固相合成基礎(chǔ)SPPS

..一、多肽合成概論1．多肽化學(xué)合成概述：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(zhǎng)肽鏈、合成蛋白質(zhì)的固相合成法,在固相法中,每步反應(yīng)后只需簡(jiǎn)單地洗滌樹脂,便可達(dá)到純化目的.克服了經(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(dòng)化合成肽奠定了基礎(chǔ).為此,Merrifield獲得1984年諾貝爾化學(xué)獎(jiǎng).

今天,固相法得到了很大發(fā)展.除了Merrifield所建立的Boc法<Boc:叔丁氧羰基>之外,又發(fā)展了Fmoc固相法<Fmoc:9-芴甲氧羰基>.以這兩種方法為基礎(chǔ)的各種肽自動(dòng)合成儀也相繼出現(xiàn)和發(fā)展,并仍在不斷得到改造和完善.

Merrifield所建立的Boc合成法［2］是采用TFA<三氟乙酸>可脫除的Boc為α-氨基保護(hù)基,側(cè)鏈保護(hù)采用芐醇類.合成時(shí)將一個(gè)Boc－氨基酸衍生物共價(jià)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(guò)Dcc活化、耦聯(lián)下一個(gè)氨基酸,最終脫保護(hù)多采用HF法或TFMSA<三氟甲磺酸>法.用Boc法已成功地合成了許多生物大分子,如活性酶、生長(zhǎng)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)。它是分子結(jié)構(gòu)介于氨基酸和蛋白質(zhì)之間的一類化合物,由多種氨基酸按照一定的排列順序通過(guò)肽鍵結(jié)合而成。到現(xiàn)在,人們已發(fā)現(xiàn)和分離出一百多種存在于人體的肽,對(duì)于多肽的研究和利用,出現(xiàn)了一個(gè)空前的繁榮景象。多肽的全合成不僅具有很重要的理論意義,而且具有重要的應(yīng)用價(jià)值。通過(guò)多肽全合成可以驗(yàn)證一個(gè)新的多肽的結(jié)構(gòu)；設(shè)計(jì)新的多肽,用于研究結(jié)構(gòu)與功能的關(guān)系；為多肽生物合成反應(yīng)機(jī)制提供重要信息；建立模型酶以及合成新的多肽藥物等。多肽的化學(xué)合成技術(shù)無(wú)論是液相法還是固相法都已成熟。近幾十年來(lái),固相法合成多肽更以其省時(shí)、省力、省料、便于計(jì)算機(jī)控制、便于普及推廣的突出優(yōu)勢(shì)而成為肽合成的常規(guī)方法并擴(kuò)展到核苷酸合成等其它有機(jī)物領(lǐng)域。本文概述了固相合成的基本原理、實(shí)驗(yàn)過(guò)程,對(duì)其現(xiàn)狀進(jìn)行分析并展望了今后的發(fā)展趨勢(shì)。從1963年Merrifield發(fā)展成功了固相多肽合成方法以來(lái),經(jīng)過(guò)不斷的改進(jìn)和完善,到今天固相法已成為多肽和蛋白質(zhì)合成中的一個(gè)常用技術(shù),表現(xiàn)出了經(jīng)典液相合成法無(wú)法比擬的優(yōu)點(diǎn)。其基本原理是：先將所要合成肽鏈的羥末端氨基酸的羥基以共價(jià)鍵的結(jié)構(gòu)同一個(gè)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(guò)脫去氨基保護(hù)基并同過(guò)量的活化羧基組分反應(yīng),接長(zhǎng)肽鏈。重復(fù)〔縮合→洗滌→去保護(hù)→中和及洗滌→下一輪縮合操作,達(dá)到所要合成的肽鏈長(zhǎng)度,最后將肽鏈從樹脂上裂解下來(lái),經(jīng)過(guò)純化等處理,即得所要的多肽。其中α-氨基用BOC〔叔丁氧羰基保護(hù)的稱為BOC固相合成法,α-氨基用FMOC〔9-芴甲氧羰基保護(hù)的稱為FMOC固相合成法,2．固相合成的基本原理多肽合成是一個(gè)重復(fù)添加氨基酸的過(guò)程,固相合成順序一般從C端〔羧基端向N端〔氨基端合成。過(guò)去的多肽合成是在溶液中進(jìn)行的稱為液相合成法。現(xiàn)在多采用固相合成法,從而大大的減輕了每步產(chǎn)品提純的難度。為了防止副反應(yīng)的發(fā)生,參加反應(yīng)的氨基酸的側(cè)鏈都是保護(hù)的。