多肽合成基礎(chǔ)知識大全

多肽合成根底學(xué)問大全RevisedbyBETTYonDecember25,2023多肽合成根底學(xué)問匯編編制: 合成部1．多肽化學(xué)合成概述：1963［1］創(chuàng)立了將氨基酸的C蛋白質(zhì)的固相合成法，在固相法中，每步反響后只需簡潔地洗滌樹脂，便可到達(dá)純化目的.抑制了經(jīng)典液相合成法中的每一步產(chǎn)物都需純化的困難，為自動化合成肽奠定了根底.為此，Merrifield1984MerrifieldBoc(Boc:叔丁氧羰基)之外，又進(jìn)展了Fmoc(Fmoc:9-芴甲氧羰基).以這兩種方法為根底的各種肽自動合成儀也相繼消滅和進(jìn)展，并仍在不斷得到改造和完善.MerrifieldBoc［2］TFA(Bocα-氨基保護(hù)基，側(cè)鏈保護(hù)承受芐Boc－氨基酸衍生物共價交聯(lián)到樹脂上，用TFA脫除Boc，用三乙胺中和游離的氨基末端，然后通過DccHFTFMSABoc子，如活性酶、生長因子、人工蛋白等.多肽是涉及生物體內(nèi)各種細(xì)胞功能的生物活性物質(zhì)。它是分子構(gòu)造介于氨基酸和蛋白質(zhì)之間的一類化合物,由多種要信息；建立模型酶以及合成的多肽藥物等。多肽的化學(xué)合成技術(shù)無論是液相法還是固相法都已成熟。近幾十年來，固相法合成多肽更以其省時、省力、省概述了固相合成的根本原理、試驗過程，對其現(xiàn)狀進(jìn)展分析并展望了今后的進(jìn)展趨勢。1963Merrifield蛋白質(zhì)合成中的一個常用技術(shù)，表現(xiàn)出了經(jīng)典液相合成法無法比較的優(yōu)點。其根本原理是：先將所要合成肽鏈的羥末端氨基酸的羥基以共價鍵的構(gòu)造同一個不溶性的高分子樹脂相連，然后以此結(jié)合在固相載體上的氨基酸作為氨基組份經(jīng)過脫去氨基保護(hù)基并同過量的活化羧基組分反響，接長肽鏈。重復(fù)〔縮合→洗滌→去保護(hù)→中和及洗滌→下一輪縮合〕操αBOC〔叔丁氧羰基〕BOC，α-氨基用FMOC〔9-芴甲氧羰基〕保護(hù)的稱為FMOC2．固相合成的根本原理C〔羧基端〕向N〔氨基端〕合成。過去的多肽合成是在溶液中進(jìn)展的稱為液相合成法?，F(xiàn)在多承受固相合成法，從而大大的減輕了每步產(chǎn)品提純的難度。為了防止副反響的發(fā)生，參與反響的氨基酸的側(cè)鏈都是保護(hù)的。羧基端是游離的，并且在反響之前必需活化?；瘜W(xué)合成方法有兩FmoctBocFmoctBocFmoc具體合成由以下幾個循環(huán)組成：一、去保護(hù)：Fmoc〔piperidine〕去除氨基的保護(hù)基團(tuán)。鍵。在此步驟使用大量的超濃度試劑驅(qū)使反響完成。循環(huán)：這兩步反響反復(fù)循環(huán)直到合成完成。三、洗脫和脫保護(hù)：多肽從柱上洗脫下來，其保護(hù)基團(tuán)被一種脫保護(hù)劑〔TFA〕樹脂的選擇及氨基酸的固定將固相合成與其他技術(shù)分開來的最主要的特征是固相載體，能用于多肽合成的固相載體必需滿足如下要求：必需包含反響位點〔或反響基團(tuán)〕，以使肽鏈連在這些位點上，并在以后除去；必需對合成過程中的物理和化學(xué)條件穩(wěn)定；載體必需允許在不斷增長的肽鏈和試劑之間快速的、不受阻礙的接觸；另外，載體必需允許供給足夠的連接點，以使每單位體積的載體給出有用產(chǎn)量的肽，并且必需盡量削減被載體束縛的肽鏈之間的相互作用。用于固相法合成多肽的高分子載體主要有三類：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)入反響基團(tuán)，才能直接連上〔第一個〕氨基酸。依據(jù)所導(dǎo)入反響基團(tuán)的不同，又把這些樹脂及樹脂衍生物分為氯甲基樹脂、羧基樹脂、氨基樹脂或酰肼型樹脂。BOCMerrifieldFMOC如王氏樹脂。氨基酸的固定主要是通過保護(hù)氨基酸的羧基同樹脂的反響基團(tuán)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(hù)氨基酸的四甲銨鹽或鈉鹽、鉀鹽、銫鹽，然后在適當(dāng)溫度下，直接同樹脂反響或在適宜的有機(jī)溶劑如二氧六環(huán)、DMFDMSODCCDCC氨基酸與樹脂之間形成的酰胺鍵來完成氨基酸的固定。氨基、羧基、側(cè)鏈的保護(hù)及脫除要成功合成具有特定的氨基酸挨次的多肽，需要對暫不參與形成酰胺鍵的氨基和羧基加以保護(hù)，同時對氨基酸側(cè)鏈上的活性基因也要保護(hù)，反響完成后再將保護(hù)基因除去。同液相合成一樣，固相合成中多承受烷氧羰基類型作為α氨基的保護(hù)基，由于這樣不易發(fā)生消旋。最早是用芐氧羰基，由于它需要較強(qiáng)的酸解條件才能脫除，所以后來改為叔丁氧羰基〔BOC〕保護(hù)，用TFA〔三氟乙酸〕脫保護(hù)，但不適用含有色氨酸等對酸不穩(wěn)定的肽類的合成。