核糖体按照信使RNA指定的氨基酸序列来构建蛋白质。在这里，研究人员设计、合成了一个小分子机器，该机器沿着的分子链按照序列特异性方式进行多肽合成。该小分子机器的化学结构是基于一个轮烷，分子环拧到一个分子桥。环带有硫醇盐基团，可以反复按照特定顺序删除氨基酸，并将它们运送到的多肽 生长延长位点。实验结果表明大约有10 18 的小分子机器在并行进行合成；这一过程会产生毫克量级多肽。串联质谱实验确认只产生了单一多肽序列。 英文全文见： http://www.sciencemag.org/content/339/6116/189.full Sequence-Specific Peptide Synthesis by an Artificial Small-Molecule Machine Bartosz Lewandowski 1 , Guillaume De Bo 1 , John W. Ward 1 , Marcus Papmeyer 1 , Sonja Kuschel 1 , María J. Aldegunde 2 , Philipp M. E. Gramlich 2 , Dominik Heckmann 2 , Stephen M. Goldup 2 , Daniel M. D’Souza 2 , Antony E. Fernandes 2 , David A. Leigh 1 , 2 , * The ribosome builds proteins by joining together amino acids in an order determined by messenger RNA. Here, we report on the design, synthesis, and operation of an artificial small-molecule machine that travels along a molecular strand, picking up amino acids that block its path, to synthesize a peptide in a sequence-specific manner. The chemical structure is based on a rotaxane, a molecular ring threaded onto a molecular axle. The ring carries a thiolate group that iteratively removes amino acids in order from the strand and transfers them to a peptide-elongation site through native chemical ligation. The synthesis is demonstrated with ~10 18 molecular machines acting in parallel; this process generates milligram quantities of a peptide with a single sequence confirmed by tandem mass spectrometry.
百度上down到好文，正好给自己用的，特特抄来 ----------------------------------------------------------------------------------------- 多 肽 合 成 基础知识汇编 编制 : 合成部 ----------------------------------------------------------------------------------------- 一 、多 肽 合 成 概 论 1 ．多肽化学合成概述： 1963 年， R.B.Merrifield ［ 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， chang Meienlofer 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. HPLC 分析和纯化 分析 HPLC 使用柱子和泵系统，可以经受传递高压，这样可以用极细的微粒（ 3-10μ m ）做填料。由此多肽要在几分钟内高度被分析。 HPLC 分两类：离子交换和反相。 离子交换 HPLC 依靠多肽和固相间的直接电荷相互作用。柱子在一定 PH 范围带有特定电荷衍变成一种离子体，而多肽或多肽混合物，由其氨基酸组成表现出相反电荷。 分离是一种电荷相互作用，通过可变 PH ， 离子强度， 或两者洗脱出多肽，通常， 先用低离子强度的溶液，以后逐渐加强或一步一步加强，直到多肽火柱中洗脱出。离子交换分离的一个例子使用强阳离子交换柱。如 sulfoethylaspartimide 通过酸性 PH 中带正电来分离。 反相 HPLC 条件与正常层析正相反。多肽通过疏水作用连到柱上，用降低离子强度洗脱， 如增加洗脱剂的疏水性。通常柱子由共价吸附到硅上的碳氢烷链构成，这种链长度为 G4-G8 碳原子。 由于洗脱是一种疏水作用。大的疏水肽用短链柱洗脱好。 然而，总体实践中， 这两类柱互变无多少显著差别，别类载体由碳水化合物构成， 比如苯基。 典型的操作常由两绶冲剂组成， 0.1%TFA-H2o 和 80% acetonitrile 0.1%TFA--H2o 稀 acetonitrile 。用线型梯变以每分钟 0.5% 到 1.0% 改变的速度混合。常见分析和纯化用柱为 4.6×250mm(3-10μ m) 和 22×250mm(10μ m). 如果用径向填柱，那么大小是 8×100 （ 3-10μ m ）和 25×250mm(10μ m) 大量各种缓冲剂含许多不同试剂，比如 heptafluorobutyric 酸， 0.1% 磷酸， 稀 He formic 酸 (5-6%, pH2-4), 10-100mM NH4HCO3, 醋酸钠 / 氨， TFA/TEA ，磷酸钠或钾，异戊酚。这样许多不同组合可形成缓冲剂，但要注意一点：硅反相柱料不能长时间暴露于高 pH ，甚至微碱 pH ， 因为这样会破坏柱子。 7. 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 . 7.1 Asp 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 . 7.2 Ser 、 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 . 7.3 Asn 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 . 7.4 His 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 . 7.5 Cys 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 . 7.6 Arg 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 . 