羧基端是游離的,并且在反應(yīng)之前必須活化?；瘜W(xué)合成方法有兩種,即Fmoc和tBoc。由于Fmoc比tBoc存在很多優(yōu)勢(shì),現(xiàn)在大多采用Fmoc法合成,如圖：具體合成由下列幾個(gè)循環(huán)組成：一、去保護(hù)：Fmoc保護(hù)的柱子和單體必須用一種堿性溶劑〔piperidine去除氨基的保護(hù)基團(tuán)。二、激活和交聯(lián)：下一個(gè)氨基酸的羧基被一種活化劑所活化?；罨膯误w與游離的氨基反應(yīng)交聯(lián),形成肽鍵。在此步驟使用大量的超濃度試劑驅(qū)使反應(yīng)完成。循環(huán)：這兩步反應(yīng)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(lái),其保護(hù)基團(tuán)被一種脫保護(hù)劑〔TFA洗脫和脫保護(hù)。2.1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(lái)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須滿足如下要求：必須包含反應(yīng)位點(diǎn)〔或反應(yīng)基團(tuán),以使肽鏈連在這些位點(diǎn)上,并在以后除去；必須對(duì)合成過(guò)程中的物理和化學(xué)條件穩(wěn)定；載體必須允許在不斷增長(zhǎng)的肽鏈和試劑之間快速的、不受阻礙的接觸；另外,載體必須允許提供足夠的連接點(diǎn),以使每單位體積的載體給出有用產(chǎn)量的肽,并且必須盡量減少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)入反應(yīng)基團(tuán),才能直接連上〔第一個(gè)氨基酸。根據(jù)所導(dǎo)入反應(yīng)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。BOC合成法通常選擇氯甲基樹脂,如Merrifield樹脂；FMOC合成法通常選擇羧基樹脂如王氏樹脂。氨基酸的固定主要是通過(guò)保護(hù)氨基酸的羧基同樹脂的反應(yīng)基團(tuán)之間形成的共價(jià)鍵來(lái)實(shí)現(xiàn)的,形成共價(jià)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,直接同樹脂反應(yīng)或在合適的有機(jī)溶劑如二氧六環(huán)、DMF或DMSO中反應(yīng)；羧基樹脂,則通常加入適當(dāng)?shù)目s合劑如DCC或羧基二咪唑,使被保護(hù)氨基酸與樹脂形成共酯以完成氨基酸的固定；氨基樹脂或酰肼型樹脂,卻是加入適當(dāng)?shù)目s合劑如DCC后,通過(guò)保護(hù)氨基酸與樹脂之間形成的酰胺鍵來(lái)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順序的多肽,需要對(duì)暫不參與形成酰胺鍵的氨基和羧基加以保護(hù),同時(shí)對(duì)氨基酸側(cè)鏈上的活性基因也要保護(hù),反應(yīng)完成后再將保護(hù)基因除去。同液相合成一樣,固相合成中多采用烷氧羰基類型作為α氨基的保護(hù)基,因?yàn)檫@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(lái)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(duì)酸不穩(wěn)定的肽類的合成。1978年,changMeienlofer和Atherton等人采用Carpino報(bào)道的Fmoc<9-芴甲氧羰基>作為α氨基保護(hù)基,Fmoc基對(duì)酸很穩(wěn)定,但能用哌啶-CH2CL2或哌啶-DMF脫去,近年來(lái),Fmoc合成法得到了廣泛的應(yīng)用。羧基通常用形成酯基的方法進(jìn)行保護(hù)。甲酯和乙酯是逐步合成中保護(hù)羧基的常用方法,可通過(guò)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(duì)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(lái)說(shuō),為了避免由于側(cè)鏈功能團(tuán)所帶來(lái)的副反應(yīng),一般也需要用適當(dāng)?shù)谋Ｗo(hù)基將側(cè)鏈基團(tuán)暫時(shí)保護(hù)起來(lái)。