固相合成基础SPPS

..一、多肽合成概论1．多肽化学合成概述：1963年,［1］创立了将氨基酸的C末端固定在不溶性树脂上,然后在此树脂上依次缩合氨基酸,延长肽链、合成蛋白质的固相合成法,在固相法中,每步反应后只需简单地洗涤树脂,便可达到纯化目的.克服了经典液相合成法中的每一步产物都需纯化的困难,为自动化合成肽奠定了基础.为此,Merrifield获得1984年诺贝尔化学奖.

今天,固相法得到了很大发展.除了Merrifield所建立的Boc法<Boc:叔丁氧羰基>之外,又发展了Fmoc固相法<Fmoc:9-芴甲氧羰基>.以这两种方法为基础的各种肽自动合成仪也相继出现和发展,并仍在不断得到改造和完善.

Merrifield所建立的Boc合成法［2］是采用TFA<三氟乙酸>可脱除的Boc为α-氨基保护基,侧链保护采用苄醇类.合成时将一个Boc－氨基酸衍生物共价交联到树脂上,用TFA脱除Boc,用三乙胺中和游离的氨基末端,然后通过Dcc活化、耦联下一个氨基酸,最终脱保护多采用HF法或TFMSA<三氟甲磺酸>法.用Boc法已成功地合成了许多生物大分子,如活性酶、生长因子、人工蛋白等.

