11..1984.MerrifieldBoc(Boc:)Fmoc.Boc2采用氟)脫Boc-保護(hù)保護(hù)用芐醇類.個(gè)－衍生共價(jià)交聯(lián)用脫Boc用三乙胺游離過耦下個(gè)最保護(hù)采用TFMSA(氟)用生活生是生活,通過離個(gè)用價(jià)通過個(gè)用生是時(shí)過Merrifield過個(gè)用是共價(jià)脫保護(hù)活保護(hù)最下過-用保護(hù)BOCα用9-護(hù)2多肽合成是一個(gè)重復(fù)添加氨基酸的過程，固相合成順序一般從端（羧端向（氨基）合成。去的肽合是在液中行的為液合成?，F(xiàn)多采固相成法從而大步產(chǎn)品純的度。了防副反的發(fā)，參反應(yīng)氨基的側(cè)都是護(hù)的羧基是游的，且在反之前須活。化合成法有種，即和。于比存很多優(yōu)，現(xiàn)在大多采用法合成，如圖：具體成由列幾循環(huán)組成：去保：保的柱子和單體必須用一種堿性溶劑（piperidine）去基的保護(hù)基團(tuán)。激活和聯(lián)：下個(gè)氨基的羧基一種活劑所活?；罨瘑误w與離的氨反應(yīng)交，形成肽。在此驟使用量的超度試劑使反應(yīng)成。循：這兩反應(yīng)反循環(huán)直合成完。三、洗和脫保：多肽從上洗脫來，其護(hù)基團(tuán)一種脫護(hù)劑（TFA）脫保護(hù)。2.1樹的擇氨酸固定將相成與其他術(shù)開的主的征固載，于肽成的固相載必下要必應(yīng)應(yīng)團(tuán)，使肽鏈在這，在須成程中的化體須肽和試劑之、載體須使單載用量肽并必量被體的鏈相用于相合多肽的分子載體主要三二交聯(lián)樹脂、-樹及生這樹脂有反應(yīng)基團(tuán)直（一個(gè)氨酸。所應(yīng)團(tuán)的，樹脂樹脂生分為基樹脂羧基樹、氨基樹脂樹。成法選基脂，如Merrifield樹脂成法選擇羧基樹脂如樹脂。氨基酸的固定主要是過保護(hù)氨基酸的羧基樹脂的反應(yīng)基團(tuán)之形成的來現(xiàn)的，形成鍵的方法有多種：樹脂，保護(hù)氨基酸的、、，在度下，文檔直接同樹脂反應(yīng)或在合適的有機(jī)溶劑如二氧六環(huán)、DMF或反應(yīng)；羧樹脂，通常加適當(dāng)?shù)暮蟿┤缁螋然溥?，被保護(hù)基酸與樹脂形共酯以成氨基的固定氨基樹脂或酰型樹脂，卻加入適的縮合如后，通過保護(hù)氨酸與樹脂之間成的酰鍵來完氨基酸固定。氨基羧基、側(cè)的保護(hù)及脫除要成合成具有定的氨基酸順的多，需要對不參與形成酰鍵的基和羧加以護(hù)，同時(shí)對氨酸側(cè)鏈上活性因也要護(hù)，應(yīng)完成后將保護(hù)基因除。同相合成樣，相合成中采用烷羰基類型為α氨基的保基，為這樣不易生消。最早用芐氧羰基由于它需要較強(qiáng)的酸解條件才脫除所以后來為叔丁氧基（BOC）保，用三氟乙酸）脫保，但不適用含色氨酸等對不穩(wěn)定的肽類合成1978年changMeienlofer和等人采用報(bào)道的芴氧)作為α基護(hù)Fmoc基對酸很定但能用哌-CH2CL2哌啶-DMF脫去近年Fmoc合成得到廣泛應(yīng)用羧基常用成酯的方進(jìn)行護(hù)。酯和乙是逐合成保護(hù)基的常用方，通除或?yàn)橐杂煤鲜宥≡谒釛l件除去；酯常除去。于合含有氨酸、氨、氨等側(cè)功能的氨基的肽，為由側(cè)鏈能所的反，一需要適當(dāng)保護(hù)將側(cè)基暫保護(hù)。?；囊?cè)基不與形酰胺反應(yīng)要在合成中同時(shí)要保在后肽解時(shí)被除。如用甲保護(hù)氨酸的、除氨酸用2,2,2-三氧羰和氟-1-丁氧羰乙基保通或三氟乙脫去氨用烷氧羰）保護(hù)用三氟酸脫去。固相的接反應(yīng)液相的基，應(yīng)的基被護(hù)的羧基保護(hù)氨基在溶液形成肽鍵，要成酰鍵，用的將羧活成酸活、酰用強(qiáng)的劑（如氨形成對等法來形成酰胺鍵。中用或HOBT/DCC的對酸法、活法接肽用最廣。解及成肽鏈BOC用解脫側(cè)鏈護(hù)基，直接用，有時(shí)條件不同，它、解、氟和等脫保護(hù)方法被采用。合成肽進(jìn)一步的、與通常采用液色、和等。