多肽合成基礎(chǔ)知識匯編編制:合成部-----------------------------------------------------------------------------------------一、多肽合成概論1963年，[1]創(chuàng)立了將氨基酸的C末端固定在不溶性樹脂上，然后在此樹脂上依次縮合氨基酸，延長肽學(xué)獎.和完善.eldBocTFABocc是分子結(jié)構(gòu)介于氨基酸和蛋白質(zhì)之間的一類化合物,的多肽，用于研究結(jié)構(gòu)與功能的關(guān)系；為多幾十年來，固相法合成多肽更以其省時、省、便于普及推廣的突出優(yōu)勢而成為肽合成的常規(guī)方法并擴展到核苷酸合成等其它有機BOCBOC氨基用FMOC(9-芴甲氧羰基)多肽合成是一個重復(fù)添加氨基酸的過程，固相合成順序一般從C端(羧基端)向N端(氨基端)合成。過圖：具體合成由下列幾個循環(huán)組成：一、去保護：Fmoc保護的柱子和單體必須用一種堿性溶劑(piperidine)去除氨基的保護基團。三、洗脫和脫保護：多肽從柱上洗脫下來，其保護基團被一種脫保護劑(TFA)洗脫和脫保護。酸的固定能用于多肽合成的固相載體必須滿足如下要求：必須包含反應(yīng)位點(或反應(yīng)基團)，以使肽鏈連在這些位點上，并在以后除去；必須對合成過程中的物理和化學(xué)夠間的相互作用。用子載體主要有三類：聚苯乙烯-苯二乙烯交聯(lián)樹脂、聚丙烯酰胺、聚乙烯-乙二醇類樹脂及衍生物，這些樹脂只有導(dǎo)入反應(yīng)基團，才能直接連上(第一個)氨基酸。根據(jù)所導(dǎo)入反應(yīng)基團的不同，又把這的反應(yīng)基團之間形成的共價鍵來實現(xiàn)的，形成共價鍵的方法有多種：氯甲基樹脂，通常先制得保護氨基酸的四甲中多采用烷氧羰基類后來改為叔丁氧羰基(BOC)保護，用TFA(三氟乙酸)脫保護，但不適用含有色氨酸等對酸不穩(wěn)定的肽類的合免由于側(cè)鏈功能團所帶來的副反應(yīng)，一般也需要用適當(dāng)?shù)谋Ｗo基將側(cè)鏈基團，同氨酸用金剛烷氧羰基(Adoc)保護，用冷的三氟乙酸脫去。物料重復(fù)轉(zhuǎn)移而產(chǎn)生的損失；固相載體共價相聯(lián)的肽鏈處于適宜的物理狀態(tài)，可通過快速的間的純化，避免了液相肽合成中冗長的重結(jié)晶或分柱步驟，可避免中間體分離純化時大量的物，迫使個別反應(yīng)完全，以便最終產(chǎn)物得到高產(chǎn)率；增加溶劑化，減少中間的產(chǎn)物聚焦；固交聯(lián)的聚合鏈緊密相混，彼此產(chǎn)生一種相互的溶劑效應(yīng)，這對肽自聚集熱力學(xué)不利而對反應(yīng)手段純化。等人采用Carpino[3]報道的Fmoc(9-芴甲氧羰基)基團作為α-氨基保護基，成功地進行了多肽的Fmoc固相合了強酸處理.分析HPLC使用柱子和泵系統(tǒng)，可以經(jīng)受傳遞高壓，這樣可以用極細(xì)的微粒(3-10μm)做填料。由此多肽要度混合。常見分析和純化用柱為×250mm(3-10μm)和22×250mm(10μm).如果用徑向填柱，那么大小是8×100(3-10μm)和25×250mm(10μm)的Fmoc-氨基酸的側(cè)鏈保護基應(yīng)在堿性條件下穩(wěn)定，在酸性條件下脫除.下面對其做一介紹.uTMSOTf(三氟甲磺酸三甲硅烷酯)除去.等保護，這四種基因均可用TFA脫除.性，抑制了消旋，成功地進行了一些合成.但是當(dāng)反復(fù)地用堿處理時，也表現(xiàn)出一定的不穩(wěn)定性.哌啶羰基在堿保護時要用很強的親核試劉如.rgg.NDMF護無消旋問題，可以采用?；Ｗo基，其它常用的保護基有芐氧碳基和Boc.不同，可根據(jù)實際條件選用.及羧基被保護的氨基酸放在溶液內(nèi)，并要形成酰胺鍵，經(jīng)常用的手段是將羧基活化，其方法是將它變成混合酸酐，或者將它變?yōu)榛顫婖?、酰其他一些如二異丙基碳二亞?DCI)、甲基叔丁基碳二亞胺也用于固相合成中，它們形成的脲酐法解度更差.同時還有些副反應(yīng)，如形成二肽、消旋等.法最常用試劑是氯甲酸的異丙基酯和異丁基酯.前者得到的酸酐穩(wěn)定性好.只產(chǎn)生很少消旋，在適當(dāng)?shù)幕瘜W(xué)酯等.類似的反應(yīng)性和消旋性能，它還有一個優(yōu)越之處，在酰化時有亮黃色、耦聯(lián)結(jié)束時顏色消失，有利于監(jiān)測反應(yīng)；fc分子量A-AlaMetI-IleV-ValK-Lys名稱名稱分子量名稱分子量密度(g/ml)DIPEA(DIA)AcO2HATUZZNMNMM分子量Mz-Trt-Ac--三、常用氨基酸結(jié)構(gòu)及性質(zhì)ThreeLetterCodeOneLetterCodenineAginineRAsparticAcidNDCAcidEQGHILonineValineValVKFPSTWYructureResidueWtAcetamidomethylcmC3H6NOAcetylC2H3Olyloxycarbonylexanoateidomethylcoumaryl7-Amido-4-trifluoromethyl-yl5-[(2-Aminoethyl)amino]-halenesulfonicacidCbzzyloxycarbonyltert-Butyltert-Butyloxycarbonyltert-ButylthioChlorobenzyloxycarbonylnzyl4-Dimethylaminophenyl-nylrenylmethylnylIsothiocyanateminefonylethoxybenzyl7-Methoxycoumarin-4-acetyl4-Methoxy-2,3,6-trimethyl-fonyltylethylbenzylthyltritylcarbonyl2,2,4,6,7-Pentamethyldihydro-ransulfonyl2,2,5,7,8-Pentamethyl-romansulfonylluenesulfonylhyluBzlOS五、多肽常識ReconstitutionandStorageofPeptidesPeptidesareusuallysuppliedasafluffy,freeze-driedmaterialinserumvials.Storepeptidesinafreezeraftertheyhavebeenreceived.Inordertoreconstitutethepeptide,distilledwaterorabuffersolutionshouldbeutilized.Somepeptideshavelowsolubilityinwaterandmustbedissolvedinothersolventssuchas10%aceticacidforapositivelychargedpeptideor10%ammoniumbicarbonatesolutionforanegativelychargedpeptide.Othersolventsthatcanbeusedfordissolvingpeptidesareacetonitrile,DMSO,DMF,orisopropanol.Usetheminimalamountofthesenon-aqueoussolventsandaddwaterorbuffertomakeupthedesiredvolume.Afterpeptidesarereconstituted,theyshouldbeusedassoonaspossibletoavoiddegradationinsolution.Unusedpeptideshouldbealiquotedintosingleuseportionsrelyophilizedifpossible,andstoredat-20°C.Repeatedthawingandrefreezingshouldbeavoided.solvePeptidesThebestwaytodissolveapeptideistousewater.Forpeptidesthatarenotsolubleinwater,usethefollowing1.Foracidicpeptides,useasmallamountofbasesuchas10%ammoniumbicarbonatetodissolvethepeptide,dilutewithwatertothedesiredconcentration.Donotusebaseforcysteine-containingpeptides.2.Forbasicpeptides,useasmallamountof30%aceticacid,dilutewithwatertothedesiredconcentration.3.Foraveryhydrophobicpeptide,trydissolvingthepeptideinaverysmallamountofDMSO,dilutewithwatertothedesiredconcentration.4.Forpeptidesthattendtoaggregate(usuallypeptidescontainingcysteines),add6Murea,6Mureawith20%aceticacid,or6Mguanidine?HCltothepeptide,thenproceedwiththenecessarydilutions.PreparationofHBTU/HOBtSolutionforthePeptideSynthesizer1.PreparationofMHOBtinDMF:oWeighganhydrousHOBtmol,MW[100g,AnaSpecCatalog#21003;500g,AnaSpecCatalog#21004]intoa250mLgraduatedcylinder.oAddDMFuntilthe200mLlevelisreached.2.PreparationofMHBTU/HOBtsolution:oAddthesolutionpreparedinstep1togHBTUmol,MW[100g,AnaSpecCatalog#21001;500g,AnaSpecCatalog#21002]containedinabeakeroranErlenmayerflask.3.Stirforabout15minwithamagneticstirringbaruntilHBTUisdissolved.4.Filterthesolutionthroughafineporesizesinteredglassfunnel.5.Pourthefilteredsolutionintoanappropriatebottleforattachmenttoapeptidesynthesizer.*Thissolutionisstableatroomtemperatureforatleastsixweeks.BiotinylationofAminoGroup1.WashmmolresinwithDMF.2.Dissolveg(+)-biotin(1mmol,MW[1g,AnaSpecCatalog#21100;5g,AnaSpecCatalog#21101]in5mLDMF-DMSO(1:1)solution.Alittlewarmingisnecessary.3.AddmLMHBTU/HOBtsolutionandmLDIEAtothesolutionpreparedinstep2.4.Addtheactivatedbiotinsolutiontotheresinandletstirovernight.5.Checkresintomakesurecouplingiscompleteasevidencedbynegativeninhydrintest(colorless).6.WashresinwithDMF-DMSO(1:1)(2x)toremoveexcess(+)-biotin.7.WashresinwithDMF(2x)andDCM(2x).8.Lettheresindrybeforeproceedingtocleavage.ProcedureforLoadingFmoc-AminoAcidto2-ChlorotritylChlorideResin1.Weigh10g2-chlorotritylchlorideresin(15mmol)[1g,AnaSpecCatalog#22229;5g,AnaSpecCatalog#22230]inareactionvessel,washwithDMF(2x),swelltheresinin50mLDMFfor10min,drainvessel.2.Weigh10mmolFmoc-aminoacidinatesttube,dissolveFmoc-aminoacidin40mLDMF,transferthesolutionintothereactionvesselabove,addmLDIEA(50mmol),swirlmixturefor30minatroomtemperature.3.Add5mLmethanolintothereactionvesselandswirlfor5min.4.DrainandwashwithDMF(5x).5.Checksubstitution.6.Add50mL20%piperidinetoremovetheFmocgroup.Swirlmixturefor30min.7.WashwithDMF(5x),DCM(2x),putresinontissuepaperoverafoampadandletdryatroomtemperatureovernightunderthehood.Covertheresinwithanotherpieceoftissuepaper,presslightlytobreakaggregates.8.Weighloadedresin.9.Packinappropriatecontainer.ProcedureforCheckingSubstitutionofFmoc-AminoAcidLoadedResins1.Weighduplicatesamplesof5to10mgloadedresininaneppendorftube,addmL20%piperidine/DMF,shakefor20min,centrifugedowntheresin.2.Transfer100μLoftheabovesolutionintoatubecontaining10mLDMF,mixwell.3.Pipette2mLDMFintoeachofthetwocells(referencecellandsamplecell),setspectrophotometertozero.Emptythesamplecell,transfer2mLofthesolutionfromstep2intothesamplecell,checkabsorbance.4.Subs=101(A)/(w)A=absorbancew=mgofresin5.Checkabsorbancethreetimesat301nm,calculateaveragesubstitution.ManualFmocSynthesismmol)1.WashresinwithDMF(4x)andthendraincompletely.2.Addapproximately10mL20%piperidine/DMFtoresin.Shakeforoneminanddrain.3.Addanother10mL20%piperidine/DMF.Shakefor30min.4.DrainreactionvesselandwashresinwithDMF(4x).Makesurethereisnopiperidineremaining.Checkbeadsusingninhydrintest,beadsshouldbeblue.5.CouplingStep-Preparethefollowingsolution:6.1mmolFmoc-aminoacid7.mLMHBTU/HOBT(1mmol)348μLDIEA(2mmol)Addabovesolutiontotheresinandshakeforaminimumof30min.Thiscouplingstepcanbelongerifdesired.8.DrainreactionvesselandwashresinwithDMF(4x).9.PerformNinhydrintest:oIfnegative(colorless),proceedtostep2andcontinuesynthesis.oIfpositive(blue),returntostep5andre-couplethesameFmoc-aminoacid.Increasethecouplingtimeifnecessary.SynthesisofPhosphotyrosineContainingPeptidesUsingFmocPhosphotyrosineReagentNFmocOphosphotyrosinegAnaSpecCataloggAnaSpecCatalog#20255]1.Formmolormmolsynthesis,usegFmoc-Tyr(PO3H2)-OH(1mmol,MW.ForABIsynthesizers,packFmoc-Tyr(PO3H2)-OHinacartridge.2.ThecycleprogramforcouplingFmoc-Tyr(PO3H2)-OHisthesameasforotherFmoc-aminoacidsexceptforthecouplingtime(seestep3).(Note:ABIsynthesizersuseHBTU/HOBTastheactivatingreagent.)3.ThecouplingtimeforFmoc-Tyr(PO3H2)-OHneedstobeincreased.ForABImodel430Apeptidesynthesizer,insertseveralsteps.,vortexon,wait990sec,vortexoff,toincreasethecouplingtime).ForABImodel431Apeptidesynthesizer,addadditional"I"s.Overnightcouplingmaybenecessaryforsomesequences.4.AfterthecouplingstepforFmoc-Tyr(PO3H2)-OH,performninhydrintesttoensurecompletecoupling.Negative(colorless)ninhydrintestindicatescompletecoupling,whileapositive(blue)ninhydrintestindicatesincompletecoupling.5.Increasethecouplingtimeoftheaminoacidresiduesafterthephosphotyrosineorperformdoublecoupling.(Note:Thecouplingofaminoacidsafterthephosphotyrosinecanbedifficult.)6.Thereisalimitonthenumberofaminoacidresiduesthatcanbecoupledafterthehosphotyrosine.Sincethephosphogroupisunprotected,sidereactionsarelikelytoccur.