多肽是涉及生物体内各种细胞功能的生物活性物质。它是分子结构介于氨基酸和蛋白质之间的一类化合物,由多种氨基酸按照一定的排列顺序通过肽键结合而成。到现在,人们已发现和分离出一百多种存在于人体的肽,对于多肽的研究和利用,出现了一个空前的繁荣景象。多肽的全合成不仅具有很重要的理论意义,而且具有重要的应用价值。通过多肽全合成可以验证一个新的多肽的结构；设计新的多肽,用于研究结构与功能的关系；为多肽生物合成反应机制提供重要信息；建立模型酶以及合成新的多肽药物等。多肽的化学合成技术无论是液相法还是固相法都已成熟。近几十年来,固相法合成多肽更以其省时、省力、省料、便于计算机控制、便于普及推广的突出优势而成为肽合成的常规方法并扩展到核苷酸合成等其它有机物领域。本文概述了固相合成的基本原理、实验过程,对其现状进行分析并展望了今后的发展趋势。从1963年Merrifield发展成功了固相多肽合成方法以来,经过不断的改进和完善,到今天固相法已成为多肽和蛋白质合成中的一个常用技术,表现出了经典液相合成法无法比拟的优点。其基本原理是：先将所要合成肽链的羟末端氨基酸的羟基以共价键的结构同一个不溶性的高分子树脂相连,然后以此结合在固相载体上的氨基酸作为氨基组份经过脱去氨基保护基并同过量的活化羧基组分反应,接长肽链。重复〔缩合→洗涤→去保护→中和及洗涤→下一轮缩合操作,达到所要合成的肽链长度,最后将肽链从树脂上裂解下来,经过纯化等处理,即得所要的多肽。其中α-氨基用BOC〔叔丁氧羰基保护的称为BOC固相合成法,α-氨基用FMOC〔9-芴甲氧羰基保护的称为FMOC固相合成法,2．固相合成的基本原理多肽合成是一个重复添加氨基酸的过程,固相合成顺序一般从C端〔羧基端向N端〔氨基端合成。过去的多肽合成是在溶液中进行的称为液相合成法。现在多采用固相合成法,从而大大的减轻了每步产品提纯的难度。为了防止副反应的发生,参加反应的氨基酸的侧链都是保护的。羧基端是游离的,并且在反应之前必须活化。化学合成方法有两种,即Fmoc和tBoc。由于Fmoc比tBoc存在很多优势,现在大多采用Fmoc法合成,如图：具体合成由下列几个循环组成：一、去保护：Fmoc保护的柱子和单体必须用一种碱性溶剂〔piperidine去除氨基的保护基团。二、激活和交联：下一个氨基酸的羧基被一种活化剂所活化。活化的单体与游离的氨基反应交联,形成肽键。在此步骤使用大量的超浓度试剂驱使反应完成。循环：这两步反应反复循环直到合成完成。三、洗脱和脱保护：多肽从柱上洗脱下来,其保护基团被一种脱保护剂〔TFA洗脱和脱保护。2.1树脂的选择及氨基酸的固定将固相合成与其他技术分开来的最主要的特征是固相载体,能用于多肽合成的固相载体必须满足如下要求：必须包含反应位点〔或反应基团,以使肽链连在这些位点上,并在以后除去；必须对合成过程中的物理和化学条件稳定；载体必须允许在不断增长的肽链和试剂之间快速的、不受阻碍的接触；另外,载体必须允许提供足够的连接点,以使每单位体积的载体给出有用产量的肽,并且必须尽量减少被载体束缚的肽链之间的相互作用。用于固相法合成多肽的高分子载体主要有三类：聚苯乙烯-苯二乙烯交联树脂、聚丙烯酰胺、聚乙烯-乙二醇类树脂及衍生物,这些树脂只有导入反应基团,才能直接连上〔第一个氨基酸。根据所导入反应基团的不同,又把这些树脂及树脂衍生物分为氯甲基树脂、羧基树脂、氨基树脂或酰肼型树脂。BOC合成法通常选择氯甲基树脂,如Merrifield树脂；FMOC合成法通常选择羧基树脂如王氏树脂。氨基酸的固定主要是通过保护氨基酸的羧基同树脂的反应基团之间形成的共价键来实现的,形成共价键的方法有多种：氯甲基树脂,通常先制得保护氨基酸的四甲铵盐或钠盐、钾盐、铯盐,然后在适当温度下,直接同树脂反应或在合适的有机溶剂如二氧六环、DMF或DMSO中反应；羧基树脂,则通常加入适当的缩合剂如DCC或羧基二咪唑,使被保护氨基酸与树脂形成共酯以完成氨基酸的固定；氨基树脂或酰肼型树脂,却是加入适当的缩合剂如DCC后,通过保护氨基酸与树脂之间形成的酰胺键来完成氨基酸的固定。氨基、羧基、侧链的保护及脱除要成功合成具有特定的氨基酸顺序的多肽,需要对暂不参与形成酰胺键的氨基和羧基加以保护,同时对氨基酸侧链上的活性基因也要保护,反应完成后再将保护基因除去。同液相合成一样,固相合成中多采用烷氧羰基类型作为α氨基的保护基,因为这样不易发生消旋。最早是用苄氧羰基,由于它需要较强的酸解条件才能脱除,所以后来改为叔丁氧羰基〔BOC保护,用TFA〔三氟乙酸脱保护,但不适用含有色氨酸等对酸不稳定的肽类的合成。1978年,changMeienlofer和Atherton等人采用Carpino报道的Fmoc<9-芴甲氧羰基>作为α氨基保护基,Fmoc基对酸很稳定,但能用哌啶-CH2CL2或哌啶-DMF脱去,近年来,Fmoc合成法得到了广泛的应用。羧基通常用形成酯基的方法进行保护。甲酯和乙酯是逐步合成中保护羧基的常用方法,可通过皂化除去或转变为肼以便用于片断组合；叔丁酯在酸性条件下除去；苄酯常用催化氢化除去。对于合成含有半胱氨酸、组氨酸、精氨酸等带侧链功能基的氨基酸的肽来说,为了避免由于侧链功能团所带来的副反应,一般也需要用适当的保护基将侧链基团暂时保护起来。保护基的选择既要保证侧链基团不参与形成酰胺的反应,又要保证在肽合成过程中不受破坏,同时又要保证在最后肽链裂解时能被除去。如用三苯甲基保护半胱氨酸的S-,用酸或银盐、汞盐除去；组氨酸的咪唑环用2,2,2-三氟-1-苄氧羰基和2,2,2-三氟-1-叔丁氧羰基乙基保护,可通过催化氢化或冷的三氟乙酸脱去。精氨酸用金刚烷氧羰基〔Adoc保护,用冷的三氟乙酸脱去。固相中的接肽反应原理与液相中的基本一致,将两个相应的氨基被保护的及羧基被保护的氨基酸放在溶液内并不形成肽键,要形成酰胺键,经常用的手段是将羧基活化,变成混合酸酐、活泼酯、酰氯或用强的失去剂〔如碳二亚氨形成对称酸酐等方法来形成酰胺键。其中选用DCC、HOBT或HOBT/DCC的对称酸酐法、活化酯法接肽应用最广。裂解及合成肽链的纯化BOC法用TFA+HF裂解和脱侧链保护基,FMOC法直接用TFA,有时根据条件不同,其它碱、光解、氟离子和氢解等脱保护方法也被采用。合成肽链进一步的精制、分离与纯化通常采用高效液相色谱、亲和层析、毛细管电泳等。4．固相合成的特点及存在的主要问题固相合成法对于肽合成的显著的优点：简化并加速了多步骤的合成；因反应在一简单反应器皿中便可进行,可避免因手工操作和物料重复转移而产生的损失；固相载体共价相联的肽链处于适宜的物理状态,可通过快速的抽滤、洗涤未完成中间的纯化,避免了液相肽合成中冗长的重结晶或分柱步骤,可避免中间体分离纯化时大量的损失；使用过量反应物,迫使个别反应完全,以便最终产物得到高产率；增加溶剂化,减少中间的产物聚焦；固相载体上肽链和轻度交联的聚合链紧密相混,彼此产生一种相互的溶剂效应,这对肽自聚集热力学不利而对反应适宜。固相合成的主要存在问题是固相载体上中间体杂肽无法分离,这样造成最终产物的纯度不如液相合成物,必需通过可靠的分离手段纯化。5．固相合成的研究发展前景固相多肽合成已经有40年的历史了,然而到现在,人们还只能合成一些较短的肽链,更谈不上随心所欲地合成蛋白质了,同时合成中的试剂毒性,昂贵费用,副产物等一直都是令人头痛的问题,而在生物体内,核糖体上合成肽链的速度和产率都是惊人的,那么,是否能从生物体合成蛋白质的原理上得到一些启发,应用在固相多肽合成〔树脂上,这是一个令人感兴趣的问题,也许是今后多肽合成的发展。在Boc合成法中,反复地用酸来脱保护,这种处理带来了一些问题：如在肽与树脂的接头处,当每次用50%TFA脱Boc基时,有约1.4%的肽从树脂上脱落,合成的肽越大,这样的丢失越严重；此外,酸催化会引起侧链的一些副反应.Boc合成法尤其不适于合成含有色氨酸等对酸不稳定的肽类.1978年,Chang、Meienlofer和Atherton等人采用Carpino［3］报道的Fmoc<9-芴甲氧羰基>基团作为α-氨基保护基,成功地进行了多肽的Fmoc固相合成.Fmoc法与Boc法的根本区别在于采用了碱可脱除的Fmoc为α-氨基的保护基.侧链的保护采用TFA可脱除的叔丁氧基等,树脂采用90%TFA可切除的对烷氧苄醇型树脂和1%TFA可切除的二烷氧苄醇型树脂,最终的脱保护避免了强酸处理.6.