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應(yīng),又要保證在肽合成過(guò)程中不受破壞,同時(shí)又要保證在最后肽鏈裂解時(shí)能被除去。如用三苯甲基保護(hù)半胱氨酸的S-,用酸或銀鹽、汞鹽除去；組氨酸的咪唑環(huán)用2,2,2-三氟-1-芐氧羰基和2,2,2-三氟-1-叔丁氧羰基乙基保護(hù),可通過(guò)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。固相中的接肽反應(yīng)原理與液相中的基本一致,將兩個(gè)相應(yīng)的氨基被保護(hù)的及羧基被保護(hù)的氨基酸放在溶液內(nèi)并不形成肽鍵,要形成酰胺鍵,經(jīng)常用的手段是將羧基活化,變成混合酸酐、活潑酯、酰氯或用強(qiáng)的失去劑〔如碳二亞氨形成對(duì)稱酸酐等方法來(lái)形成酰胺鍵。其中選用DCC、HOBT或HOBT/DCC的對(duì)稱酸酐法、活化酯法接肽應(yīng)用最廣。裂解及合成肽鏈的純化BOC法用TFA+HF裂解和脫側(cè)鏈保護(hù)基,FMOC法直接用TFA,有時(shí)根據(jù)條件不同,其它堿、光解、氟離子和氫解等脫保護(hù)方法也被采用。合成肽鏈進(jìn)一步的精制、分離與純化通常采用高效液相色譜、親和層析、毛細(xì)管電泳等。4．固相合成的特點(diǎn)及存在的主要問(wèn)題固相合成法對(duì)于肽合成的顯著的優(yōu)點(diǎn)：簡(jiǎn)化并加速了多步驟的合成；因反應(yīng)在一簡(jiǎn)單反應(yīng)器皿中便可進(jìn)行,可避免因手工操作和物料重復(fù)轉(zhuǎn)移而產(chǎn)生的損失；固相載體共價(jià)相聯(lián)的肽鏈處于適宜的物理狀態(tài),可通過(guò)快速的抽濾、洗滌未完成中間的純化,避免了液相肽合成中冗長(zhǎng)的重結(jié)晶或分柱步驟,可避免中間體分離純化時(shí)大量的損失；使用過(guò)量反應(yīng)物,迫使個(gè)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；固相載體上肽鏈和輕度交聯(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(duì)肽自聚集熱力學(xué)不利而對(duì)反應(yīng)適宜。固相合成的主要存在問(wèn)題是固相載體上中間體雜肽無(wú)法分離,這樣造成最終產(chǎn)物的純度不如液相合成物,必需通過(guò)可靠的分離手段純化。5．固相合成的研究發(fā)展前景固相多肽合成已經(jīng)有40年的歷史了,然而到現(xiàn)在,人們還只能合成一些較短的肽鏈,更談不上隨心所欲地合成蛋白質(zhì)了,同時(shí)合成中的試劑毒性,昂貴費(fèi)用,副產(chǎn)物等一直都是令人頭痛的問(wèn)題,而在生物體內(nèi),核糖體上合成肽鏈的速度和產(chǎn)率都是驚人的,那么,是否能從生物體合成蛋白質(zhì)的原理上得到一些啟發(fā),應(yīng)用在固相多肽合成〔樹脂上,這是一個(gè)令人感興趣的問(wèn)題,也許是今后多肽合成的發(fā)展。在Boc合成法中,反復(fù)地用酸來(lái)脫保護(hù),這種處理帶來(lái)了一些問(wèn)題：如在肽與樹脂的接頭處,當(dāng)每次用50%TFA脫Boc基時(shí),有約1.4%的肽從樹脂上脫落,合成的肽越大,這樣的丟失越嚴(yán)重；此外,酸催化會(huì)引起側(cè)鏈的一些副反應(yīng).Boc合成法尤其不適于合成含有色氨酸等對(duì)酸不穩(wěn)定的肽類.1978年,Chang、Meienlofer和Atherton等人采用Carpino［3］報(bào)道的Fmoc<9-芴甲氧羰基>基團(tuán)作為α-氨基保護(hù)基,成功地進(jìn)行了多肽的Fmoc固相合成.Fmoc法與Boc法的根本區(qū)別在于采用了堿可脫除的Fmoc為α-氨基的保護(hù)基.側(cè)鏈的保護(hù)采用TFA可脫除的叔丁氧基等,樹脂采用90%TFA可切除的對(duì)烷氧芐醇型樹脂和1%TFA可切除的二烷氧芐醇型樹脂,最終的脫保護(hù)避免了強(qiáng)酸處理.6.

