Solid Phase Peptide Synthesis, Strategies and Resins

1 Solid Phase Peptide synthesis , Solid Phase Peptide synthesis , StrategiesStrategiesand and ResinsResins((FmocFmocstrategystrategy)) Bettina QuadeBettina QuadeOutlineOutline IntroductionIntroduction FmocFmoc--basebaselabile Nlabile N protectingprotectinggroupgroup ProtectionProtectionof of thethesidesidechainschains ResinsResins(Wang and (Wang and RinkRinkAmideAmidelinker)linker) CycleCycle::1. 1. attachmentattachmentof of thethefirstfirstNN protectedprotectedaminoaminoacidacid2. 2. deprotectingdeprotectingNN 3. 3. activationactivationand and couplingcouplingof of thetheaminoaminoacidacid4.

2 4. cleavagecleavagestepsstepsSolid Phase Peptide synthesis (SPPS) Solid Phase Peptide synthesis (SPPS)FmocFmoc SPPS SPPS isismoremoreefficientefficient, , fasterfasterand and thethepurificationpurificationisiseasier easier methodmethodoftenoftenusedusedin in chemistrychemistryand and biochemstrybiochemstry fullyfullyautomatedautomatedpeptidepepti desynthesizersynthesizer ititisispossiblepossibleto to synthesizesynthesizepeptidespeptideswith withan an lengthlengthof 50 of 50 aaaa synthesissynthesisfromfromthetheCC--to to thetheNN--terminusterminus a a labellabelgroupgroupprotectsprotectsthet he --aminoaminogroupgroupforforaminesamines FmocFmocisisa a basebaselabile Nlabile N

3 Protectingprotectinggroupgroup all permanent all permanent protectingprotectinggroups groups forforthethesideside--chainschainsareare acidacid--labilelabileFmocFmocHOHNROI ntroduction: Fmoc-Cl or Fmoc-Succinimide exceptionalexceptionalacidacidstabilitys tability high ultraviolett high ultraviolett absorptionabsorption permitspermitsmonitoringmonitoringof of acylationacylationand and deprotectingdeprotectingreactionreaction compatiblecompatiblewithwithterttert--bu tylbutyl--basedbasedsideside--chainchain protectionprotection FmocFmocprotectedprotectedaminoaminoacid sacidsarearegenerallygenerallyeasyeasyto to prepareprepare labile to labile to organicorganicbasesbases.

4 Particularyparticularyto to secondarysecondaryaminesaminesFluorenyl- 9-methoxycarbonyl group (Fmoc)Side Chain Side Chain ProtectingProtectingGroupsGroups SerSer, , ThrThr, Tyr: Ether, Tyr: Etherterttert--ButyletherButylether AspAspand and GluGlu::terttert--ButylesterButylester LysLys: : bocboc--protectingprotectinggroupgroup protectsprotectsthethereactivereactivefu nctionalfunctionalgroupgroupof of thetheaminoaminoacidacid preventspreventsformationformationof of branchedbranchedchainschains permanent, orthogonal, permanent, orthogonal, compatiblecompatiblewithwithNN --protectionprotection remainremainattachedattachedtroughouttro ughoutthethesynthesissynthesis easyeasyto to removeremove((withwithacidacid) ) afteraftercompletioncompletionof of thethesynthesissynthesis His: N : BocortritylN : Benzyloxymethyl Cys.

5 TritylResinsResins insolubleand chemicallyunreactive polystyrol thepolymer supportisconstructedof 20 50 m diameterparticles swellextensivelyin solvens linker armsareaccessible firstaminoacidiscovalentlybound cleavageaftercompletionof synthesisOOHOONHFmocWang-resinRinkAmidRe sin11ststStepStep: : AttachmentAttachmentof of thetheFirst NFirst N --ProtectedProtectedAminoAminoAcidAcid22 nd nd StepStep: : DeprotectionDeprotectionof of thetheNN HONHRONHONHROONHRONHHH+NCO2NH2R exceptionalexceptionallabilitylabilityto to secondarysecondaryaminesamines activationactivationof of thethering ring protonproton carbanioncarbanion EE1cb1cb--mechanismmechanism primaryprimaryproductsproductsof of thethecleavagecleavagereactionreactionar earecarbamatecarbamatesaltsaltand and dibenzofulvenedibenzofulvene 20 % 20 % piperidinepiperidinein DMFin DMF33rdrdStepStep.

6 CouplingCouplingand and ActivationActivationof of thetheSecond Second AminoAminoAcidAcid a mixtureof DCC and HOBtistheclassicaltechnique DCC activatesthecarbonacid formationof a acylisourea HOBt+ acylisourea activeesterFmocHNROHNCNOFmocHNROOR NHCNNNNOHOHNCNHR NHFmocROONNN active esterNH2 FmocHNROHNRpepRpep44ththStepStep: : DeprotectionDeprotectionof of thetheSide CSide Chainshains reactionmechansimof thesidechaindeprotectionwithtrifluoroace ticacid: protectedaminoacid:44ththStepStep: : CleavageCleavagefromfromthetheResinResin deprotectionwithTFA (95 %) result: Full-length, deprotectedpeptidein solutionseparatedfromtheinsolubleresinsu pportbyfiltration cleavagesolutioncontainsscavengersto minimizeanyalkylatingsidereactionsLitera tureLiterature Kates S.

7 A., Kates S. A., AlbericioAlbericioF.(edF.(ed.), Solid .), Solid -- Phase synthesis , A Phase synthesis , A practicalpracticalguideguide, Marcel , Marcel DekkerDekker, Inc., , Inc., 20002000(86/VK 5500 K19)(86/VK 5500 K19) http:// http:// http:// http:// http:// http://