Fmoc―氨基酸的制备和侧链保护Fmoc基团是在有NaHCO3或Na2CO3存在的二氧六环溶液中,通过以下反应引入到氨基酸中的：理想的Fmoc-氨基酸的侧链保护基应在碱性条件下稳定,在酸性条件下脱除.下面对其做一介绍.

6.1Asp和Glu

Asp和Glu侧链羧基常用t-Bu保护.可用TFA、TMSBr等脱除.但是用t-Bu保护仍有侧链环化形成酰亚胺的副反应发生.近年来,发展了一些新的保护基如环烷醇酯、金刚烷醇酯等可减轻这一副反应,这些保护基可用TMSOTf<三氟甲磺酸三甲硅烷酯>除去.

6.2Ser、Thr和Tyr

ser、Thr的羟基及Tyr的酚羟基通常用t-Bu保护.叔丁基的引入比较麻烦,首先ser制成苄氧羰基酯,再在酸催化下与异丁烯反应.Ser和Thr还可用苄基保护,Ser用苄醇引入苄基、Thr用溴苄引入苄基.

6.3Asn和Gln

Asn和Gln侧链的酰胺键在肽合成中一般不加以保护.但合成大肽时,Asn和Gln的α-羧基活化时可能会发生分子内脱氢反应生成氰基化合物.碱性时Gln的侧链可以环化生成酰胺.而且不保护的Fmoc-Gln和Fmoc-Asn在DCM中溶解度很差.为了避免这些问题,可以用9-咕吨基,2,4,6-三甲氧苄基,4,4′―二甲氧二苯甲基或三苯甲基等保护,这四种基因均可用TFA脱除.