Fmoc―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(guò)以下反應(yīng)引入到氨基酸中的：理想的Fmoc-氨基酸的側(cè)鏈保護(hù)基應(yīng)在堿性條件下穩(wěn)定,在酸性條件下脫除.下面對(duì)其做一介紹.

6.1Asp和Glu

Asp和Glu側(cè)鏈羧基常用t-Bu保護(hù).可用TFA、TMSBr等脫除.但是用t-Bu保護(hù)仍有側(cè)鏈環(huán)化形成酰亞胺的副反應(yīng)發(fā)生.近年來(lái),發(fā)展了一些新的保護(hù)基如環(huán)烷醇酯、金剛烷醇酯等可減輕這一副反應(yīng),這些保護(hù)基可用TMSOTf<三氟甲磺酸三甲硅烷酯>除去.

6.2Ser、Thr和Tyr

ser、Thr的羥基及Tyr的酚羥基通常用t-Bu保護(hù).叔丁基的引入比較麻煩,首先ser制成芐氧羰基酯,再在酸催化下與異丁烯反應(yīng).Ser和Thr還可用芐基保護(hù),Ser用芐醇引入芐基、Thr用溴芐引入芐基.

6.3Asn和Gln

Asn和Gln側(cè)鏈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Gln的α-羧基活化時(shí)可能會(huì)發(fā)生分子內(nèi)脫氫反應(yīng)生成氰基化合物.堿性時(shí)Gln的側(cè)鏈可以環(huán)化生成酰胺.而且不保護(hù)的Fmoc-Gln和Fmoc-Asn在DCM中溶解度很差.為了避免這些問(wèn)題,可以用9-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),這四種基因均可用TFA脫除.

6.4His

His是最容易發(fā)生消旋化的氨基酸,必須加以保護(hù).

對(duì)咪唑環(huán)的非π-N開始用芐基<Bzl>和甲基磺酰基<TOS>保護(hù).但這兩種保護(hù)基均不太理想.TOS對(duì)親核試劑不穩(wěn)定,Bzl需要用氫解或Na/NHs除去,并且產(chǎn)生很大程度消旋.Boc基團(tuán)是一個(gè)較理想的保護(hù)基,降低了咪唑環(huán)的堿性,抑制了消旋,成功地進(jìn)行了一些合成.但是當(dāng)反復(fù)地用堿處理時(shí),也表現(xiàn)出一定的不穩(wěn)定性.哌啶羰基在堿中穩(wěn)定,但是沒(méi)能很好地抑制消旋,而且脫保護(hù)時(shí)要用很強(qiáng)的親核試劉如.

對(duì)咪唑環(huán)π-N保護(hù),可以完全抑制消旋,π-N可以用芐氧甲基<Bom>和叔丁氧甲基<Bum>保護(hù),<Bum>可以用TFA脫除,Bom更穩(wěn)定些,需用催化氫解或強(qiáng)酸脫保護(hù),Bum是目前很有發(fā)展前途的His側(cè)鏈保護(hù)基,其不足之處在于Fmoc<His>Bum在DCM和DMF中的溶解度較差.

6.5Cys

Cys的－SH具有強(qiáng)親核性,易被?；闪蛎?也易被氧化為二硫鍵,必須加以保護(hù).常用保護(hù)基有三類：一類用TFA可脫除,如對(duì)甲芐基、對(duì)甲氧芐基和三苯甲基等；第二類可用<CF3CO>3T1／TFA脫除,對(duì)TFA穩(wěn)定.如t-Bu、Bom和乙酰胺甲基等.第三類對(duì)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還原,Cys<Bzl>可用Na/NH3<1>脫保護(hù).

6.6Arg

Arg的胍基具有強(qiáng)親核性和堿性,必須加以保護(hù).理想的情況是三個(gè)氮都加以保護(hù),實(shí)際上保護(hù)1或2個(gè)胍基氮原子.保護(hù)基分四類：<1>硝基<2>烷氧羰基<3>磺酰基<4>三苯甲基.

硝基在制備、?；呀庵挟a(chǎn)生很多副反應(yīng),應(yīng)用不廣.烷氧羰基應(yīng)主要有Boc和二金剛烷氧羰基<Adoc>2、Fmoc<Arg>Boc的耦聯(lián)反效率不高,哌啶理時(shí)不處穩(wěn)定,會(huì)發(fā)生副反應(yīng)；Adoc保護(hù)了兩個(gè)非π－N,但有同樣的副反應(yīng)發(fā)生.對(duì)磺?；Ｗo(hù),其中TOS應(yīng)用最廣,但它較難脫除.近年來(lái)2,3,6-三甲基-4-甲氧苯橫酰基<Mtr>較受歡迎,在TFA作用下,30分鐘即可脫除,但是它們都不能完全抑制側(cè)鏈的?；l(fā)生.三苯甲基保護(hù)基可用TFA脫除.缺點(diǎn)是反應(yīng)較慢,側(cè)鏈仍有酰化反應(yīng),且其在DCM、DMF中溶解度不好.

6.7Lys

Lys的ε-NH2必須加以保護(hù).但與α－NH2的保護(hù)方式應(yīng)不同,該保護(hù)基要到肽鏈合成后除去.ε－NH2的保護(hù)無(wú)消旋問(wèn)題,可以采用酰基保護(hù)基,其它常用的保護(hù)基有芐氧碳基和Boc.

