplicationofFmoc/tBustrategyGáborMezőResearchGroupofPeptideChemistryHungarianAcademyofSciencesEöt

vösL.UniversityBudapest,HungaryOutline✓Resins;✓Protectinggroups;✓Syntheticprotocol;✓Monitoring;✓Clea

vagetechnics;✓Sidereactions;Fmoc/tBu:NHCCH2OCNHCHCH2COOCOHCCH3CH3CH3NHCHCOOCH2CH2OCH2OOCCH3CH3CH3CRFmoctert-b

utyl..piperidineTFAWang-resinFmoc-Asp(OtBu)-Tyr(tBu)-WangresinTypeofresinsforFmoc-chemistryTherearem

anydifferentresinsandmostofthemareusedforspecialcasesandinindividuallaboratories.Hereonlythemostwidelyappliedresinswillbepresented.Re

sinsarebasedonPS-DVB(1%)copolymer.4-Alkoxybenzylalcohol(Wang)resin:CH2OPCH2HOAttachmentofthefirstaminoacid:Fmoc-Aaa(X)-OH:DIC:D

MAP(2:2:0.2equivtotheresinOHcontent)inDMF,1hatRT.ThefinalcleavageresultsinpeptideswithCOOHgroupattheC-terminusTheresinisnotavailableforthesyn

thesisofpeptideswithasequenceontheC-terminalthatissensitivefordiketopiperazineformation!SASRIN(SuperAcidS

ensitiveResIN)(2-methoxy-4-alkoxybenzyl-alcoholresin)CH2OPCH2HOOCH3Peptideiscleavablewith0.5-1.0%TFAinDCMresultedinprotectedpeptidefragment

s.CH2OCH2HOCOOH4-Hydroxymethylphenoxyaceticacid(HMPA)linker:AttachtoaminomethylPS-DVBresin(CH2)3OCH2HO

COOHOCH34-(4-Hydroxymethyl-3-methoxyphenoxy)butyricacid(HMPB)linker:RemovalofthepeptidewithTFARemovalofthepeptidewithdilutedTFAAttachtoaminomet

hylPS-DVBresin2-Chlorotritylchloride(ClTrt)resin:ClClPAttachmentofthefirstaminoacid:Thefinalcleavageresultsinpeptideswit

hCOOHgroupattheC-terminusCleavagewith90-95%TFA+scavangersresultsinfreepeptidesCleavagewithAcOH:MeOH(TFE):DCM(1:1:8or2:2:6)

resultsinprotectedpeptides(availableforfragmentcondensation).ClTrtresinpreventsthediketopiperazineforma

tion!AttachmentofCysandHisderivativestotheresinisfreefromenantiomerisation!1gClTrt-resin+2mmolFmoc-Aaa(X)-OH+8mmolDIEAin3-5

mLDCM,for1.5hthen0.8mLMeOHtoblocktheunreactedgroupswashingwithDCM,iPrOH,MeOH,ether(Calculationoftheresincapacity)Determinationofloa

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

k;✓Themixtureisshakenthoroughlyandleftfor25-30min;✓Theresinisfilteredoffandtheabsorbanceofthefiltrateis

measuredat301nm(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%piperid

ine/DMF30minatRT,thenfiltration30minatRTdilutewithMeOHto25mLdilutewithMeOHto25mLCapacityoftheresin(mm

ol/g)=1000.mgly.Aresin301Mgly.mresin.Agly301Mgly=297RinkAmideResin:synthesisofpeptideswithCONH2C-terminusCH

NH2PCH3CHNFmoc-HOCH2-POCH3COH3CleavagewithhighconcentrationofTFAcanleadtothebreakdownofthelinkerbyproducts.UselowT

FAconcentrationand/ortrialkylsilanesinthecleavagemixture.Peptide-resinbondcanbedetachedwith5%TFA.Removalofprotectinggroupsneedsaseparatest

ep.CHNFmoc-HOCH2-CO-Nle-ROCH3COH3RinkAmide-AMandRinkAmide-MBHAresins:CHNH2P2Aminomethyl-PS-DVB4-methylbenzhydrylamine-P

S-DVBPeptidecleavagewith90-95%TFAsolution.Nleisareferenceforquantitation.Pegylatedresins:compositionofpolyethyle