7.7 Lys 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. 7.8 Fmoc 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 . 7.9 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 . 7.10 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 . 7.11 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 . 7.12 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 . 7.13 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 . 7.14 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 . 7.15 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 缩写 名称 分子量 A-Ala Fmoc- Ala 329.3 M-Met Fmoc-Met 371.5 R-Arg Fmoc-Arg(Pbf) 648.77 F-Phe Fmoc- Phe 387.4 N-Asn Fmoc- Asn (Trt) 596.7 (D)F-Phe Fmoc-D-Phe 387.4 D-Asp Fmoc-Asp(OtBu) 411.46 P-Pro Fmoc- Pro 337.4 C-Cys Fmoc-Cys(Trt) 585.7 S-Ser Fmoc- Ser (tBu) 383.4 Q-Gln Fmoc- Gln (Trt) 610.7 T-Thr Fmoc-Thr(tBu) 397.5 E-Glu Fmoc- Glu (OtBu) 425.48 W-Trp Fmoc- Trp (Boc) 526.59 G-Gly Fmoc- Gly 297.3 (D)W-Trp Fmoc-D-Trp(Boc) 526.59 H-His Fmoc- His (Trt) 619.7 D-Tyr Fmoc-Tyr(tBu) 459.5 I-Ile Fmoc- Ile 353.4 V-Val Fmoc-Val 339.4 L-Leu Fmoc- Leu 353.4 pGlu PyroGlu 129.1 K-Lys Fmoc-Lys (Boc) 468.5 常用试剂种类及数据 名称 分子量 名称 分子量 密度（ g/ml ） HOBt 135.1 DIPCDI(DIC) 126.3 0.806 TBTU 321.1 DIPEA(DIA) 129.4 0.76 HATU 380.3 Ac2O 102.1 1.08 DMAP 122.1 Pyridine 79.1 0.983 HOAT 136.1 TFA 114.2 1.44 TMP 121.18 0.92 EDT 94.24 1.123 TIS 158.4 NMM 101.15 0.9168 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- 222 tBu- 56 Acm- 57 Pbf- 253 OtBu- 72 Mz- 165.1 Trt- 242.3 Ac- 43 Boc- 101.1 Z- 134.1 三、常用氨基酸结构及性质 Name Symbol MW MW (-H2O) Structure Three Letter Code One Letter Code Alanine Ala A 89.09 71.08 Arginine Arg R 174.20 156.19 Asparagine Asn N 132.12 114.10 Aspartic Acid Asp D 133.10 115.09 Cysteine Cys C 121.15 103.15 Glutamic Acid Glu E 147.13 129.12 Glutamine Gln Q 146.15 128.13 Glycine Gly G 75.07 57.05 Histidine His H 155.16 137.14 Isoleucine Ile I 131.17 113.16 Leucine Leu L 131.17 113.16 Lysine Lys K 146.19 128.17 Methionine Met M 149.21 131.20 Phenylalanine Phe F 165.19 147.18 Proline Pro P 115.13 97.12 Serine Ser S 105.09 87.08 Threonine Thr T 119.12 101.11 Tryptophan Trp W 204.23 186.21 Tyrosine Tyr Y 181.19 163.18 Valine Val V 117.15 99.13 四、常见保护基团结构 Name Structure Symbol Formula Residue Wt Acetamidomethyl Acm C3H6NO 72.1 Acetyl Ac C2H3O 43.0 Allyloxycarbonyl Aloc C4H5O2 85.1 6-Amidohexanoate LC C4H7NO 85.1 7-Amido-4-methylcoumaryl AMC C10H8NO2 174.2 7-Amido-4-trifluoromethyl-coumaryl AFC C10H5F3NO2 228.2 5- -naphthalene-1-sulfonic acid EDANS C12H13N2O3S 265.3 Benzoyl Bz C7H5O 105.1 Benzyl Bzl C7H7 91.1 Benzyloxycarbonyl Z (Cbz) C8H7O2 135.1 Benzyloxymethyl Bom C8H9O 121.2 (+)-Biotinyl Biotin C10H15N2O2S 227.3 2-Bromobenzyloxycarbonyl 2-Br-Z C8H6BrO2 214.0 tert-Butyl tBu C4H9 57.1 tert-Butyloxycarbonyl Boc C5H9O2 101.1 tert-Butylthio StBu C4H9S 89.2 2-Chlorobenzyloxycarbonyl 2-Cl-Z C8H6ClO2 169.6 Cyclohexyl cHex C6H11 83.1 2,6-Dichlorobenzyl 2,6-di-Cl-Bzl C7H5Cl2 160.0 4-(4-Dimethylaminophenyl-azo)benzoyl DABCYL C15H14N3O 252.3 2,4-Dinitrophenyl Dnp C6H3N2O4 167.1 9-Fluorenylmethyl Fm C14H11 179.1 9-Fluorenylmethyloxy-carbonyl Fmoc C15H11O2 223.3 Fluorescein