1978changMeienloferAthertonCarpinoFmoc(9-芴甲氧羰基)作為α氨基保護(hù)基，F(xiàn)mocFmoc乙酯是逐步合成中保護(hù)羧基的常用方法，可通過皂化除去或轉(zhuǎn)變?yōu)殡乱员阌糜谄瑪嘟M合；叔丁酯在酸性條件下除去；芐酯常用催化氫化除去。對于合成含有半胱氨酸、組氨酸、精氨酸等帶側(cè)鏈功能基的氨基酸的肽來說，為了避開由于側(cè)鏈功能團(tuán)所帶來的副反響，一般也需要用適當(dāng)?shù)谋Ｗo(hù)基將側(cè)鏈基團(tuán)臨時保護(hù)起來。保護(hù)基的選擇既要保證側(cè)鏈基團(tuán)不參與形成酰胺的反響，又要保證在肽合成過程中不受破壞，同時又要保證在最終肽鏈裂解時能被除去。如用三苯甲基保護(hù)半S2,2,2-三氟-12,2,2-三氟-1-叔丁氧羰基乙基保護(hù)，可通過催化氫化或冷的三氟乙酸脫去。精氨酸用金剛烷氧羰基〔Adoc〕保護(hù)，用冷的三氟乙酸脫去?！踩缣级啺薄承蜠CC、HOBTHOBT/DCCBOCTFA+HFFMOCTFA，有時依據(jù)條件不同，其它堿、光解、氟離子和氫解等脫保護(hù)方法也被承受。合成肽鏈進(jìn)一步的精制、分別與純化通常承受高效液相色譜、親和層析、毛細(xì)管電泳等。固相合成的特點及存在的主要問題固相載體上中間體雜肽無法分別，這樣造成最終產(chǎn)物的純度不如液相合成物，必需通過牢靠的分別手段純化。固相合成的爭論進(jìn)展前景質(zhì)了，同時合成中的試劑毒性，昂貴費用，副產(chǎn)物等始終都是令人頭痛的問題，而在生物體內(nèi)，核糖體上合成肽鏈的速〔樹脂〕上，這是一個令人感興趣的問題，或許是今后多肽合成的進(jìn)展。在Boc合成法中，反復(fù)地用酸來脫保護(hù)，這種處理帶來了一些問題：如在肽與樹脂的接頭處，當(dāng)每次用50%TFA脫.Boc法尤其不適于合成含有色氨酸等對酸不穩(wěn)定的肽類.1978，Chang、MeienloferAthertonCarpino［3］Fmoc(9-芴甲氧羰基)基團(tuán)作為α-氨基保護(hù)基，成功地進(jìn)展了多肽的Fmoc.FmocBocFmocα-氨基的保護(hù)基.側(cè)鏈的保護(hù)承受TFA90%TFA對1%TFAHPLC〔3-10μm〕做填料。由此多肽要在幾分鐘內(nèi)高度被分析。HPLCHPLCPH定電荷衍變成一種離子體，而多肽或多肽混合物，由其氨基酸組成表現(xiàn)出相反電荷。分別是一種電荷相互作用，通過可sulfoethylaspartimidePHHPLCG4-G8鏈柱洗脫好。然而，總體實踐中，這兩類柱互變無多少顯著差異，別類載體由碳水化合物構(gòu)成，比方苯基。典型的操作常由兩綬沖劑組成，%TFA-H2o80%acetonitrile%TFA--H2oacetonitrile。用線型梯變以每分鐘%到%轉(zhuǎn)變的速度混合。常見分析和純化用柱為×250mm(3-10μm)22×250mm(10μm).假設(shè)用徑向填柱，那么大小是8×100〔3-10μm〕25×250mm(10μm)heptafluorobutyricHeformic(5-6%,pH2-4),10-100mMNH4HCO3,/氨，TFA/TEA，磷酸鈉或鉀，異戊酚。這樣很多不同組合可形成緩沖劑，但要留意一點：硅反相pH，甚至微堿pHFmoc―氨基酸的制備和側(cè)鏈保護(hù)Fmoc基團(tuán)是在有NaHCO3或Na2CO3存在的二氧六環(huán)溶液中，通過以下反響引入到氨基酸中的：AspGluAspGlut-BuTFA、TMSBrt-BuTMSOTf(三氟甲磺酸三甲硅烷酯)除去.Ser、Thr和Tyrser、ThrTyrt-Buser下與異丁烯反響.SerThrSerThrAsnGln，AsnGlnα-羧基活化時可能會發(fā)生分子內(nèi)GlnFmoc-GlnFmoc-Asn在DCM9-咕噸基，2，4，6-三甲氧芐基，4，4′―二甲氧二苯甲基或三苯甲基等保護(hù)，這四種基TFAHisπ-N(Bzl)和甲基磺?；?TOS)保護(hù).但這兩種保護(hù)基均不太抱負(fù).TOS.Boc消旋，成功地進(jìn)展了一些合成.但是當(dāng)反復(fù)地用堿處理時，也表現(xiàn)出肯定的不穩(wěn)定性.哌啶羰基在堿中穩(wěn)定，但是沒能很好地抑制消旋，而且脫保護(hù)時要用很強(qiáng)的親核試劉如.π-Nπ-N(Bom)和叔丁氧甲基(Bum)保護(hù)，(Bum)可以用TFA除，BomBumHisFmoc(His)Bum在DCMDMFCysTFA(CF3CO)3T1／TFATFAt-Bu、Bom胺甲基等.第三類對弱酸穩(wěn)定，如芐基和叔丁硫基(stBu)等，Cys(StBu)可用巰基試劑和磷試劑復(fù)原，Cys(Bzl)可用Na/NH3(1)脫保護(hù).Arg12保護(hù)基分四類：(1)硝基(2)烷氧羰基(3)磺?；?4)三苯甲基.Boc(Adoc)2、Fmoc(Arg)BocAdocπ－N，但有同樣的副反響發(fā)TOS2，3，6-三甲基-4-甲氧苯橫酰基(Mtr)較受歡送，在TFATFADCM、DMFLysα－NH2.