6.8Fmoc基團(tuán)的脫除Fmoc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,反應(yīng)條件很溫和.反應(yīng)過(guò)程可表示如下：哌啶進(jìn)攻9-H,β消除形成二苯芴烯,很容易被二級(jí)環(huán)胺進(jìn)攻形成穩(wěn)定的加成物.Fmoc基團(tuán)對(duì)不同的堿穩(wěn)定性不同,可根據(jù)實(shí)際條件選用.6.9耦聯(lián)反應(yīng)固相中的接肽反應(yīng)原理與液相中基本一致.將兩個(gè)相應(yīng)的氨基被保護(hù)的及羧基被保護(hù)的氨基酸放在溶液內(nèi),并不形成肽鍵.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?或者用強(qiáng)的失去劑<碳二亞胺>也可形成酰胺鍵,耦聯(lián)反應(yīng)可表示如下：<A：羰基活潑試劑>碳二亞胺是常用的活化試劑,其中Dcc使用范圍最廣,其缺點(diǎn)是形成了不溶于DCM的DCH,過(guò)濾時(shí)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,它們形成的脲溶于DCM中,經(jīng)洗滌可以除去.其他活化試劑,還有Bop<Bop－C1>、氯甲酸異丙酯、氯甲酸異丁酯、SOC12等.其中Dcc、Bop活化形成對(duì)稱酸酐、SOC12形成酰氯,其余三種形成不對(duì)稱酸酐.

6.10對(duì)稱酸酐法用Dcc形成對(duì)稱酸酐的方法使用較廣.其缺點(diǎn)是有些氨基酸在DCM中不易溶解,生成的Fmoc氨基酸酐溶解度更差.同時(shí)還有些副反應(yīng),如形成二肽、消旋等.

6.11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(jì)量及溶劑條件下,耦聯(lián)反應(yīng)很快.而且,在此反應(yīng)中使用的N-甲基嗎啉和N-甲基哌啶對(duì)Fmoc基團(tuán)無(wú)影響.

6.12酰氯法在Boc法中不常用的酰氯,因?yàn)楸容^激烈,一些保護(hù)基如Boc不穩(wěn)定.但是,Fmoc基團(tuán)可以耐受酰氯處理,生成的Fmoc氨基酰氯也很穩(wěn)定.在三甲基乙酸／三胺或苯并三氮唑／二異丙基乙二胺中,反應(yīng)速度很快,消旋很少.酰氯法在固相合成中應(yīng)用還不多,但已表明,Fmoc－氨基酰氯適用于合成有立體障礙的肽序列.

6.13活化酯法活化酯法在固相合成中應(yīng)用最為廣泛.采用過(guò)的試劑也很多,近來(lái)最常用的有HOBt酯、ODhbt酯、OTDO酯等.

HOBt酯反應(yīng)快,消旋少,用碳二亞胺很容易制得；ODhbt酯很穩(wěn)定,容易進(jìn)行分離純化,與HOBt酯具有類似的反應(yīng)性和消旋性能,它還有一個(gè)優(yōu)越之處,在?；瘯r(shí)有亮黃色、耦聯(lián)結(jié)束時(shí)顏色消失,有利于監(jiān)測(cè)反應(yīng)；OTDO酯與ODhbt酯類似,消旋化極低,易分離,酰化時(shí)伴有顏色從桔紅色到黃色的變化等.

6.14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)行反應(yīng)叫做原位法.