(Note:Peptideshavebeensuccessfullycoupledwithsequencescontainingupotenadditionalaminoacidsfollowingthephosphotyrosineresidue.)SimultaneousSynthesisofPeptidesWhichDifferintheC-TerminiUsing2-ChlorotritylResinandWangPeptideswhichdifferintheC-terminicanbesimultaneouslysynthesizedinonereactionvesselbyemployingresinsthatpossessdifferentcleavageproperties.Theresinsusedweretheweakacidlabile2-chlorotritylresinsandtheTFAlabileWangresins.Thesuccessofthisapproachwasshownbytheco-synthesisofACTH(4-10)withACTH(4-11)andNeuropeptideY,aC-terminalamidepeptidewithitscorrespondingC-terminalfreeacidanalog.*HongA.,LeT.,andPhanT.TechniquesinProteinChemistryVI,531-562(1995).CleavageProtocoltoProduceFullyProtectedPeptideStartingResin:ChlorotritylresinsReagentsfor1gPeptide-Resin:1mLaceticacid(AcOH)2mLtrifluoroethanol(TFE)7mLdichloromethane1.Prepareabovemixture.2.Addpeptide-resintothemixtureandletitstiratroomtemperaturefor1h.3.Filterandwashresinwith10mLTFE:DCM(2:8)(2x)toensurethatalloftheproductisrecovered.4.Evaporatethesolventuntilthereislessthan5mLofliquid.5.Addethertoatesttubecontainingabout100Loftheabovesolution.Checksolubilityofthefullyprotectedpeptideinether.Iftheproductprecipitates,proceedtostep6.Ifnoprecipitateisobserved,proceedtostep7.6.Addcoldethertotheresidualliquidinstep4toprecipitatethefullyprotectedpeptide.Filterthroughafinesinteredfunneltoobtaintheproduct.7.Somefullyprotectedpeptidesaresolubleinether.Inthiscase,addwatertoprecipitatethemout.Filterthroughafinesinteredfunneltoobtaintheproduct.ProcedureforFITCLabelingofPeptidesFITC[1g,AnaSpecCatalog#20151]Fmoc--Ahx-OH[1g,AnaSpecCatalog#20957;5g,AnaSpecCatalog#20958]1.CoupleFmoc--Ahx-OHtotheaminoterminalofthepeptide-resinusingstandardcouplingconditions.2."De-Fmoc"withpiperidineusingthestandard20%piperidineprocedure.3.WashresinwithDMF(3-4x).4.SwellresinwithDCManddrain.5.PreparesolutionofequivalentofFITCinpyridine/DMF/DCM(12:7:5).Usejustenoughsolutiontoformaslurrywiththeresin.Donotusetoomuchsolutionsincetherateofthereactionisproportionatetotheconcentrationofthesolution.6.Addthesolutionpreparedinstep2totheresin.7.Letmixovernight.8.Checkthecompletionofthereactionusingninhydrintest.9.IfthecouplingofFITCtotheaminogroupisnotcomplete,ninhydrintestwillgiveabluecolor.RepeatthecouplingwithFITC(steps5-7)ifnecessary.WashresinwithDMF2x),isopropanol(2x),andDCM(2x).upfromFmocLysMttonSolidPhasegAnaSpecCataloggAnaSpecCatalog1.SwellresininDCM.2.Washresinwith3%TFA/DCM(2x)(sincetheresinisswolleninDCM,thisstepofwashingtheresinquicklywith3%TFA/DCMensuresthattheactualconcentrationofTFAis3%).3.Shaketheresinin3%TFAfor10min.4.Repeatstep3.5.WashresinwithDCM(3x),DMF(3x),isopropanol(3x),andDCM(3x).6.Lettheresindryinair.ProcedureforFluoresceinLabelingofPeptides5-carboxyfluorescein(5-FAM)[g,AnaSpecCatalog#24623;g,AnaSpecCatalog#24624)Usestandardcouplingmethodtocouple5-carboxyfluoresceintotheaminogroupofthepeptide.Forcostsavingpurposes,use2xexcesscomparedtothemmolofresin,insteadofthestandard4xexcessusedforFmoc-aminoacids.Formmolsynthesis,use75mg5-carboxyfluorescein,76mgHBTU,and70mLDIEA.