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

ellingproperties(alsoinwater)highdiffusionratesavailablewithmanytypesoffunctionalgroupslowcapacity(0.2-0.6mmol/g),suitableforthesynthesisofaggre

gatingpeptides,foronresincyclisationandfragmentcondansation.ThebasicpolymersupportisaminomethylPEG-PS-DVB(NovaSynRTG)PEGNH2

CH2OCH2HOCOOH4-hydroxymethylphenoxyaceticacidlinkerNovaSynRTGAresinSimilartoWangresinHOCOOH4-carboxytrityllinkerNovaSynRTGT

alcoholresinBeforeusetheresinmustbeconvertedtothechlorideformbyheatingwithAcClorSOCl2intoluene.Simila

rtoClTrtresin.CHNH2OCH2OCH3COH32,4-dimethoxy-benzhydryllinkerNovaSynRTGRresinSimilartoRinkAmideMBHAresinCOOH

AppliedsidechainprotectinggroupsinFmoc-chemistry-OH(Ser,Thr,Tyr)Sidechainfunctionalgroupprotectinggroupname(abbreviation)

CH3CCH3CH3tert-butyl(tBu)Trtgroupcanbeusedifon-resinderivatizationisrequired(glycosylation,phosphorylation).Trtcan

becleavedwithdilutedTFA,whiletBuneeds90%TFAsolutionforeffectiveremoval.-SH(Cys)trityl(Trt)CH2NHCOCH3acetamidomethyl(Acm)Forsele

ctivedeprotectionRacemisationduringtheattachmentofCysderivativestotheresinsinthepresenceofDMAP:Fmoc-

Cys(Trt)-OH>Fmoc-Cys(Acm)-OHHowever,Fmoc-Cys(Acm)attheC-terminalresultesinsidereaction:CH2OPOCH2CCHNHCH2Acm-SCH2OPOCH2OCCNHCH2Opiperidin

epiperidineCCHNHCH2ONH2OCCHNHCH2OHOCysMcalcDAlaMcalc–34DL-Ala(Pip)Mcalc+41DL-SerMcalc–16Sidechainfunctionalgroupprotectinggro

upname(abbreviation)tert-butyloxycarbonyl(Boc)eNH2(Lys)OOCCCH3CH3CH3Selectivelyremovableprotectinggroupsforpreparationofmodifiedpeptides(lab

eled,functionalised,branchedorcyclicpeptides):eNH2(Lys)CH34-methytrityl(Mtt)Mttcanberemovedselectivelywith1%TFA/DCM

solutioninthepresenceof3-5%TES(triethylsilane)atRTin15-30min.Trtgroupsmaybenotstableenoughunderthiscondition.Sidechainfun

ctionalgroupprotectinggroupname(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)2ivDdeismorestableinbasiccleavagemix

tureappliedforFmocremovalthanDde.Bothprotectinggroupscanberemovedwith2%NH2-NH2inDMFeNH2(Lys)OOCCH2CHCH2allyloxycarbonyl(Aloc)Alocpr

otectinggroupiscompatiblewithBocaswellasFmoc-chemistry.Itisstableinacidsandbases.ItcanberemovedinP(Ph)3byPd(0)catalysis.Topreventaddition

ondoublebondunderothercleavageconditionsapplicationofallylalcoholincleavagemixturesisrecommended.wCOOH(Asp,Glu)Sidechainfu

nctionalgroupprotectinggroupname(abbreviation)OCCH3CH3CH3tert-butylester(OtBu)Selectivelyremovableprotectinggroupsfor

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

l-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino}Benzylester(ODmab)SimilarlytoDdeandivDde,theDmabprotectinggroupcanberem

ovedwith2%hydrazineinDMF.OCH2CHCH2allylester(OAll)ItcanberemovedinP(Ph)3byPd(0)catalysis.Succinimideringformation(Asp):Acidc

atalisedreactionresultsinaorb-Asp-peptides,however,piperidinecatalisedsidereactionunderFmoccleavageprocedureresultsinpiperidide:-A

sp-Gly-NHCHCONHCH2COCH2COtBuO-Asu-Gly-NHCHNCH2CCOCH2OCONH-tBuOHNHCHCONHCH2COCH2CONHCHCH2CCOONHNCH2COpiperidinepiperidineNNM=Mcalc+57Appli

cationofothercleavagereagents(DBU,TBAF,DEA,morpholine)eliminatethepiperidideformation,butnotthesuccinimideformation.AdditionofHOBttothecleavagemi