4固相合成的特及成法于合加固，肽合中間時(shí)的；使過反，使反應(yīng)，以最得到；加溶劑，中間；固相上鏈和的合相，生一相溶劑應(yīng)這文檔5原理啟樹脂）個(gè)感趣也許今后Boc復(fù)酸來脫保護(hù)處理帶來：與樹脂處當(dāng)每次脫約樹脂脫落大丟失越重；外催會引側(cè).Boc其色酸穩(wěn)、Atherton采3］報(bào)道甲羰基基團(tuán)作為-氨基護(hù)功行.Fmoc與根本區(qū)別于采堿脫除Fmoc為-?；FA脫除叔丁氧基樹采切除烷氧芐醇型樹脂1%TFA切除二烷芐醇型樹脂保護(hù)避強(qiáng)酸處.6.―氨基制護(hù)團(tuán)二氧六環(huán)溶以下引入氨基酸：理想基酸側(cè)護(hù)基堿件下穩(wěn)酸條件下脫除面其做紹.6.1AspGluAspGlu羧常t-Bu護(hù).TFATMSBr脫除但t-Bu保護(hù)仍側(cè)形酰亞胺.來新保護(hù)基環(huán)烷醇酯金剛烷醇酯輕保護(hù)基TMSOTf(氟甲磺酸甲硅酯)去.6.2ThrserThr基酚羥常護(hù)丁基引入比麻煩先ser制芐氧羰基再催與異烯.SerThr芐保護(hù)Ser芐醇入芐基、Thr溴芐引入芐基6.3AsnGlnAsn和側(cè)的酰胺鍵在肽合成中一般不加以保護(hù)但合成大肽時(shí)，Asn和的基活化可能發(fā)生分內(nèi)脫氫應(yīng)生成基化合.堿性時(shí)的側(cè)鏈可以環(huán)化生成酰胺且不護(hù)的Fmoc-Gln和在溶解度很差了避免這些題，可用9-咕噸，24，6-甲氧基，44′―甲氧二甲基三苯基等保護(hù)，這種基因均可用TFA脫除6.4HisHis是最容易發(fā)生旋化的氨基酸，必須加以保護(hù)對的非π-N開始用基(Bzl)甲基基TOS)保護(hù).這兩護(hù)基太理.TOS對核試劑穩(wěn)定要用解或除去，且產(chǎn)生大消旋.基團(tuán)是一個(gè)較理想的保護(hù)基降低了咪唑環(huán)堿性抑制了消旋成功地進(jìn)行了一些合.但是當(dāng)反地用堿處理時(shí)，也表現(xiàn)出一定的不定性.哌啶羰基在堿中穩(wěn)，但是沒能很好地抑制消旋，而且保護(hù)時(shí)要用很強(qiáng)的親核試劉如對環(huán)保護(hù)完全抑可以用芐氧甲(和叔丁氧基保，(Bum)可用TFA脫除更穩(wěn)定些用催化氫解強(qiáng)酸脫護(hù)是目前很發(fā)展前的側(cè)保護(hù)基，其足之處于Fmoc(His)Bum在和中溶解度較差6.5CysCys的－SH有強(qiáng)親核性被酰成硫醚易被化為二硫須加以.用保護(hù)基有三一用TFA脫除，對甲芐對甲芐基和苯甲基二可CF3CO)3T1TFA脫除，對TFA穩(wěn)定如和酰胺甲基.三對酸穩(wěn)定，叔丁硫基等用基試劑和試劑，Cys(Bzl)可用脫保護(hù).6.6ArgArg的基具有強(qiáng)親核性和堿性，必須加以保護(hù)理想是三個(gè)加以保護(hù)，保護(hù)或2個(gè)基護(hù)基四(1)基氧基磺基基基制?；庵猩芊磻?yīng)，應(yīng)用不.氧羰基應(yīng)要有和二氧羰基Fmoc(Arg)Boc的反不哌啶時(shí)不穩(wěn)定會發(fā)反保護(hù)兩個(gè)非π－N但有的反應(yīng)生.磺?；o(hù)，其中應(yīng)最，但較脫.2，，6-三基4-甲酰(，在用，30分可除但是不能完全制側(cè)鏈?；l(fā)生.三苯基保護(hù)基可用TFA除.是反應(yīng)較，側(cè)鏈有?；磻?yīng)，且其在DMF溶解不好.6.7LysLys的-NH2必加以護(hù).但α－NH2的保護(hù)應(yīng)護(hù)基鏈合除去－的保消旋題可以?；o(hù)基其的護(hù)基芐氧和Boc.6.8Fmoc基的脫團(tuán)的環(huán)的子用9-H有酸性，易被較堿除去，反應(yīng)很和.應(yīng)程可表如文檔哌啶進(jìn)攻β消除形成二苯烯，很容易被二級環(huán)胺進(jìn)攻形成穩(wěn)定的加成.基團(tuán)對不的穩(wěn)定性不同，可根據(jù)實(shí)際條件選6.9