Isothiocyanate FITC C21H12NO5S 390.4 Lissamine Rhodamine LR C31H38N2O6S2 598.8 Mesitylene-2-sulfonyl Mts C9H11O2S 183.3 4-Methoxybenzyl Mob C8H9O 121.2 (7-Methoxycoumarin-4-yl)acetyl Mca C12H9O4 217.2 4-Methoxy-2,3,6-trimethyl-benzenesulfonyl Mtr C10H13O3S 213.3 4-Methoxytrityl Mmt C20H17O 273.4 4-Methylbenzyl MBzl C8H9 105.2 4-Methyltrityl Mtt C20H17 257.4 4-Morpholinecarbonyl Mu C5H8NO2 114.1 p-Nitroanilide pNA C6H5N2O2 137.1 2,2,4,6,7-Pentamethyldihydro-benzofuran-5-sulfonyl Pbf C13H17O3S 253.3 2,2,5,7,8-Pentamethyl-chroman-6-sulfonyl Pmc C14H19O3S 267.4 Rhodamine 110 R110 C20H13N2O3 329.3 Succinyl Suc C4H5O3 101.1 4-Toluenesulfonyl Tos C7H7O2S 155.2 Trityl Trt C17H15 243.3 Xanthyl Xan C13H9O 181.2 五、多肽常识 Reconstitution and Storage of Peptides Peptides are usually supplied as a fluffy, freeze-dried material in serum vials. Store peptides in a freezer after they have been received. In order to reconstitute the peptide, distilled water or a buffer solution should be utilized. Some peptides have low solubility in water and must be dissolved in other solvents such as 10% acetic acid for a positively charged peptide or 10% ammonium bicarbonate solution for a negatively charged peptide. Other solvents that can be used for dissolving peptides are acetonitrile, DMSO, DMF, or isopropanol. Use the minimal amount of these non-aqueous solvents and add water or buffer to make up the desired volume. After peptides are reconstituted, they should be used as soon as possible to avoid degradation in solution. Unused peptide should be aliquoted into single-use portions, relyophilized if possible, and stored at -20°C. Repeated thawing and refreezing should be avoided. Methods to Dissolve Peptides The best way to dissolve a peptide is to use water. For peptides that are not soluble in water, use the following procedure: 1. For acidic peptides, use a small amount of base such as 10% ammonium bicarbonate to dissolve the peptide, dilute with water to the desired concentration. Do not use base for cysteine-containing peptides. 2. For basic peptides, use a small amount of 30% acetic acid, dilute with water to the desired concentration. 3. For a very hydrophobic peptide, try dissolving the peptide in a very small amount of DMSO, dilute with water to the desired concentration. 4. For peptides that tend to aggregate (usually peptides containing cysteines), add 6 M urea, 6 M urea with 20% acetic acid, or 6 M guanidine•HCl to the peptide, then proceed with the necessary dilutions. Preparation of HBTU/HOBt Solution for the Peptide Synthesizer 1. Preparation of 0.5 M HOBt in DMF: o Weigh 13.5 g anhydrous HOBt (0.1 mol, MW 135.1) into a 250 mL graduated cylinder. o Add DMF until the 200 mL level is reached. 2. Preparation of 0.45 M HBTU/HOBt solution: o Add the solution prepared in step 1 to 37.9 g HBTU (0.1 mol, MW 379.3) contained in a beaker or an Erlenmayer flask. 3. Stir for about 15 min with a magnetic stirring bar until HBTU is dissolved. 4. Filter the solution through a fine pore size sintered glass funnel. 