xturecanreducethesuccinimideringclosure.ButthebestresultsmaygetwiththeuseofFmoc-(Hmb)-aminoacidderivatives:Hmb:2-hydroxy-4-methoxybenzyl(removab

lewithTFA)NHCHCONCH2COCH2COtBuO(Hmb)aminoacidderivativesaresecundaryamines:RemovalofFmocgroupandtheattachem

entofthenextAspderivativeisdifficult,needslongertime.Ninhydrintestcan’tdetecttheefficacyofthecoupling.Fmoc-(Fmoc-Hmb)Gly-OH1g=370EUR(NovaBioc

hem)Theincreasingofthesolubilityofprotectedpeptidefragmentsaswellaspreventingofaggregationof”difficult”sequencescanbereachbytheapplicati

onofHmbgroups.Sidechainfunctionalgroupprotectinggroupname(abbreviation)wCONH2(Asn,Gln)trityl(Trt)ThesolubilityofF

moc-Asn-OHandFmoc-Gln-OHisextremelybad.TheTrtprotectinggroupincreasesthesolubilityandpreventsthedehydratationaswellasringclosuresidereacti

onsduringthesynthesis.N-terminalGlnorAsn-Gly(Arg,Ser,Ala,Asn)sequencemaycauseproblemsafterthecleavageoftheprotectinggroup.NNH(Hi

s)ptimidazolgrouptert-butyloxymethyl(Bum)(p)CH2OCCH3CH3CH3ThesameproblemasincaseofBominBocstartegy.Don’tuseitforthesynthesisofpeptidescontain

ingCysattheN-terminal!Sidechainfunctionalgroupprotectinggroupname(abbreviation)NNH(His)ptimidazolgrouptrityl(Trt)(t)Trtgro

upprotectsthetN.However,itsapplicationpreventsbothepimerisaton(notincaseofattachmenttoresins)andalkyla

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

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

ativecleavagemixturethatcanremoveMtrmoreeffectively.Sidechainfunctionalgroupprotectinggroupname(abbreviatio

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

ihydrobenzofurane-6-sulfonyl(Pbf)SOOCH3CH3CH3OCH3CH3SOOCH3CH3CH3OCH3CH3PmccanbecleavedwithTFAin2-3hrs,butPbfprotectinggr

oupcanberemoved1.5-2timesfasterthanPmc.PbfalsogivesrisetolesssulfonatedTrpbyproductthanPmcorMtr.UseFmoc-Arg(Pbf)-OHforthesynthes

isofoligo-Argasacellpenetratingpeptide!Sidechainfunctionalgroupprotectinggroupname(abbreviation)indoleNH(T

rp)tert-butyloxycarbonyl(Boc)OOCCCH3CH3CH3TheprotectionofindolesidechainofTrpisnotnecessary,buttheapplicationofBocgroupisrecommen

ded.UnderTFAcleavagetheappearanceofinN-carboxyindoleprotectsTrpvsalkylationandsulfonation.inN-carboxygroupisrem

ovedunderaqueousconditioninworkingupprocedure.ProtectionofthesidechainofMetisnotneededinFmoc-strategy.Fmoc/Bzl(benzyltyp

eprotectinggroupsforblockingofsidechains)strategyisappliedforthesynthesisofprotectedpeptidefragments,becauseofth

ebettersolubilityofbenzylprotectedfragmentsovertert-butylandtritylprotectedfragments.1)Washtheresin3xwithDMF;0.5-1.0mineach2)Cle

avageofFmocprotectionwith3)2%piperidine+2%DBU/DMF;2+2+5+10min*3)Washtheresin8xwithDMF;0.5-1.0mineach**4)Coupling:Fmoc-aminoa

cidderivative-DIC-HOBtinDMF***(3equiveachcalculatedtotheresincapacity);60min5)Washtheresin2xwithDMF;0.5-1.0mineach6)Washtheresin2xwithDCM;0.5-1.0m

ineach7)NinhydrinmonitoringSyntheticprotocolofFmoc-strategy(-)yellow(+)blue*DBUisthecleavagereagent,piperidineisforthecaptureofdibenzofulvene20%or5