耦固本一.及羧液，不成鍵.要形成酰鍵，經(jīng)用的手是將羧活化，方法將它變混合酸，或者它變活潑酯酰氯，者用強(qiáng)失去(碳二胺)可形成酰胺鍵，耦聯(lián)反應(yīng)可表示如下試劑)碳中Dcc使用范圍最廣，其缺點(diǎn)是形成了不溶于DCH，濾時(shí)又難于除盡.他一些如二異丙基碳二亞(DCI)甲叔基二胺用固合中它形的溶于，洗可除去其他化劑還有Bop(Bop－C1)、氯甲酸丙酯、氯甲酸異丁酯、SOC12等.中、Bop活化形成對稱酸酐、SOC12形成酰氯，其余三種形成對稱酸酐.6.10

對文檔Dcc..6.11

..N-N-.6.12

..Fmoc于立體礙序列.6.13

.過HOBtODhbtOTDO.碳亞容制ODhbt容行分離純與具類能它個(gè)優(yōu)之亮黃色結(jié)顏失利于監(jiān)測；與ODhbt類似極低離伴顏從桔色色.6.14

亞α－N直樹脂進(jìn)叫原位.代替效.活BopBop-C1.位操作DIC次憾Bop致癌六限制.6.15

裂側(cè)脫Fmoc裂脫側(cè)鏈采弱.TFA弱脫除t-BuMtr溫.足Arg鏈Mtr難脫除除掉t-Bu.采強(qiáng)脫：HF脫弱AspGluSerThr)脫除吸電子會引起環(huán)TMSOTf硫醚存脫.外據(jù)堿光離子氫.于經(jīng)了十年歷史殘?zhí)貎?yōu)越將Boc互補(bǔ)會分子發(fā)揮。22、縮寫