5. Pour the filtered solution into an appropriate bottle for attachment to a peptide synthesizer. * This solution is stable at room temperature for at least six weeks. Biotinylation of Amino Group 1. Wash 0.1 mmol resin with DMF. 2. Dissolve 0.244 g (+)-biotin (1 mmol, MW 244.3) in 5 mL DMF-DMSO (1:1) solution. A little warming is necessary. 3. Add 2.1 mL 0.45 M HBTU/HOBt solution and 0.3 mL DIEA to the solution prepared in step 2. 4. Add the activated biotin solution to the resin and let stir overnight. 5. Check resin to make sure coupling is complete as evidenced by negative ninhydrin test (colorless). 6. Wash resin with DMF-DMSO (1:1) (2x) to remove excess (+)-biotin. 7. Wash resin with DMF (2x) and DCM (2x). 8. Let the resin dry before proceeding to cleavage. Procedure for Loading Fmoc-Amino Acid to 2-Chlorotrityl Chloride Resin 1. Weigh 10 g 2-chlorotrityl chloride resin (15 mmol) in a reaction vessel, wash with DMF (2x), swell the resin in 50 mL DMF for 10 min, drain vessel. 2. Weigh 10 mmol Fmoc-amino acid in a test tube, dissolve Fmoc-amino acid in 40 mL DMF, transfer the solution into the reaction vessel above, add 8.7 mL DIEA (50 mmol), swirl mixture for 30 min at room temperature. 3. Add 5 mL methanol into the reaction vessel and swirl for 5 min. 4. Drain and wash with DMF (5x). 5. Check substitution. 6. Add 50 mL 20% piperidine to remove the Fmoc group. Swirl mixture for 30 min. 7. Wash with DMF (5x), DCM (2x), put resin on tissue paper over a foam pad and let dry at room temperature overnight under the hood. Cover the resin with another piece of tissue paper, press lightly to break aggregates. 8. Weigh loaded resin. 9. Pack in appropriate container. Procedure for Checking Substitution of Fmoc-Amino Acid Loaded Resins 1. Weigh duplicate samples of 5 to 10 mg loaded resin in an eppendorf tube, add 1.00 mL 20% piperidine/DMF, shake for 20 min, centrifuge down the resin. 2. Transfer 100 μL of the above solution into a tube containing 10 mL DMF, mix well. 3. Pipette 2 mL DMF into each of the two cells (reference cell and sample cell), set spectrophotometer to zero. Empty the sample cell, transfer 2 mL of the solution from step 2 into the sample cell, check absorbance. 4. Subs = 101(A)/7.8(w) A = absorbance w = mg of resin 5. Check absorbance three times at 301 nm, calculate average substitution. Manual Fmoc Synthesis (0.25 mmol) 1. Wash resin with DMF (4x) and then drain completely. 2. Add approximately 10 mL 20% piperidine/DMF to resin. Shake for one min and drain. 3. Add another 10 mL 20% piperidine/DMF. Shake for 30 min. 4. Drain reaction vessel and wash resin with DMF (4x). Make sure there is no piperidine remaining. Check beads using ninhydrin test, beads should be blue. 5. Coupling Step - Prepare the following solution: 1 mmol Fmoc-amino acid 2.1 mL 0.45 M HBTU/HOBT (1mmol) 348 μL DIEA (2 mmol) Add above solution to the resin and shake for a minimum of 30 min. This coupling step can be longer if desired. 6. Drain reaction vessel and wash resin with DMF (4x). 