ε－NH2Boc.FmocFmoc基團(tuán)的芴環(huán)系的吸電子作用使9-H具有酸性，易被較弱堿除去，反響條件很溫順.反響過程可表示如下：.Fmoc可依據(jù)實際條件選用.耦聯(lián)反響的失去劑(碳二亞胺)也可形成酰胺鍵，耦聯(lián)反響可表示如下：(A：羰基活潑試劑)DccDCMDCH，過濾時又難于除盡.其他一些如二異丙基碳二亞胺(DCI)、甲基叔丁基碳二亞胺也用于固相合成中，它們形成的脲Bop(Bop－C1)、氯甲酸異丙酯、氯甲酸異丁酯、SOC12SOC12對稱酸酐法DccDCMFmoc差.同時還有些副反響，如形成二肽、消旋等.混合酸酐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)計量及N-甲基嗎啉和N-甲基哌啶對Fmoc酰氯法BocBocFmoc固相合成中應(yīng)用還不多，但已說明，F(xiàn)moc－氨基酰氯適用于合成有立體障礙的肽序列.活化酯法活化酯法在固相合成中應(yīng)用最為廣泛.承受過的試劑也很多，近來最常用的有HOBt酯、ODhbt酯、OTDO酯等.HOBtODhbtHOBt反響性和消旋性能，它還有一個優(yōu)越之處，在?；瘯r有亮黃色、耦聯(lián)完畢時顏色消逝，有利于監(jiān)測反響；OTDO原位法將碳二亞胺和α－N保護(hù)氨基酸直接加到樹脂中進(jìn)展反響叫做原位法.DICDccBopBop-C1DICBop、Bop-C1Bop裂解及側(cè)鏈保護(hù)基脫除Fmoc.TFAt-Bu、Boc、Adoc、MtrArgMtrTFACyst-BuHFAsp、Glu、Ser、ThrBzl(芐基)保護(hù)基等，但是當(dāng)脫除Asp的吸電子保護(hù)基時，會引起環(huán)化副反響.而TMSBr和TMSOTf在有苯甲硫醚存在時，脫保護(hù)速度很快.此外，依據(jù)條件不同，堿、光解、氟離子和氫解等脫保護(hù)方法也有應(yīng)用.FmocBoc二、常用保護(hù)氨基酸數(shù)據(jù)縮寫縮寫A-AlaR-ArgN-AsnD-AspC-CysQ-GlnE-Glu名稱Fmoc-AlaFmoc-Arg(Pbf)Fmoc-Asn(Trt)Fmoc-Asp(OtBu)Fmoc-Cys(Trt)Fmoc-Gln(Trt)Fmoc-Glu(OtBu)分子量縮寫M-MetF-Phe(D)F-PheP-ProS-SerT-ThrW-Trp名稱Fmoc-MetFmoc-PheFmoc-D-PheFmoc-ProFmoc-Ser(tBu)Fmoc-Thr(tBu)Fmoc-Trp(Boc)分子量G-GlyG-GlyH-HisI-IleL-LeuK-LysFmoc-GlyFmoc-His(Trt)Fmoc-IleFmoc-LeuFmoc-Lys(Boc)(D)W-TrpD-TyrV-ValpGluFmoc-D-Trp(Boc)Fmoc-Tyr(tBu)Fmoc-ValPyroGlu常用試劑種類及數(shù)據(jù)名稱分子量名稱分子量密 度〔g/ml〕HOBtDIPCDI(DIC)TBTUDIPEA(DIA)HATUAcODMAPPyridineHOATTFAPyBOPTMP2EDTTIS2EDTTISNMM縮寫分子量縮寫分子量縮寫分子量Fmoc-222tBu-56Acm-57Pbf-253OtBu-72Mz-Trt-Ac-43Boc-Z-eSymboleSymbolThreeLetterCodeOneLetterCodeMWMW(-H2O)StructureanineAlaAginineArgRparagineparticAcidsteineAsnAspCysNDCNameNameStructureSymbolFormulaResidueWtutamicAcid Glu Eutamine Gln QycineGlyGycineGlyGstidineHisHoleucineIleIucineLeuLsineLysKthionineMetMenylalaninePheFolineProPrineSerSreonineThrTrosine Tyr Yline Val V四、常見保護(hù)基團(tuán)構(gòu)造Acetamidomethyl Acm C3H6NOAcetyl Ac C2H3OAllyloxycarbonyl6-Amidohexanoate7-Amido-4-methylcoumaryl7-Amido-4-trifluoromethyl-coumarylBenzoylBzC7H5OBenzylBzlC7H7BenzoylBzC7H5OBenzylBzlC7H7BenzyloxycarbonylZ(Cbz)C8H7O2BenzyloxymethylBomC8H9O(+)-BiotinylBiotinC10H15N2O2S2-Bromobenzyloxycarbonyl2-Br-ZC8H6BrO2tert-ButyltBuC4H9tert-ButyloxycarbonylBocC5H9O2tert-ButylthioStBuC4H9S2-Chlorobenzyloxycarbonyl2-Cl-ZC8H6ClO2CyclohexylcHexC6H112,6-Dichlorobenzyl2,6-di-Cl-BzlC7H5Cl2