用DIC代替Dcc效果更好.其他的活化試劑還有Bop和Bop-C1等.原位法反應(yīng)快、副反應(yīng)少、易操作.其中DIC最有效,其次是Bop、Bop-C1等.遺憾的是Bop?；瘯r(shí)生成致癌的六甲基磷酰胺,限制了其應(yīng)用.6.15裂解及側(cè)鏈保護(hù)基脫除Fmoc法裂解和脫側(cè)鏈保護(hù)基時(shí)可采用弱酸.TFA為應(yīng)用最廣泛的弱酸試劑,它可以脫除t-Bu、Boc、Adoc、Mtr等；條件溫和、副反應(yīng)較少.不足之處：Arg側(cè)鏈的Mtr很難脫除,TFA用量較大；無(wú)法除掉Cys的t-Bu等基因.也有采用強(qiáng)酸脫保護(hù)的方法：如用HF來(lái)脫除一些對(duì)弱酸穩(wěn)定的保護(hù)基,如Asp、Glu、Ser、Thr的Bzl<芐基>保護(hù)基等,但是當(dāng)脫除Asp的吸電子保護(hù)基時(shí),會(huì)引起環(huán)化副反應(yīng).而TMSBr和TMSOTf在有苯甲硫醚存在時(shí),脫保護(hù)速度很快.此外,根據(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.Fmoc基團(tuán)用于固相合成多肽已經(jīng)有了十多年的歷史,在合成一些含有在酸性條件下不穩(wěn)定的氨基的殘基的肽時(shí),具有特別優(yōu)越之處.將Fmoc法和Boc法互相補(bǔ)充,定會(huì)在合成更多、更大的生物分子中發(fā)揮。二、常用保護(hù)氨基酸數(shù)據(jù)縮寫名稱分子量殘基縮寫名稱分子量殘基A-AlaFmoc-Ala311.371.08M-MetFmoc-Met371.5131.20R-ArgFmoc-Arg<Pbf>648.77155.18F-PheFmoc-Phe387.4147.18N-AsnFmoc-Asn<Trt>596.7114.11P-ProFmoc-Pro337.497.19D-AspFmoc-Asp<OtBu>411.46115.09S-SerFmoc-Ser<tBu>383.487.08C-CysFmoc-Cys<Trt>585.7103.15T-ThrFmoc-Thr<tBu>397.5101.11Q-GlnFmoc-Gln<Trt>610.7128.13W-TrpFmoc-Trp<Boc>526.59186.22E-GluFmoc-Glu<OtBu>425.48129.12<D>W-TrpFmoc-Tyr<tBu>459.5163.18G-GlyFmoc-Gly297.357.05D-TyrFmoc-Val339.499.13H-HisFmoc-His<Trt>619.7137.14V-ValPyroGlu129.1I-IleFmoc-Ile353.4113.16pGluL-LeuFmoc-Leu353.4113.16K-LysFmoc-Lys<Boc>468.5128.18常用試劑種類及數(shù)據(jù)名稱分子量名稱分子量密度〔g/mlHOBt135.1DIPCDI<DIC>126.30.806TBTU321.1DIPEA<DIA>129.40.76HATU380.3Ac2O102.11.08DMAP122.1Pyridine79.10.983HOAT136.1TFA114.21.44Cl-HOBt169.57TMP121.180.92HBTU379.3EDT94.241.123HMPA182.18TIS158.40.773PyBoc520.4NMM101.150.9168常見保護(hù)基團(tuán)結(jié)構(gòu)及數(shù)據(jù)縮寫分子量縮寫分子量縮寫分子量Fmoc-222tBu-56Acm-57Pbf-253OtBu-72Mz-165.1Trt-242.3Ac-43Boc-101.1Z-134.1三、常用氨基酸結(jié)構(gòu)及性質(zhì)NameSymbolMWMW<-H2O>StructureThreeLetterCodeOneLetterCodeAlanineAlaA89.0971.08