7. Perform Ninhydrin test: o If negative (colorless), proceed to step 2 and continue synthesis. o If positive (blue), return to step 5 and re-couple the same Fmoc-amino acid. Increase the coupling time if necessary. Synthesis of Phosphotyrosine-Containing Peptides Using Fmoc-Phosphotyrosine Reagent: N- a -Fmoc-O-phosphotyrosine 1. For 0.1 mmol or 0.25 mmol synthesis, use 0.483 g Fmoc-Tyr(PO3H2)-OH (1 mmol, MW 483.4) . For ABI synthesizers, pack Fmoc-Tyr(PO3H2)-OH in a cartridge. 2. The cycle program for coupling Fmoc-Tyr (PO3H2)-OH is the same as for other Fmoc-amino acids except for the coupling time (see step 3). (Note: ABI synthesizers use HBTU/HOBT as the activating reagent.) 3. The coupling time for Fmoc-Tyr(PO3H2)-OH needs to be increased. For ABI model 430A peptide synthesizer, insert several steps (i.e., vortex on, wait 990 sec, vortex off, to increase the coupling time). For ABI model 431A peptide synthesizer, add additional "I"s. Overnight coupling may be necessary for some sequences. 4. After the coupling step for Fmoc-Tyr(PO3H2)-OH, perform ninhydrin test to ensure complete coupling. Negative (colorless) ninhydrin test indicates complete coupling, while a positive (blue) ninhydrin test indicates incomplete coupling. 5. Increase the coupling time of the amino acid residues after the phosphotyrosine or perform double coupling. (Note: The coupling of amino acids after the phosphotyrosine can be difficult.) 6. There is a limit on the number of amino acid residues that can be coupled after the hosphotyrosine. Since the phospho group is unprotected, side reactions are likely to ccur. (Note: Peptides have been successfully coupled with sequences containing up o ten additional amino acids following the phosphotyrosine residue.) Simultaneous Synthesis of Peptides Which Differ in the C-Termini Using 2-Chlorotrityl Resin and Wang Resin* Peptides which differ in the C-termini can be simultaneously synthesized in one reaction vessel by employing resins that possess different cleavage properties. The resins used were the weak acid labile 2-chlorotrityl resins and the TFA labile Wang resins. The success of this approach was shown by the co-synthesis of ACTH (4-10) with ACTH (4-11) and Neuropeptide Y, a C-terminal amide peptide with its corresponding C-terminal free acid analog. *Hong A., Le T., and Phan T. Techniques in Protein Chemistry VI, 531-562 (1995). Cleavage Protocol to Produce Fully Protected Peptide Starting Resin: Chlorotrityl resins Reagents for 1 g Peptide-Resin: 1 mL acetic acid (AcOH) 2 mL trifluoroethanol (TFE) 7 mL dichloromethane (DCM) 1. Prepare above mixture. 2. Add peptide-resin to the mixture and let it stir at room temperature for 1 h. 3. Filter and wash resin with 10 mL TFE:DCM (2:8) (2x) to ensure that all of the product is recovered. 4. Evaporate the solvent until there is less than 5 mL of liquid. 5. Add ether to a test tube containing about 100 m L of the above solution. Check solubility of the fully protected peptide in ether. If the product precipitates, proceed to step 6. If no precipitate is observed, proceed to step 7. 6. Add cold ether to the residual liquid in step 4 to precipitate the fully protected peptide. Filter through a fine sintered funnel to obtain the product. 