AlocLCAMCAFCEDANS

C4H5O2C4H7NOC10H8NO2C10H5F3NO2C12H13N2O3S4-(4-Dimethylaminophenyl-azo)benzoyl2,4-Dinitrophenyl9-Fluorenylmethyl9-Fluorenylmethyloxy-

DABCYLDnpFmcarbonylFluoresceinIsothiocyanatecarbonylFluoresceinIsothiocyanateFITCC21H12NO5SLissamineRhodamineLRC31H38N2O6S2Mesitylene-2-sulfonylMtsC9H11O2S4-MethoxybenzylMobC8H9O

C15H14N3OC6H3N2O4C14H11C15H11O2(7-Methoxycoumarin-4-yl)acetylbenzenesulfonyl4-Methoxytritylbenzenesulfonyl4-MethoxytritylMmtC20H17O4-MethylbenzylMBzlC8H94-MethyltritylMttC20H174-MorpholinecarbonylMuC5H8NO2p-NitroanilidepNAC6H5N2O2

McaMtr

C12H9O4C10H13O3S2,2,4,6,7-Pentamethyldihydro-benzofuran-5-sulfonyl2,2,5,7,8-Pentamethyl-

Pbf C13H17O3Schroman-6-sulfonylRhodamine110chroman-6-sulfonylRhodamine110R110C20H13N2O3SuccinylSucC4H5O34-ToluenesulfonylTosC7H7O2STritylTrtC17H15Xanthyl五、多肽常識XanC13H9OReconstitutionandStorageofPeptidesPeptidesareusuallysuppliedasafluffy,freeze-driedmaterialinserumvials.Storepeptidesinafreezeraftertheyhavebeenreceived.Inordertoreconstitutethepeptide,distilledwaterorabuffersolutionshouldbeutilized.Somepeptideshavelowsolubilityinwaterandmustbedissolvedinothersolventssuchas10%aceticacidforapositivelychargedpeptideor10%ammoniumbicarbonatesolutionforanegativelychargedpeptide.Othersolventsthatcanbeusedfordissolvingpeptidesareacetonitrile,DMSO,DMF,orisopropanol.Usetheminimalamountofthesenon-aqueoussolventsandaddwaterorbuffertomakeupthedesiredvolume.Afterpeptidesarereconstituted,theyshouldbeusedassoonaspossibletoavoiddegradationinsolution.Unusedpeptideshouldbealiquotedintosingle-useportions,relyophilizedifpossible,andstoredat-20°C.Repeatedthawingandrefreezingshouldbeavoided.MethodstoDissolvePeptidesThebestwaytodissolveapeptideistousewater.Forpeptidesthatarenotsolubleinwater,usethefollowingprocedure:Foracidicpeptides,useasmallamountofbasesuchas10%ammoniumbicarbonatetodissolvethepeptide,dilutewithwatertothedesiredconcentration.Donotusebaseforcysteine-containingpeptides.Forbasicpeptides,useasmallamountof30%aceticacid,dilutewithwatertothedesiredconcentration.Foraveryhydrophobicpeptide,trydissolvingthepeptideinaverysmallamountofDMSO,dilutewithwatertothedesiredconcentration.Forpeptidesthattendtoaggregate(usuallypeptidescontainingcysteines),add6Murea,6Mureawith20%aceticacid,or6Mguanidine?HCltothepeptide,thenproceedwiththenecessarydilutions.PreparationofHBTU/HOBtSolutionforthePeptideSynthesizerPreparationof MHOBtinDMF:Weigh ganhydrousHOBt mol,MW [100g,AnaSpecCatalog#21003;500g,AnaSpecCatalog#21004]intoa250mLgraduatedcylinder.o AddDMFuntilthe200mLlevelisreached.Preparationof MHBTU/HOBtsolution:Addthesolutionpreparedinstep1to gHBTU mol,MW [100g,AnaSpecCatalog#21001;500g,AnaSpecCatalog#21002]containedinabeakeroranErlenmayerflask.Stirforabout15minwithamagneticstirringbaruntilHBTUisdissolved.Filterthesolutionthroughafineporesizesinteredglassfunnel.Pourthefilteredsolutionintoanappropriatebottleforattachmenttoapeptidesynthesizer.*Thissolutionisstableatroomtemperatureforatleastsixweeks.BiotinylationofAminoGroupWash mmolresinwithDMF.Dissolve g(+)-biotin(1mmol,MW [1g,AnaSpecCatalog#21100;5g,AnaSpecCatalog#21101]in5mLDMF-DMSO(1:1)solution.Alittlewarmingisnecessary.Add mL MHBTU/HOBtsolutionand mLDIEAtothesolutionpreparedinstep2.Addtheactivatedbiotinsolutiontotheresinandletstirovernight.Checkresintomakesurecouplingiscompleteasevidencedbynegativeninhydrintest(colorless).WashresinwithDMF-DMSO(1:1)(2x)toremoveexcess(+)-biotin.WashresinwithDMF(2x)andDCM(2x).Lettheresindrybeforeproceedingtocleavage.ProcedureforLoadingFmoc-AminoAcidto2-ChlorotritylChlorideResinWeigh10g2-chlorotritylchlorideresin(15mmol)[1g,AnaSpecCatalog#22229;5g,AnaSpecCatalog#22230]inareactionvessel,washwithDMF(2x),swelltheresinin50mLDMFfor10min,drainvessel.Weigh10mmolFmoc-aminoacidinatesttube,dissolveFmoc-aminoacidin40mLDMF,transferthesolutionintothereactionvesselabove,addmLDIEA(50mmol),swirlmixturefor30minatroomtemperature.Add5mLmethanolintothereactionvesselandswirlfor5min.DrainandwashwithDMF(5x).Checksubstitution.Add50mL20%piperidinetoremovetheFmocgroup.Swirlmixturefor30min.WashwithDMF(5x),DCM(2x),putresinontissuepaperoverafoampadandletdryatroomtemperatureovernightunderthehood.Covertheresinwithanotherpieceoftissuepaper,presslightlytobreakaggregates.Weighloadedresin.Packinappropriatecontainer.ProcedureforCheckingSubstitutionofFmoc-AminoAcidLoadedResinsWeighduplicatesamplesof5to10mgloadedresininaneppendorftube,add