名稱

分子量

殘基

縮寫

名稱

分子量

殘基A-AlaFmoc-

Ala

71.08M-MetFmoc-Met

131.20N-AsnAsn(Trt)

Pro337.4D-AspC-CysFmoc-Cys(Trt)Q-GlnGln(Trt)E-Glu(OtBu)

(tBu)Fmoc-Thr(tBu)(Boc)(D)W-TrpFmoc-Tyr(tBu)

87.08101.11186.22163.18G-Gly

Gly

D-TyrFmoc-Val

99.13H-HisHis

V-ValPyroGlu

Ile353.4Leu353.4

pGluK-Lys

常名稱

分子量

名稱

分子量

密度）HOBt135.1126.30.806TBTUDIPEA(DIA)0.76380.3Ac1.08DMAP122.1Pyridine136.1Cl-HOBt121.18HBTU379.3EDTHMPA182.18TIS158.40.773520.4NMM?？s寫

分子量

縮寫

分子量

縮寫

分子量222tBu-Acm-57Pbf-72Mz-Trt-242.3Ac-Z-134.1文檔、Name

CodeOneCode

MW(-H2O)

A71.08Arginine

ArgR156.19

NAcid

AspD115.09Cysteine

CysC103.15Acid

E147.13129.12文檔

Q128.13Glycine

G75.0757.05

HisH155.16137.14

131.17113.16

LeuL113.16

LysK146.19

131.20文檔

PheF165.19147.18

ProP115.13Serine

SerS105.0987.08

ThrT119.12

W204.23186.21

Y181.19163.18文檔

ValV117.15文檔、C3H6NOC2H3O43.06-AmidohexanoateLCC4H7NOC10H8NO2174.2文檔

AFC228.2acid

EDANSBenzoylBzC7H5O105.1BenzylBzl91.1Z(Cbz)文檔BenzyloxymethylC8H9O121.2227.32-Bromobenzyloxycarbonyl2-Br-ZC8H6BrO2214.0C4H957.1C5H9O2101.1C4H9S文檔2-Cl-Z83.1160.0

DABCYL252.3DnpC6H3N2O4167.1文檔C14H11179.1FmocC15H11O2223.3FITC390.4LR598.8文檔Mesitylene-2-sulfonyl183.3121.2

McaC12H9O4217.2

213.3C20H17O273.4文檔MBzl105.2C20H17257.4C5H8NO2114.1C6H5N2O2137.12,2,4,6,7-Pentamethyldihydro-

Pbf253.3文檔

PmcC14H19O3S110C20H13N2O3SuccinylC4H5O3101.14-ToluenesulfonylC7H7O2S243.3文檔XanC13H9O181.2文檔、ofPeptidesusuallyasaserumvials.Storefreezerhavebeenreceived.reconstitutepeptide,solutionshouldutilized.Someandbedissolvedsolventssuchasapositivelybicarbonatesolutionnegativelypeptide.solventsacetonitrile,Usethesenon-aqueoussolventsandaddmakevolume.areshouldusedassoonasdegradationshouldbesingle-usepossible,andC.Repeatedshouldavoided.MethodsDissolvePeptidesThebestwaydissolveaisForareprocedure:Foruseasuchasusecysteine-containingForbasicpeptides,a30%acid,desiredForverydissolvingverydesiredForpeptidescysteines),6Murea,6Mor6M?HCltothenecessaryPreparationofHBTU/HOBtSolutionforthePeptide1.0.5HOBtDMF:oo