0%piperidineinDMF,50%morpholineorDEAinDMFand20mMTBAFinDMFarealsousedascleavagemixture.**After4DMFwashing,IPAwashi

ngmaybeappliedforshrinkingtheresin.AnunefficientremovalofbasefromtheresinmaycauseFmoccleavageinthenextcouplingstep.***

DICisusedinsteadofDCCinthismethod,becauseofthelimitedsolubilityofDCUintheappliedsolvents.N,N’-diisopropylcarbodiimide(DIC,DIPCDI))Coupling

agentsNNCN,N’-dicyclohexylcarbodiimide(DCC)NNCCHHCCH3CH3H3CH3CNNHCOCOCHX-NHRX:Boc,FmocO-acyl-isoureaderivativesNN

HCOCOCHX-NHRN-acyl-ureaderivativesO-NacylshiftNHNHCOureaderivatives:DCU,DIUCOCHX-NHROBtHOBtinsituactiveester+N

NNOH1-hydroxybenzotriazole(HOBt)NNNOHN1-hydroxy-7-aza-benzotriazole(HOAt)NNNOP+(CH3)2NN(CH3)2N(CH3)2PF6-benzotriazol-1-yl-ox

y-tris(dimethylamino)-phosphoniumhexafluorophosphate(BOP)NNNOP+PF6-NNNbenzotriazol-1-yl-oxy-tris(pyrro

lidino)phosphoniumhexafluorophosphate(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)-meth

ylene]-N-methanaminiumhexafluorophosphateN-oxideAccordingtoNMRandröntgendiffractionstudiesanewstructurewassuggested:HATU,TATU,HBPyU,HAPyU,etc.Guanyla

tionwithuroniumtypecouplingreagentsNNNOC+(CH3)2NN(CH3)2PF6-COCHHNHRNH-PEPTIDE+COCHNHRNH-PEPTIDEC(CH3)2NN+(CH3)2+

HOBtPF6-Don’tuseexcessofcouplingagent(cyclisation,fragmentcondensation);Makepreactivationoftheincomingaminoacid;Apply:X-Aaa-OH:HBTU:DIEA=3:2.9:3(

equiv)totheresincapacity.FmoccleavageflowchartDoesthepeptidecontainN-terminalFmocgroup?yesnoRemoveFmocDoesthepeptidecontainA

rg,Met,TrporTrt?yesnoDoesthepeptidecontainArg,Met?UsecleavagemixtureAyesnoUsecleavagemixtureBDoesthepeptideco

ntainTrporTrt?noyesUsecleavagemixtureCA:0.5mLd.i.water9.5mLTFAC:0.25mLEDT0.25mLd.i.Water9.50mLTFAB:0.75

gcryst.phenol0.25mLEDT0.50mLthioanisole0.50mLd.i.water10mLTFABoc/BzlorFmoc/tBustrategyAminoacidderivativesandresinsforBoc-strategyisstillcheaper:Bo

c-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(forpeptidesynthesis)49EUR/LDMF(forpeptidesynthesis)111EUR/LHowever,applicationofB

oc-strategyneedsaspecialHFcleavageapparatus!ManysynthesizersaredesignedforFmocchemistry.TheyareTFAsensitive.Orderingofpiperidin

emightneedallowance,becauseitisthestartingmaterialinthesynthesisofmorphine.BocFmoc➢ItisbetterforavoidingDKPformation;➢

ThereisnoproblemwiththeBoccleavage,soitisbetterincaseofpeptidesthataggregateeasily.AggregatesaredestroyedineveryTFA

cleavagestep;➢Becauseoftheextraneutralisationstep,thesyntheticcycletakeslongertime;➢ResinsforBoc-strategyareavailableforFmo

c-chemistry,too.Twostepscleavageproceduremayresultsinbettercrudeproduct.FirststepTFAcleavage(sidechain

protectinggroups)thenHF(peptide-resinbond).Moresuitableforpreparationofbranchedpeptides.➢ClTrtresinmustbeusedtopreventDKPformation;➢Incomplet

eFmocdeprotectionincaseofaggregatingpeptides;➢Itisbetterforacidsensitivepeptides(Trp,Met),oxidation,a

lkylationcanbeavoided.Asp-Probondishighlyacidsensitive.➢especiallyrecommendedforO-glycosylatedorsulfatedpeptides;➢Becauseoftheorthogonalityof

Naandsidechainprotectinggroupsfullyprotectedsequencescanbeprepared.