6.4His

His是最容易发生消旋化的氨基酸,必须加以保护.

对咪唑环的非π-N开始用苄基<Bzl>和甲基磺酰基<TOS>保护.但这两种保护基均不太理想.TOS对亲核试剂不稳定,Bzl需要用氢解或Na/NHs除去,并且产生很大程度消旋.Boc基团是一个较理想的保护基,降低了咪唑环的碱性,抑制了消旋,成功地进行了一些合成.但是当反复地用碱处理时,也表现出一定的不稳定性.哌啶羰基在碱中稳定,但是没能很好地抑制消旋,而且脱保护时要用很强的亲核试刘如.

对咪唑环π-N保护,可以完全抑制消旋,π-N可以用苄氧甲基<Bom>和叔丁氧甲基<Bum>保护,<Bum>可以用TFA脱除,Bom更稳定些,需用催化氢解或强酸脱保护,Bum是目前很有发展前途的His侧链保护基,其不足之处在于Fmoc<His>Bum在DCM和DMF中的溶解度较差.

6.5Cys

Cys的－SH具有强亲核性,易被酰化成硫醚,也易被氧化为二硫键,必须加以保护.常用保护基有三类：一类用TFA可脱除,如对甲苄基、对甲氧苄基和三苯甲基等；第二类可用<CF3CO>3T1／TFA脱除,对TFA稳定.如t-Bu、Bom和乙酰胺甲基等.第三类对弱酸稳定,如苄基和叔丁硫基<stBu>等,Cys<StBu>可用巯基试剂和磷试剂还原,Cys<Bzl>可用Na/NH3<1>脱保护.

6.6Arg

Arg的胍基具有强亲核性和碱性,必须加以保护.理想的情况是三个氮都加以保护,实际上保护1或2个胍基氮原子.保护基分四类：<1>硝基<2>烷氧羰基<3>磺酰基<4>三苯甲基.

硝基在制备、酰化裂解中产生很多副反应,应用不广.烷氧羰基应主要有Boc和二金刚烷氧羰基<Adoc>2、Fmoc<Arg>Boc的耦联反效率不高,哌啶理时不处稳定,会发生副反应；Adoc保护了两个非π－N,但有同样的副反应发生.对磺酰基保护,其中TOS应用最广,但它较难脱除.近年来2,3,6-三甲基-4-甲氧苯横酰基<Mtr>较受欢迎,在TFA作用下,30分钟即可脱除,但是它们都不能完全抑制侧链的酰化发生.三苯甲基保护基可用TFA脱除.缺点是反应较慢,侧链仍有酰化反应,且其在DCM、DMF中溶解度不好.

6.7Lys

Lys的ε-NH2必须加以保护.但与α－NH2的保护方式应不同,该保护基要到肽链合成后除去.ε－NH2的保护无消旋问题,可以采用酰基保护基,其它常用的保护基有苄氧碳基和Boc.

6.8Fmoc基团的脱除Fmoc基团的芴环系的吸电子作用使9-H具有酸性,易被较弱碱除去,反应条件很温和.反应过程可表示如下：哌啶进攻9-H,β消除形成二苯芴烯,很容易被二级环胺进攻形成稳定的加成物.Fmoc基团对不同的碱稳定性不同,可根据实际条件选用.6.9耦联反应固相中的接肽反应原理与液相中基本一致.将两个相应的氨基被保护的及羧基被保护的氨基酸放在溶液内,并不形成肽键.要形成酰胺键,经常用的手段是将羧基活化,其方法是将它变成混合酸酐,或者将它变为活泼酯、酰氯,或者用强的失去剂<碳二亚胺>也可形成酰胺键,耦联反应可表示如下：<A：羰基活泼试剂>碳二亚胺是常用的活化试剂,其中Dcc使用范围最广,其缺点是形成了不溶于DCM的DCH,过滤时又难于除尽.其他一些如二异丙基碳二亚胺<DCI>、甲基叔丁基碳二亚胺也用于固相合成中,它们形成的脲溶于DCM中,经洗涤可以除去.其他活化试剂,还有Bop<Bop－C1>、氯甲酸异丙酯、氯甲酸异丁酯、SOC12等.其中Dcc、Bop活化形成对称酸酐、SOC12形成酰氯,其余三种形成不对称酸酐.