gHOBt(0.1135.1)AnaSpec500g,AnaSpec250mLDMF200mLlevelis2.0.45HBTU/HOBto

137.9gMW379.3)[100g,500g,CatalogErlenmayer15magneticisdissolved.asizeglassPoura文檔weeks.BiotinylationofAminoGroupWash0.1DMF.Dissolve0.244g(1244.3)g,AnaSpec21100;g,AnaSpecCatalog21101]5mLDMF-DMSO(1:1)is2.10.45HBTU/HOBtand0.32.4.andcouplingasnegative(colorless).(1:1)(2x)excess(+)-biotin.Wash(2x)DCM(2x).proceedingcleavage.ProcedureLoadingAcidto2-ChlorotritylResin10gAnaSpec5AnaSpecavessel,wash(2x),swellvessel.Weighacidadissolveacid40DMF,8.7DIEA(50vessel5DMF(5x).5020%Swirl30(5x),DCM(2x),aandhood.WeighloadedPackforofAcidLoadedResinsWeigh5101.0020%shake20Transfera10mLDMF,2mLDMF(referencecellsamplecell),zero.samplecell,2cell,checkSubs101(A)/7.8(w)absorbance=5.Check301average文檔ManualFmocSynthesismmol)WashDMF(4x)completely.10mL20%andAdd10Shake30vesselDMF(4x).MakeCheckbeadsblue.CouplingPrepareFmoc-aminoacid0.45HBTU/HOBT348μL(2above30Thisdesired.vesselDMF(4x).oo

negativeproceed2synthesis.(blue),step5re-coupleacid.SynthesisofPhosphotyrosine-ContainingPeptidesCatalog20254;5g,20255]0.10.25synthesis,0.483gFmoc-Tyr(PO3H2)-OH(1MW483.4)ABIFmoc-Tyr(PO3H2)-OHThecycleFmoc-Tyrsameasacids(seeABIsynthesizersHBTU/HOBTasFmoc-Tyr(PO3H2)-OHneedsABIsynthesizer,severalsteps(i.e.,990sec,ForABI431Asynthesizer,add"I"s.maysequences.stepFmoc-Tyr(PO3H2)-OH,ensureNegativea(blue)acidTheacidsbeThereisaacidbeSinceisside(Note:acidsSimultaneousofPeptidesWhichintheC-Termini2-ChlorotritylandWangResin*beoneresinspossessTheacid文檔TFAWangThebynthesisACTH(4-10)ACTH(4-11)Y,aT.,T.531-562(1995).CleavageFullyProtectedPeptideResin:Reagents1gPeptide-Resin:(AcOH)(TFE)aboveand1and10TFE:DCM(2:8)(2xsolventless5acontaining100Checkproceedstep6.observed,7.coldstep4fullyaarecase,aProcedureforFITCofPeptidesFITCCatalog-OHAnaSpec20957;5AnaSpecCouple-OHusingcoupling20%WashresinDMF(3-4x).SwellDCM1.1FITCridine/DMF/DCMUsearesin.Dois2resin.mixFITCgiveaRepeatFITC5-7).WashDMF(2x),(2x),DCM).文檔ProcedureRemovingMttgroupfromonSolidReagent:AnaSpec20093;AnaSpec#20094]SwellTFA/DCM(2x)(sinceDCM,quickly3%TFA/DCMensuresTFAShakeTFA10Repeatstep3.DCM(3x),(3x),(3x),DCM).dryProcedureFluoresceinofPeptidesReagent:y[0.1g,Catalog24623;0.5g,24624)Usecostsaving2xexcess4xexcessFor0.1nthesis,use75fluorescein,76HBTU,70mLDIEA.文檔、一合劑．Name:Category:Reagent．-氯羥基-苯并-三氮唑Category:．(DIC)Category:PeptideCouplingReagent．環(huán)己基碳二亞胺Name:(DCC)PeptideCouplingReagents．Name:BOPCategory:CouplingReagent．六氟磷苯并三唑-基氧基三吡咯烷磷Category:CouplingReagent文檔．羰基二咪唑Name:N,N'-Carbonyldiimidazole(CDI)Category:PeptideCouplingReagent．Name:TBTUCategory:Coupling（二）鏈接劑．Name:SieberCategory:forPhaseSynthesis．Name:RinkAmideLinkerCategory:LinkersforSolidPhaseSynthesis（三）脂1．

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