ArginineArgR174.20156.19

AsparagineAsnN132.12114.10

AsparticAcidAspD133.10115.09

CysteineCysC121.15103.15

GlutamicAcidGluE147.13129.12

GlutamineGlnQ146.15128.13

GlycineGlyG75.0757.05

HistidineHisH155.16137.14

IsoleucineIleI131.17113.16

LeucineLeuL131.17113.16

LysineLysK146.19128.17

MethionineMetM149.21131.20

PhenylalaninePheF165.19147.18

ProlineProP115.1397.12

SerineSerS105.0987.08

ThreonineThrT119.12101.11

TryptophanTrpW204.23186.21

TyrosineTyrY181.19163.18

ValineValV117.1599.13

四、常見保護(hù)基團(tuán)結(jié)構(gòu)NameStructureSymbolFormulaResidueWtAcetamidomethyl

AcmC3H6NO72.1Acetyl

AcC2H3O43.0Allyloxycarbonyl

AlocC4H5O285.16-Amidohexanoate

LCC4H7NO85.17-Amido-4-methylcoumaryl

AMCC10H8NO2174.27-Amido-4-trifluoromethyl-coumaryl

AFCC10H5F3NO2228.25-[<2-Aminoethyl>amino]-naphthalene-1-sulfonicacid

EDANSC12H13N2O3S265.3Benzoyl

BzC7H5O105.1Benzyl

BzlC7H791.1Benzyloxycarbonyl

Z<Cbz>C8H7O2135.1Benzyloxymethyl

BomC8H9O121.2<+>-Biotinyl

BiotinC10H15N2O2S227.32-Bromobenzyloxycarbonyl

2-Br-ZC8H6BrO2214.0tert-Butyl

tBuC4H957.1tert-Butyloxycarbonyl

BocC5H9O2101.1tert-Butylthio

StBuC4H9S89.22-Chlorobenzyloxycarbonyl

2-Cl-ZC8H6ClO2169.6Cyclohexyl

cHexC6H1183.12,6-Dichlorobenzyl

2,6-di-Cl-BzlC7H5Cl2160.04-<4-Dimethylaminophenyl-azo>benzoyl

DABCYLC15H14N3O252.32,4-Dinitrophenyl

DnpC6H3N2O4167.19-Fluorenylmethyl

FmC14H11179.19-Fluorenylmethyloxy-carbonyl

FmocC15H11O2223.3FluoresceinIsothiocyanate

FITCC21H12NO5S390.4LissamineRhodamine

LRC31H38N2O6S2598.8Mesitylene-2-sulfonyl

MtsC9H11O2S183.34-Methoxybenzyl

MobC8H9O121.2<7-Methoxycoumarin-4-yl>acetyl

McaC12H9O4217.24-Methoxy-2,3,6-trimethyl-benzenesulfonyl

MtrC10H13O3S213.34-Methoxytrityl

MmtC20H17O273.44-Methylbenzyl

MBzlC8H9105.24-Methyltrityl

MttC20H17257.44-Morpholinecarbonyl

MuC5H8NO2114.1p-Nitroanilide

pNAC6H5N2O2137.12,2,4,6,7-Pentamethyldihydro-benzofuran-5-sulfonyl

PbfC13H17O3S253.32,2,5,7,8-Pentamethyl-chroman-6-sulfonyl

PmcC14H19O3S267.4Rhodamine110

R110C20H13N2O3329.3Succinyl

SucC4H5O3101.14-Toluenesulfonyl

TosC7H7O2S155.2Trityl

TrtC17H15243.3Xanthyl

XanC13H9O181.2五、多肽常識(shí)ReconstitutionandStorageofPeptidesPeptidesareusuallysuppliedasafluffy,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/HOBtSolutionforthePeptideSynthesizerPreparationof0.5MHOBtinDMF:Weigh13.5ganhydrousHOBt<0.1mol,MW135.1>[100g,AnaSpecCatalog#21003;500g,AnaSpecCatalog#21004]intoa250mLgraduatedcylinder.AddDMFuntilthe200mLlevelisreached.Preparationof0.45MHBTU/HOBtsolution:Addthesolutionpreparedinstep1to37.9gHBTU<0.1mol,MW379.3>[100g,AnaSpecCatalog#21001;500g,AnaSpecCatalog#21002]containedinabeakeroranErlenmayerflask.Stirforabout15minwithamagneticstirringbaruntilHBTUisdissolved.Filterthesolutionthroughafineporesizesinteredglassfunnel.Pourthefilteredsolutionintoanappropriatebottleforattachmenttoapeptidesynthesizer.*Thissolutionisstableatroomtemperatureforatleastsixweeks.BiotinylationofAminoGroupWash0.1mmolresinwithDMF.Dissolve0.244g<+>-biotin<1mmol,MW244.3>[1g,AnaSpecCatalog#21100;5g,AnaSpecCatalog#21101]in5mLDMF-DMSO<1:1>solution.Alittlewarmingisnecessary.Add2.1mL0.45MHBTU/HOBtsolutionand0.3mLDIEAtothesolutionpreparedinstep2.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,add8.7mLDIEA<50mmol>,swirlmixturefor30minatroomtemperature.Add5mLmethanolintothereactionvesselandswirlfor5min.DrainandwashwithDMF<5x>.Checksubstitution.Add50mL20%piperidinetoremovetheFmocgroup.Swirlmixturefor30min.WashwithDMF<5x>,DCM<2x>,putresinontissuepaperoverafoampadandletdryatroomtemperatureovernightunderthehood.Covertheresinwithanotherpieceoftissuepaper,presslightlytobreakaggregates.Weighloadedresin.Packinappropriatecontainer.ProcedureforCheckingSubstitutionofFmoc-AminoAcidLoadedResinsWeighduplicatesamplesof5to10mgloadedresininaneppendorftube,add1.00mL20%piperidine/DMF,shakefor20min,centrifugedowntheresin.Transfer100μLoftheabovesolutionintoatubecontaining10mLDMF,mixwell.Pipette2mLDMFintoeachofthetwocells<referencecellandsamplecell>,setspectrophotometertozero.Emptythesamplecell,transfer2mLofthesolutionfromstep2intothesamplecell,checkabsorbance.Subs=101<A>/7.8<w>

A=absorbance

w=mgofresinCheckabsorbancethreetimesat301nm,calculateaveragesubstitution.ManualFmocSynthesis<0.25mmol>WashresinwithDMF<4x>andthendraincompletely.Addapproximately10mL20%piperidine/DMFtoresin.Shakeforoneminanddrain.Addanother10mL20%piperidine/DMF.Shakefor30min.DrainreactionvesselandwashresinwithDMF<4x>.Makesurethereisnopiperidineremaining.Checkbeadsusingninhydrintest,beadsshouldbeblue.CouplingStep-Preparethefollowingsolution:

1mmolFmoc-aminoacid

2.1mL0.45MHBTU/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]For0.1mmolor0.25mmolsynthesis,use0.483gFmoc-Tyr<PO3H2>-OH<1mmol,MW483.4>.ForABIsynthesizers,packFmoc-Tyr<PO3H2>-OHinacartridge.ThecycleprogramforcouplingFmoc-Tyr<PO3H2>-OHisthesameasforotherFmoc-aminoacidsexceptforthecouplingtime<seestep3>.<Note:ABIsynthesizersuseHBTU/HOBTastheactivatingreagent.>ThecouplingtimeforFmoc-Tyr<PO3H2>-OHneedstobeincreased.ForABImodel430Apeptidesynthesizer,insertseveralsteps<i.e.,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#20151]

Fmoc--Ahx-OH[1g,AnaSpecCatalog#20957;5g,AnaSpecCatalog#20958]CoupleFmoc--Ahx-OHtotheaminoterminalofthepeptide-resinusingstandardcouplingconditions."De-Fmoc"withpiperidineusingthestandard20%piperidineprocedure.WashresinwithDMF<3-4x>.SwellresinwithDCManddrain.Preparesolutionof1.1equivalentofFITCinpyridine/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#20093;5g,AnaSpecCatalog#20094]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>[0.1g,AnaSpecCatalog#24623;0.5g,AnaSpecCatalog#24624>Usestandardcouplingmethodtocouple5-carboxyfluoresceintotheaminogroupofthepeptide.Forcostsavingpurposes,use2xexcesscomparedtothemmolofresin,insteadofthestandard4xexcessusedforFmoc-aminoacids.For0.1mmolsynthesis,use75mg5-carboxyfluorescein,76mgHBTU,and70mLDIEA.六、常用試劑〔一縮合劑1．Name:

EDC.HCl

Category:

PeptideCouplingReagent2．6-氯-1-羥基-苯并-三氮唑Name:

Cl-HOBt

Category:

PeptideCouplingReagent3．Name:N,N'-Diisopropylcarbodiimide<DIC>Category:PeptideCouplingReagent4．二環(huán)己基碳化二亞胺Name:

Dicyclohexylcarbodiimide<DCC>

Category:PeptideCouplingReagents5．Name:BOPReagent

Category:PeptideCouplingReagent6．六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷Name:PyBOP

Category:PeptideCouplingReagent7．N,N'-羰基二咪唑Name:N,N'-Carbonyldiimidazole<CDI>Category:PeptideCouplingReagent8．Name:

TBTU

Category:

PeptideCouplingReagent〔二鏈接劑1．Name:SieberLinker

Category:LinkersforSolidPhaseSynthesis2．Name:

RinkAmideLinker

Category:LinkersforSolidPhaseSynthesis〔三樹脂Resin1．Name:

2-ChlorotritylChlorideResin

Category:Resins2．Name:

AminomethylpolystyreneResin

Category:Resins3．〔用于合成肽醇Name:

DHPHMResin

Category:Resins4．Name:

HMPA-AMResin

Category:Resins5．〔用于合成肽酰胺Name:

KnorrResin

Category:Resins6．〔用于合成肽酰胺Name:

Knorr-2-ChlorotritylResin

Category:Resins7．Name:

MBHAResin

Category:Resins8．Name:

MerrifieldResin

Category:Resins9．Name:

Rinkamide-AMResin

Category:Resins10．Name:

Rinkamide-MBHAResin

Category:Resins11．Name:

SieberResin

Category:Resins12Name:

WangResin

Category:Resins13．〔用于合成肽醛Name:

WeinrebAMResin

Category:Resins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>2Cys<Acm>Lys<Boc>Arg<Mtr>Cys<Trt>Lys<Fmoc>Arg<Pbf>Gln<Tmob>Lys<Mtt>Arg<Pmc>Gln<Trt>Ser<tBu>Asn<Tmob>Glu<OtBu>Thr<tBu>Asn<Trt>His<Boc>Tyr<tBu>Asp<OtBu>His<Trt>Dependingonthetypeofprotectinggroupspresent,certaincombinationsofscavengersmustbeused.Forinstance,wheneitherBocandt-Butylgroupsarepresent,theircarbocationcounterparts<t-butylcationsandt-butyltrifluoroacetate>canreactwithTrp,Tyr,andMettoformtheirt-butylderivatives.WhileEDTisaveryefficientscavengerfort-butyltrifluoroacetate,itdoesnotprotectTrpfromt-butylation.Therefore,watermustbeaddedinordertosuppressalkylation.TheindoleringofTrpandthehydroxylgroupofTyrareespeciallysusceptibletothe