6.10对称酸酐法用Dcc形成对称酸酐的方法使用较广.其缺点是有些氨基酸在DCM中不易溶解,生成的Fmoc氨基酸酐溶解度更差.同时还有些副反应,如形成二肽、消旋等.

6.11混合酸酐法最常用试剂是氯甲酸的异丙基酯和异丁基酯.前者得到的酸酐稳定性好.只产生很少消旋,在适当的化学计量及溶剂条件下,耦联反应很快.而且,在此反应中使用的N-甲基吗啉和N-甲基哌啶对Fmoc基团无影响.

6.12酰氯法在Boc法中不常用的酰氯,因为比较激烈,一些保护基如Boc不稳定.但是,Fmoc基团可以耐受酰氯处理,生成的Fmoc氨基酰氯也很稳定.在三甲基乙酸／三胺或苯并三氮唑／二异丙基乙二胺中,反应速度很快,消旋很少.酰氯法在固相合成中应用还不多,但已表明,Fmoc－氨基酰氯适用于合成有立体障碍的肽序列.

6.13活化酯法活化酯法在固相合成中应用最为广泛.采用过的试剂也很多,近来最常用的有HOBt酯、ODhbt酯、OTDO酯等.

HOBt酯反应快,消旋少,用碳二亚胺很容易制得；ODhbt酯很稳定,容易进行分离纯化,与HOBt酯具有类似的反应性和消旋性能,它还有一个优越之处,在酰化时有亮黄色、耦联结束时颜色消失,有利于监测反应；OTDO酯与ODhbt酯类似,消旋化极低,易分离,酰化时伴有颜色从桔红色到黄色的变化等.

6.14原位法将碳二亚胺和α－N保护氨基酸直接加到树脂中进行反应叫做原位法.

用DIC代替Dcc效果更好.其他的活化试剂还有Bop和Bop-C1等.原位法反应快、副反应少、易操作.其中DIC最有效,其次是Bop、Bop-C1等.遗憾的是Bop酰化时生成致癌的六甲基磷酰胺,限制了其应用.6.15裂解及侧链保护基脱除Fmoc法裂解和脱侧链保护基时可采用弱酸.TFA为应用最广泛的弱酸试剂,它可以脱除t-Bu、Boc、Adoc、Mtr等；条件温和、副反应较少.不足之处：Arg侧链的Mtr很难脱除,TFA用量较大；无法除掉Cys的t-Bu等基因.也有采用强酸脱保护的方法：如用HF来脱除一些对弱酸稳定的保护基,如Asp、Glu、Ser、Thr的Bzl<苄基>保护基等,但是当脱除Asp的吸电子保护基时,会引起环化副反应.而TMSBr和TMSOTf在有苯甲硫醚存在时,脱保护速度很快.此外,根据条件不同,碱、光解、氟离子和氢解等脱保护方法也有应用.Fmoc基团用于固相合成多肽已经有了十多年的历史,在合成一些含有在酸性条件下不稳定的氨基的残基的肽时,具有特别优越之处.将Fmoc法和Boc法互相补充,定会在合成更多、更大的生物分子中发挥。二、常用保护氨基酸数据缩写名称分子量残基缩写名称分子量残基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常用试剂种类及数据名称分子量名称分子量密度〔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常见保护基团结构及数据缩写分子量缩写分子量缩写分子量Fmoc-222tBu-56Acm-57Pbf-253OtBu-72Mz-165.1Trt-242.3Ac-43Boc-101.1Z-134.1三、常用氨基酸结构及性质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、常见保护基团结构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、多肽常识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、常用试剂〔一缩合剂1．Name:

EDC.HCl

Category:

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

Cl-HOBt

Category:

PeptideCouplingReagent3．Name:N,N'-Diisopropylcarbodiimide<DIC>Category:PeptideCouplingReagent4．二环己基碳化二亚胺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〔二链接剂1．Name:SieberLinker

Category:LinkersforSolidPhaseSynthesis2．Name:

RinkAmideLinker

Category:LinkersforSolidPhaseSynthesis〔三树脂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