mL20%piperidine/DMF,shakefor20min,centrifugedowntheresin.Transfer100μLoftheabovesolutionintoatubecontaining10mLDMF,mixwell.Pipette2mLDMFintoeachofthetwocells(referencecellandsamplecell),setspectrophotometertozero.Emptythesamplecell,transfer2mLofthesolutionfromstep2intothesamplecell,checkabsorbance.Subs=101(A)/(w)A=absorbancew=mgofresinCheckabsorbancethreetimesat301nm,calculateaveragesubstitution.ManualFmocSynthesismmol)WashresinwithDMF(4x)andthendraincompletely.Addapproximately10mL20%piperidine/DMFtoresin.Shakeforoneminanddrain.Addanother10mL20%piperidine/DMF.Shakefor30min.DrainreactionvesselandwashresinwithDMF(4x).Makesurethereisnopiperidineremaining.Checkbeadsusingninhydrintest,beadsshouldbeblue.CouplingStep-Preparethefollowingsolution:1mmolFmoc-aminoacid mL MHBTU/HOBT(1mmol)348μLDIEA(2mmol)Addabovesolutiontotheresinandshakeforaminimumof30min.Thiscouplingstepcanbelongerifdesired.DrainreactionvesselandwashresinwithDMF(4x).PerformNinhydrintest:Ifnegative(colorless),proceedtostep2andcontinuesynthesis.Ifpositive(blue),returntostep5andre-couplethesameFmoc-aminoacid.Increasethecouplingtimeifnecessary.SynthesisofPhosphotyrosine-ContainingPeptidesUsingFmoc-PhosphotyrosineReagent:N--Fmoc-O-phosphotyrosine[1g,AnaSpecCatalog#20254;5g,AnaSpecCatalog#20255]For mmolor mmolsynthesis,use gFmoc-Tyr(PO3H2)-OH(1mmol,MW .ForABIsynthesizers,packFmoc-Tyr(PO3H2)-OHinacartridge.ThecycleprogramforcouplingFmoc-Tyr(PO3H2)-OHisthesameasforotherFmoc-aminoacidsexceptforthecouplingtime(seestep3).(Note:ABIsynthesizersuseHBTU/HOBTastheactivatingreagent.)ThecouplingtimeforFmoc-Tyr(PO3H2)-OHneedstobeincreased.ForABImodel430Apeptidesynthesizer,insertseveralsteps.,vortexon,wait990sec,vortexoff,toincreasethecouplingtime).ForABImodel431Apeptidesynthesizer,addadditional“I“s.Overnightcouplingmaybenecessaryforsomesequences.AfterthecouplingstepforFmoc-Tyr(PO3H2)-OH,performninhydrintesttoensurecompletecoupling.Negative(colorless)ninhydrintestindicatescompletecoupling,whileapositive(blue)ninhydrintestindicatesincompletecoupling.Increasethecouplingtimeoftheaminoacidresiduesafterthephosphotyrosineorperformdoublecoupling.(Note:Thecouplingofaminoacidsafterthephosphotyrosinecanbedifficult.)Thereisalimitonthenumberofaminoacidresiduesthatcanbecoupledafterthehosphotyrosine.Sincethephosphogroupisunprotected,sidereactionsarelikelytoccur.(Note:Peptideshavebeensuccessfullycoupledwithsequencescontainingupotenadditionalaminoacidsfollowingthephosphotyrosineresidue.)SimultaneousSynthesisofPeptidesWhichDifferintheC-TerminiUsing2-ChlorotritylResinandWangResin*PeptideswhichdifferintheC-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)7mLdichloromethane(DCM)Prepareabovemixture.Addpeptide-resintothemixtureandletitstiratroomtemperaturefor1h.Filterandwashresinwith10mLTFE:DCM(2:8)(2x)toensurethatalloftheproductisrecovered.Evaporatethesolventuntilthereislessthan5mLofliquid.Addethertoatesttubecontainingabout100Loftheabovesolution.Checksolubilityofthefullyprotectedpeptideinether.Iftheproductprecipitates,proceedtostep6.Ifnoprecipitateisobserved,proceedtostep7.Addcoldethertotheresidualliquidinstep4toprecipitatethefullyprotectedpeptide.Filterthroughafinesinteredfunneltoobtaintheproduct.Somefullyprotectedpeptidesaresolubleinether.Inthiscase,addwatertoprecipitatethemout.Filterthroughafinesinteredfunneltoobtaintheproduct.ProcedureforFITCLabelingofPeptidesReagents:FITC[1g,AnaSpecCatalog#20231]Fmoc--Ahx-OH[1g,AnaSpecCatalog#20957;5g,AnaSpecCatalog#20958]CoupleFmoc--Ahx-OHtotheaminoterminalofthepeptide-resinusingstandardcouplingconditions.