六、常用試劑及非天然氨基酸 (一)縮合劑2．6-氯-1-羥基-苯并-三氮唑yName:N,N'-Diisopropylcarbodiimide二環(huán)己基碳化二亞胺exylcarbodiimideDCC六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷N,N'-羰基二咪唑yldiimidazoleCDI (二)鏈接劑 (三)樹脂Resinn (用于合成肽醇) (用于合成肽酰胺) (用于合成肽酰胺)l(四)保護劑9-芴甲醇Boc-酸酐4,4'-二甲氧基三苯基氯甲烷芴甲氧羰酰氯芴甲氧羰酰琥珀酰亞胺叔丁基二甲基氯硅烷10．苯甲氧羰酰琥珀酰亞胺(Cbz-OSu)(五)非天然氨基酸HeLoLoLorye(六)標(biāo)記試劑 (七)修飾及標(biāo)記TetramethylrhodamineAcetylationdationCterminal蛋白載體聯(lián)接KLH(KeyhOlelimpetlleiiiocyanin)BSA(BovineserumalbUnin)MAP)icbranchesicbranchesProteinsarepresentineverylivingcellandpossessavarietyofbiochemicalactivities.Theyappearasenzymes,hormones,antibiotics,andreceptors.Theycomposeamajorportionofmuscle,hair,andskin.Consequently,scientistshavebeenveryinterestedinsynthesizingtheminthelaboratory.ThisinteresthasdevelopedintoamajorsyntheticfieldknownasPeptideSynthesis.Themajorobjectivesinthisfieldarefour-fold:1.Toverifythestructureofnaturallyoccurringpeptidesasdeterminedbydegradationtechniques.2.Tostudytherelationshipbetweenstructureandactivityofbiologicallyactiveproteinandpeptidesandestablishtheirmolecularmechanisms.3.Tosynthesizepeptidesthatareofmedicalimportancesuchashormonesandvaccines.4.Todevelopnewpeptide-basedimmunogens.hasePeptideSynthesisSPPSThefundamentalpremiseofthistechniqueinvolvestheincorporationofN--aminoacidsintoapeptideofanydesiredsequencewithoneendofthesequenceremainingattachedtoasolidsupportmatrix.Whilethepeptideisbeingsynthesizedusuallybystepwisemethodsallsolublereagentscanberemovedfromthepeptide-solidsupportmatrixbyfiltrationandwashedawayattheendofeachcouplingstep.Afterthedesiredsequenceofaminoacidshasbeenobtained,thepeptidecanberemovedfromthepolymericsupport.ThegeneralschemeforsolidphasepeptidesynthesisisoutlinedinFigure1.Thesolidsupportisasyntheticpolymerthatbearsreactivegroupssuchas-OH.ThesegroupsaremadesothattheycanreacteasilywiththecarboxylgroupofanNprotectedaminoacid,therebycovalentlybindingittothepolymer.TheaminoprotectinggroupXcanthenberemovedandasecondN--protectedaminoacidcanbecoupledtotheattachedaminoacidThesestepsarerepeateduntilthedesiredsequenceisobtained.Attheendofthesynthesis,adifferentreagentisappliedtocleavethebondbetweentheC-terminalaminoacidandthepolymersupport;thepeptidethengoesintosolutionandcanbeobtainedfromthesolution.Thecruciallinkinanypolypeptidechainistheamidebond,whichisformedbythecondensationofanaminegroupofoneaminoacidandacarboxylgroupofanother.Generally,anaminoacidconsistsofacentralcarbonatomcalledthecarbonthatisattachedtofourothergroups:ahydrogen,anaminogroup,acarboxylgroup,andasidechaingroupThesidechaingroup,designatedR,definesthedifferentstructuresofaminoacids.Certainsidechainscontainfunctionalgroupsthatcaninterferewiththeformationoftheamidebond.Therefore,itisimportanttomaskthefunctionalgroupsoftheaminoacidsidechain.ThegeneralschemewhichoutlinesthestrategyofFmocsynthesisisshowninFigure2.Initially,thefirstFmocaminoacidisattachedtoaninsolublesupportresinviaanacidlabilelinker.DeprotectionofFmoc,isaccomplishedbytreatmentoftheaminoacidwithabaseusuallypiperidine.ThesecondFmocaminoacidiscoupledutilizingapreactivatedspeciesorinsituactivationAfterthedesiredpeptideissynthesized,theresinboundpeptideisdeprotectedanddetachedfromthesolidsupportviaTFAcleavage.Theremovalofpeptidesinsolidphasepeptidesynthesisisprimarilydonebyacidolysis.TheFmocchemistryemploystheuseofweakacidssuchasTFAorTMSBr.Variousscavengersareincludedtoprotectthepeptidefromcarbocationsgeneratedduringcleavagewhichcanleadtosidereactions.Theseadditivesusuallyincludethiolcompounds,phenol,andwater.ThefollowingprotectinggroupsarecompatiblewithTFAandTMSBrcleavage:ArgBoc)2ArgMtr)ArgPbf)ArgPmc)AsnTmob)AsnTrt)AspOtBu)bBuThrtBuTyrtBuDependingonthetypeofprotectinggroupspresent,certaincombinationsofscavengersmustbeused.Forinstance,wheneitherBocandt-Butylgroupsarepresent,theircarbocationcounterparts(t-butylcationsandt-butyltrifluoroacetate)canreactwithTrp,Tyr,andMettoformtheirt-butylderivatives.WhileEDTisaveryefficientscavengerfort-butyltrifluoroacetate,itdoesnotprotectTrpfromt-butylation.Therefore,watermustbeaddedinordertosuppressalkylation.TheindoleringofTrpandthehydroxylgroupofTyrareespeciallysusceptibletothereactivityofthecleavedPmcgroup.Again,waterhasbeenshowntobeeffectiveinsuppressingthisreactionSimilaroccurrencescanhappenwiththeTrtandMtrgroups.Therefore,scavengersintheappropriatecombinationwillgreatlyreducetheamountofsidereactions.SThegeneralschemewhichoutlinesthestrategyofBocsynthesisisshowninFigure3.Initially,thefirstBocaminoacidisattachedtoaninsolublesupportresinviaaHFcleavablelinker.DeprotectionofBoc,isaccomplishedbytreatmentoftheaminoacidwithTFAThesecondBocaminoacidiscoupledutilizingapre-activatedspeciesorinsituactivationAfterthedesiredpeptideissynthesized,theresinboundpeptideisdeprotectedanddetachedfromthesolidsupportviaHFcleavage.TheBocchemistryemploystheuseofstrongacidssuchasHF,TFMSOTf,orTMSOTf.Variousadditives,usuallythiolcompoundsareaddedtoprotectthepeptidefromthecarbocationsgeneratedduringcleavage.ThefollowingprotectinggroupsarecompatiblewithHFcleavage:ArgMts)sMeOBzlArgTos)lAspOBzl)HexAsp(OcHex)ThrBzlTrp(CHO)sMeBzlTyrBrZ)Asp(OtBu)ThefollowingprotectinggroupsarecompatiblewithTFMSOTfcleavage:ArgMts)AspOBzl)rBzlBzlTrp(CHO)Lys(Cl-Z)Tyr(Br-Z)ThefollowingprotectinggroupsarecompatiblewithTMSOTfcleavage:rgMtsrgMbsspOBzlspOcHexpCHOpMtsyrBrZrBzlTyr(Cl-Bzl)sBomThrBzlGeneralCouplingMethodsinSPPSCouplingreactionsinSPPSrequiretheacylationreactionstobehighlyefficienttoyieldhigh-puritypeptides.gMethodsinFmocSPPSusingactivatingreagentssuchasDCCHBTUTBTUBOPorBOPClThedirectadditionofcarbodiimideisconsideredtobethebestchoice3).HBTUandTBTUwouldranksecond,followedbyBOPandfinallyBOP-ClWithregardstoestercouplingthefollowingorderwasfound:BOP/HOBt>carbodiimide/HOBt~carbodiimide/ODhbT>DCC/OPfp(14-15).MorerecentlyhydroxyazabenzotriazoleHOAtanditscorrespondinguroniumsaltanalogOazabenzotrizolyl,1,3,3,tetra-methyluroniumhexafluorophosphate(HATU)havebeendevelopedandfoundtohaveagreatercatalyticactivitythantheirHOBtandHBTUcounterparts.TheuseofHOAtandHATUenhancescouplingyieldsshortenscouplingtimes,andreducesracemization.Consequently,thesereagentsaresuitableforthecouplingofstericallyhinderedaminoacids,therebyensuringgreatersuccessinthesynthesisofdifficultpeptides(16-17).lingMethodsinBocSPPSrsamixedcarbonicanhydrideortheNcarboxyanhydridemethods57).ThecarbodiimidemethodinvolvescouplingNandCprotectedaminoacidsbyusingDCCasthecouplingreagent.Essentially,thiscouplingreagentpromotesdehydrationbetweenthefreecarboxylgroupofanN-protectedaminoacidandthefreeaminogroupoftheCprotectedaminoacid,resultingintheformationofanamidebondwithprecipitationofthebyproductNN'-dicylcohexylurea.Thismethod,however,ishamperedbysidereactionswhichcanresultinracemization59)orinthepresenceofastrongbase,theformationof5(4H)-oxazolones(60)andN-acylureasFortunately,thesesidereactionscanbeminimized,ifnotaltogethereliminated,byaddingacouplingcatalystsuchasNhydroxysuccinimide(HOSu)or1-hydroxybenzotriazole(HOBt).Inaddition,thismethodcanbeemployedtosynthesizetheactiveesterderivativesofN-protectedaminoacids(62).Inturn,theresultingactivatedesterwillreactspontaneouslywithanyotherC-protectedaminoacidorpeptidetoformanewpeptide.reseparationoftheactivatedesterfromthebyproductdicyclohexylureaprovestobedifficultthemixedcarbonicanhydridemethodcanbeemployed.Thismethodconsistsoftwostages:thefirststageinvolvesactivatingthecarboxylgroupofanN--protectedaminoacidwithanappropriatealkylchlorocarbonate,suchasethylchlorocarbonateorpreferablyisobutylchlorocarbonate(64).Activationoccursinanorganicsolventinthepresenceofatertiarybase.Thesecondstageinvolvesreactingthecarbonicanhydridewithafreeaminecomponentofanaminoacidorapeptideunit.Thecarbonicanhydrideisusuallyaddedata14-foldexcessovertheaminecomponent.Themixedcarbonicanhydridemethodisnotedforbeinghighlyeffectiveatlowtemperatures,resultinginhighyieldsandpureproductsHowever,itdoeshaveitsshort-comings.Forinstance,thereisatendencyfortheanhydridederivativetoundergoracemizationasaresultofthestrongactivationofthecarbonylgroup.ThisproblemdoesnotoccurwhenN--urethaneprotectinggroups,suchasCbzort-Boc,areemployed(65-66).Furthermoreasaresultoftheirhighreactivity,mixedcarbonicanhydridesarepronetotheformationof5(4H)-oxazolonesurethanes(68-69),diacyimides(70-71),esters(72),andaresubjecttodisproportion(73-74).Conditionswhichpromptsuchsidereactionstooccurarehightemperatures,prolongedactivationtimes(thetimeintervalbetweentheadditiontothealkylchlorocarbonateandtheaminecomponentafterthemixedanhydrideisformedstericbulkoftheaminecomponent,andincompleteformationofthemixedanhydride.Fortunatelymostofthesesidereactions,exceptforoxazaloneandurethaneformation,canbesubstantiallyreducedbyperformingthereactionatlowtemperature(~-15°C)andallowingforshorteractivationtimes(~1-2minTominimizetheformationofoxazoloneandurethanederivatives,thefollowingconditionsmustbeimplementeddriedorganicsolventssuchasethylacetate,tetrahydrofuran,t-butanol,oracetonitrilemustbeused;2)thetertiarybase,N-methylmorpholine,shouldbeused(76);and3)Cbz-orBoc-N--protectedaminoacidsmustbeutilized(77).Althoughisobutylandethylchlorocarbonatearetypicallyusedtoformcarbonicanhydrides,othercouplingreagentsdoexistForexampleNethyloxycarbonyl2-ethyloxy-1,2-dihydroquinoline(EEDQ)(78)andN-isobutyloxycarbonylisobutyloxydihydroquinoline(IIDQ)(79)weredevelopedtoreactwiththecarboxylcomponenttoformtheethylorisobutylcarbonatederivativeUnliketheclassicalanhydrideprocedure,EEDQandIIDQdonotrequirebasenorlowreactiontemperatures.Typically,theprocedureinvolvesreactingequimolaramountsofthecarboxylandaminecomponentsinanorganicsolvent(awidevarietyofsolventscanbeusedatMtoMconcentrations.ThenEEDQorIIDQisaddedin5-10%excessandthemixtureisallowedtostirfor4hoursatroomtemperature.Afterremovalofthesolvent,invacuo,theresidueisdissolvedinethylacetateandwashedwithNNaHCO3,10%citricacid,andsaltwater,thendriedwithNa2SO4anhydrousandevaporatedTheproductcanthenberecrystallizedorpurifiedbychromatography.Whilethismethodcircumventstheuseofbase,itisstillsubjecttoracemizationandurethanesideproductformationatelscomparabletothosefoundintheclassicalanhydrideapproachConsequentlyitsonlyadvantagemaybethatitiseasyandconvenienttouse.Itshouldbenotedthatadetailedcomparisonofthetwomethodshasnotbeencarriedouttothisdate.HPLCAnalysisandPurification(81-84)AnalyticalHPLCutilizescolumnsandpumpingsystemsthatcanwithstandanddeliververyhighpressuresenablingtheuseofveryfineparticles(3-10microns)aspackingmaterial.Consequently,peptidescanberesolvedwithahighdegreeofresolutioninashorttimeinterval.,minutes).TwocommonHPLCpurificationmethodsare,ionexchangeandreversephase.IonexchangeHPLCisbasedondirectchargeinteractionsbetweenthepeptideandthestationaryphase.ThecolumnsupportisderivatizedwithanionicspeciesthatmaintainsaparticularchargeoveracertainpHrange,whilethepeptideorpeptidemixtureexhibitsanoppositechargewhichisdependentonitsaminoacidcomposition.Separationisdependentonchargeinteractions.ThepeptideiselutedbychangingthepH,theionicstrength,orboth.Typically,asolutionoflowionicstrengthisused;theionicstrengthofthesolutionisthengraduallyorstep-wiseincreaseduntilthepeptideiselutedfromthecolumn.OneexampleofionexchangeseparationincorporatestheuseofstrongcationexchangecolumnssuchassulfoethylaspartimidewhichseparatesonthebasisofpositivechargeatanacidicpH.ReversephaseHPLCconditionsareessentiallythereverseofnormalphasechromotography.Thepeptidebindsonthecolumnthroughhydrophobicinteractionsandiselutedbydecreasingthe