“De-Fmoc“withpiperidineusingthestandard20%piperidineprocedure.WashresinwithDMF(3-4x).SwellresinwithDCManddrain.Preparesolutionof equivalentofFITCinpyridine/DMF/DCM(12:7:5).Usejustenoughsolutiontoformaslurrywiththeresin.Donotusetoomuchsolutionsincetherateofthereactionisproportionatetotheconcentrationofthesolution.Addthesolutionpreparedinstep2totheresin.Letmixovernight.Checkthecompletionofthereactionusingninhydrintest.IfthecouplingofFITCtotheaminogroupisnotcomplete,ninhydrintestwillgiveabluecolor.RepeatthecouplingwithFITC(steps5-7)ifnecessary.WashresinwithDMF(2x),isopropanol(2x),andDCM(2x).ProcedureforRemovingMttgroupfromFmoc-Lys(Mtt)onSolidPhaseReagent:Fmoc-Lys(Mtt)-OH[1g,AnaSpecCatalog#20233;5g,AnaSpecCatalog#20234]SwellresininDCM.Washresinwith3%TFA/DCM(2x)(sincetheresinisswolleninDCM,thisstepofwashingtheresinquicklywith3%TFA/DCMensuresthattheactualconcentrationofTFAis3%).Shaketheresinin3%TFAfor10min.Repeatstep3.WashresinwithDCM(3x),DMF(3x),isopropanol(3x),andDCM(3x).Lettheresindryinair.ProcedureforFluoresceinLabelingofPeptidesReagent:5-carboxyfluorescein(5-FAM)[g,AnaSpecCatalog#24623; g,AnaSpecCatalog#24624)Usestandardcouplingmethodtocouple5-carboxyfluoresceintotheaminogroupofthepeptide.Forcostsavingpurposes,use2xexcesscomparedtothemmolofresin,insteadofthestandard4xexcessusedforFmoc-aminoacids.For mmolsynthesis,use75mg5-carboxyfluorescein,76mgHBTU,and70mLDIEA.六、常用試劑及非自然氨基酸1．Name:Category:PeptideCouplingReagent2．6-氯-1-羥基-苯并-三氮唑Name:Cl-HOBtCategory:PeptideCouplingReagent3．Name:N,N”-Diisopropylcarbodiimide(DIC)Category:PeptideCouplingReagent4．二環(huán)己基碳化二亞胺Name:Dicyclohexylcarbodiimide(DCC)Category:PeptideCouplingReagents5．Name:BOPReagentCategory:PeptideCouplingReagent6．六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷Name:PyBOPCategory:PeptideCouplingReagent7．N,N”-羰基二咪唑Name:N,N”-Carbonyldiimidazole(CDI)Category:PeptideCouplingReagent8．Name:DEPBTCategory:PeptideCouplingReagent9．Name:4,5-DicyanoimidazoleCategory:PeptideCouplingReagent10．Name:HBTUCategory:PeptideCouplingReagent11．Name:HOBt(anhydrous)Category:PeptideCouplingReagent12．Name:HOOBtCategory:PeptideCouplingReagent13．Name:TBTUCategory:PeptideCouplingReagent〔二〕鏈接劑1．Name:SieberLinkerCategory:LinkersforSolidPhaseSynthesis2．Name:WeinrebLinkerCategory:LinkersforSolidPhaseSynthesis3．Name:DHPLinkerCategory:LinkersforSolidPhaseSynthesis4．Name:HMPLinkerCategory:LinkersforSolidPhaseSynthesis5．Name:RinkAmideLinkerCategory:LinkersforSolidPhaseSynthesis〔三〕Resin1．Name:2-ChlorotritylChlorideResinCategory:Resins2．Name:AminomethylpolystyreneResinCategory:Resins3．〔用于合成肽醇〕Name:DHPHMResinCategory:Resins4．Name:HMPA-AMResinCategory:Resins5．〔用于合成肽酰胺〕Name:KnorrResinCategory:Resins6．〔用于合成肽酰胺〕Name:Knorr-2-ChlorotritylResinCategory:Resins7．Name:MBHAResinCategory:Resins8．Name:MerrifieldResinCategory:Resins9．Name:OximeResinCategory:Resins10．Name:PAMResinCategory:Resins11．Name:Rinkamide-AMResinCategory:Resins12．Name:Rinkamide-MBHAResinCategory:Resins13．Name:SieberResinCategory:Resins14．Name:WangResinCategory:Resins15．〔用于合成肽醛〕Name:WeinrebAMResinCategory:Resins1．9-芴甲醇Name:9-FluorenylmethanolCategory:N-ProtectingReagents2．Boc-酸酐Name:BocAnhydrideCategory:N-ProtectingReagentsName:DMT-ClCategory:N-ProtectingReagents4．Name:N,N”-Disuccinimidylcarbonate(DSC)Category:N-ProtectingReagents5．芴甲氧羰酰氯Name:Fmoc-ClCategory:N-ProtectingReagents6．芴甲氧羰酰琥珀酰亞胺Name:Fmoc-OSuCategory:N-ProtectingReagents叔丁基二甲基氯硅烷Name:tert-ButyldimethylsilylChlorideCategory:N-ProtectingReagents8．Name:TritylChlorideCategory:N-ProtectingReagents苯甲氧羰酰氯(Cbz-Cl)Name:Z-ClCategory:N-ProtectingReagents苯甲氧羰酰琥珀酰亞胺(Cbz-OSu)Name:Z-OSuCategory:N-ProtectingReagents11．Category:N-ProtectingReagents12．Name:Z(2-Br)-OSuCategory:N-ProtectingReagents1．Name:Fmoc-Dap(Boc)-OHCategory:UnusualAminoAcids2．Name:3-Chloro-L-PhenylalanineCategory:UnusualAminoAcids3．Name:3-Cl-Tyr-OHCategory:UnusualAminoAcids4．Name:4-Amino-L-PhenylalanineCategory:UnusualAminoAcids5．Name:Category:UnusualAminoAcids16．Name:3,4-Dichloro-phenylalanine.10.

Category:UnusualAminoAcidsName:4-Fluoro-L-PhenylalanineCategory:UnusualAminoAcidsCategory:UnusualAminoAcidsName:4-MethoxyphenylalanineCategory:UnusualAminoAcidsName:3-NH-Tyr-OH2Category:UnusualAminoAcids4-Nitro-L-PhenylalanineHydrateCategory:UnusualAminoAcids11.Name:3-(3-Pyridyl)Category:UnusualAminoAcids12.Name:DL-m-Tyrosinecategory:UnusualAminoAcids13.Name:Fmoc-Tyr(3-NO

)-OH2Category:UnusualAminoAcids14.Name:Boc-Homo-L-TyrosineCategory:UnusualAminoAcids15.Name:Sarcosinetert-butylCategory:UnusualAminoAcids〔六〕標(biāo)記試劑1．7-氨基-4-甲基香豆素Name:7-Amino-4-methylcoumarin(AMC)Category:LabelingReagents1.標(biāo)記AminocoumarinBodipyLissamineRhodaliiineNBDFluoresceinTetramethylrhodamine2.修飾AcetylationFormylationAmidation(C—terminal)NitronationFattyacidPhosphorylation-SerinePhosphorylation-ThreoninePhosphorylation-TyrosineBenzyloxycarbonylationBiotinDansvlationSuccinylationDinitrobenzoylationSulfonation蛋白載體聯(lián)接KLH(KeyhOlelimpetlleiiiocyanin)BSA(BovineserumalbUnin)復(fù)合抗原多(MAP)Asymmetric4branchesAsymmetric8branchesSymmetric4branchesSymmetric8branches七、OverviewofPeptideSynthesisIntroductionProteinsarepresentineverylivingcellandpossessavarietyofbiochemicalactivities.Theyappearasenzymes,hormones,antibiotics,andreceptors.Theycomposeamajorportionofmuscle,hair,andskin.Consequently,scientistshavebeenveryinterestedinsynthesizingtheminthelaboratory.ThisinteresthasdevelopedintoamajorsyntheticfieldknownasPeptideSynthesis.Themajorobjectivesinthisfieldarefour-fold:Toverifythestructureofnaturallyoccurringpeptidesasdeterminedbydegradationtechniques.Tostudytherelationshipbetweenstructureandactivityofbiologicallyactiveproteinandpeptidesandestablishtheirmolecularmechanisms.Tosynthesizepeptidesthatareofmedicalimportancesuchashormonesandvaccines.Todevelopnewpeptide-basedimmunogens.SolidPhasePeptideSynthesis(SPPS)ThefundamentalpremiseofthistechniqueinvolvestheincorporationofN--aminoacidsintoapeptideofanydesiredsequencewithoneendofthesequenceremainingattachedtoasolidsupportmatrix.Whilethepeptideisbeingsynthesizedusuallybystepwisemethods,allsolublereagentscanberemovedfromthepeptide-solidsupportmatrixbyfiltrationandwashedawayattheendofeachcouplingstep.Afterthedesiredsequenceofaminoacidshasbeenobtained,thepeptidecanberemovedfromthepolymericsupport.ThegeneralschemeforsolidphasepeptidesynthesisisoutlinedinFigure1.Thesolidsupportisasyntheticpolymerthatbearsreactivegroupssuchas-OH.ThesegroupsaremadesothattheycanreacteasilywiththecarboxylgroupofanN--protectedaminoacid,therebycovalentlybindingittothepolymer.Theaminoprotectinggroup(X)canthenberemovedandasecondN--protectedaminoacidcanbecoupledtotheattachedaminoacid.Thesestepsarerepeateduntilthedesiredsequenceisobtained.Attheendofthesynthesis,adifferentreagentisappliedtocleavethebondbetweentheC-terminalaminoacidandthepolymersupport;thepeptidethengoesintosolutionandcanbeobtainedfromthesolution.FmocStrategyinSPPSThecruciallinkinanypolypeptidechainistheamidebond,whichisformedbythecondensationofanaminegroupofoneaminoacidandacarboxylgroupofanother.Generally,anaminoacidconsistsofacentralcarbonatom(calledthe-carbon)thatisattachedtofourothergroups:ahydrogen,anaminogroup,acarboxylgroup,andasidechaingroup.Thesidechaingroup,designatedR,definesthedifferentstructuresofaminoacids.Certainsidechainscontainfunctionalgroupsthatcaninterferewiththeformationoftheamidebond.Therefore,itisimportanttomaskthefunctionalgroupsoftheaminoacidsidechain.ThegeneralschemewhichoutlinesthestrategyofFmocsynthesisisshowninFigure2.Initially,thefirstFmocaminoacidisattachedtoaninsolublesupportresinviaanacidlabilelinker.DeprotectionofFmoc,isaccomplishedbytreatmentoftheaminoacidwithabase,usuallypiperidine.ThesecondFmocaminoacidiscoupledutilizingapre-activatedspeciesorinsituactivation.Afterthedesiredpeptideissynthesized,theresinboundpeptideisdeprotectedanddetachedfromthesolidsupportviaTFAcleavage.FmocCleavageTheremovalofpeptidesinsolidphasepeptidesynthesisisprimarilydonebyacidolysis.TheFmocchemistryemploystheuseofweakacidssuchasTFAorTMSBr.Variousscavengersareincludedtoprotectthepeptidefromcarbocationsgeneratedduringcleavagewhichcanleadtosidereactions.Theseadditivesusuallyincludethiolcompounds,phenol,andwater.ThefollowingprotectinggroupsarecompatiblewithTFAandTMSBrcleavage:Arg(Boc)2Arg(Mtr)Arg(Pbf)Arg(Pmc)Asn(Tmob)Asn(Trt)Asp(OtBu)

Cys(Acm)Cys(Trt)Gln(Tmob)Gln(Trt)Glu(OtBu)His(Boc)His(Trt)

Lys(Boc)Lys(Fmoc)Lys(Mtt)Ser(tBu)Thr(tBu)Tyr(tBu)Dependingonthetypeofprotectinggroupspresent,certaincombinationsofscavengersmustbeused.Forinstance,wheneitherBocandt-Butylgroupsarepresent,theircarbocationcounterparts(t-butylcationsandt-butyltrifluoroacetate)canreactwithTrp,Tyr,andMettoformtheirt-butylderivatives.WhileEDTisaveryefficientscavengerfort-butyltrifluoroacetate,itdoesnotprotectTrpfromt-butylation.Therefore,watermustbeaddedinordertosuppressalkylation.TheindoleringofTrpandthehydroxylgroupofTyrareespeciallysusceptibletothereactivityofthecleavedPmcgroup.Again,waterhasbeenshowntobeeffectiveinsuppressingthisreaction.SimilaroccurrencescanhappenwiththeTrtandMtrgroups.Therefore,scavengersintheappropriatecombinationwillgreatlyreducetheamountofsidereactions.BocStrategyinSPPSThegeneralschemewhichoutlinesthestrategyofBocsynthesisisshowninFigure3.Initially,thefirstBocaminoacidisattachedtoaninsolublesupportresinviaaHFcleavablelinker.DeprotectionofBoc,isaccomplishedbytreatmentoftheaminoacidwithTFA.ThesecondBocaminoacidiscoupledutilizingapre-activatedspeciesorinsituactivation.Afterthedesiredpeptideissynthesized,theresinboundpeptideisdeprotectedanddetachedfromthesolidsupportviaHFcleavage.BocCleavageTheBocchemistryemploystheuseofstrongacidssuchasHF,TFMSOTf,orTMSOTf.Variousadditives,usuallythiolcompoundsareaddedtoprotectthepeptidefromthecarbocationsgeneratedduringcleavage.ThefollowingprotectinggroupsarecompatiblewithHFcleavage:Arg(Mts)Arg(Tos)Asp(OBzl)Asp(OcHex)Cys(Acm)Cys(4-MeBzl)Asp(OtBu)

Cys(4-MeOBzl)Glu(OBzl)Glu(OcHex)His(Bom)His(Dnp)His(Tos)His(Trt)

His(Z)Lys(Cl-Z)Ser(Bzl)Thr(Bzl)Trp(CHO)Tyr(Br-Z)ThefollowingprotectinggroupsarecompatiblewithTFMSOTfcleavage:Arg(Mts)Asp(OBzl)Cys(Acm)Cys(4-MeBzl)Glu(OBzl)

His(Bom)His(Dnp))His(Tos)His(Z)Lys(Cl-Z)

Met(O)Ser(Bzl)Thr(Bzl)Trp(CHO)Tyr(Br-Z)ThefollowingprotectinggroupsarecompatiblewithTMSOTfcleavage:Arg(Mts)Arg(Mbs)Asp(OBz