7. Some fully protected peptides are soluble in ether. In this case, add water to precipitate them out. Filter through a fine sintered funnel to obtain the product. Procedure for FITC Labeling of Peptides Reagents: FITC Fmoc- e -Ahx-OH 1. Couple Fmoc- e -Ahx-OH to the amino terminal of the peptide-resin using standard coupling conditions. 2. "De-Fmoc" with piperidine using the standard 20% piperidine procedure. 3. Wash resin with DMF (3-4x). 4. Swell resin with DCM and drain. 5. Prepare solution of 1.1 equivalent of FITC in pyridine/DMF/DCM (12:7:5). Use just enough solution to form a slurry with the resin. Do not use too much solution since the rate of the reaction is proportionate to the concentration of the solution. 6. Add the solution prepared in step 2 to the resin. 7. Let mix overnight. 8. Check the completion of the reaction using ninhydrin test. 9. If the coupling of FITC to the amino group is not complete, ninhydrin test will give a blue color. Repeat the coupling with FITC (steps 5-7) if necessary. 10. Wash resin with DMF (2x), isopropanol (2x), and DCM (2x). Procedure for Removing Mtt group from Fmoc-Lys(Mtt) on Solid Phase Reagent: Fmoc-Lys(Mtt)-OH 1. Swell resin in DCM. 2. Wash resin with 3% TFA/DCM (2x) (since the resin is swollen in DCM, this step of washing the resin quickly with 3% TFA/DCM ensures that the actual concentration of TFA is 3%). 3. Shake the resin in 3% TFA for 10 min. 4. Repeat step 3. 5. Wash resin with DCM (3x), DMF (3x), isopropanol (3x), and DCM (3x). 6. Let the resin dry in air. Procedure for Fluorescein Labeling of Peptides Reagent: 5-carboxyfluorescein (5-FAM) [0.1 g, AnaSpec Catalog # 24623; 0.5 g, AnaSpec Catalog # 24624) Use standard coupling method to couple 5-carboxyfluorescein to the amino group of the peptide. For cost saving purposes, use 2x excess compared to the mmol of resin, instead of the standard 4x excess used for Fmoc-amino acids. For 0.1 mmol synthesis, use 75 mg 5-carboxyfluorescein, 76 mg HBTU, and 70 mL DIEA. 六、常用试剂 及非天然氨基酸 （一）缩合剂 1 ． Name: EDC.HCl Category: Peptide Coupling Reagent 2 ． 6- 氯 -1- 羟基 - 苯并 - 三氮唑 Name: Cl-HOBt Category: Peptide Coupling Reagent 3 ． Name: N,N'-Diisopropylcarbodiimide (DIC) Category: Peptide Coupling Reagent 4 ． 二环己基碳化二亚胺 Name: Dicyclohexylcarbodiimide (DCC) Category: Peptide Coupling Reagents 5 ． Name: BOP Reagent Category: Peptide Coupling Reagent 6 ． 六氟磷酸苯并三唑 -1- 基 - 氧基三吡咯烷基磷 Name: PyBOP Category: Peptide Coupling Reagent 7 ． N,N'- 羰基二咪唑 Name: N,N'-Carbonyldiimidazole (CDI) Category: Peptide Coupling Reagent 8 ． Name: DEPBT Category: Peptide Coupling Reagent 9 ． Name: 4,5-Dicyanoimidazole Category: Peptide Coupling Reagent 10 ． Name: HBTU Category: Peptide Coupling Reagent 11 ． Name: HOBt (anhydrous) Category: Peptide Coupling Reagent 12 ． Name: HOOBt Category: Peptide Coupling Reagent 13 ． Name: TBTU Category: Peptide Coupling Reagent （二）链接剂 1 ． Name: Sieber Linker Category: Linkers for Solid Phase Synthesis 2 ． Name: Weinreb Linker Category: Linkers for Solid Phase Synthesis 3 ． Name: DHP Linker Category: Linkers for Solid Phase Synthesis 4 ． Name: HMP Linker Category: Linkers for Solid Phase Synthesis 5 ． Name: Rink Amide Linker Category: Linkers for Solid Phase Synthesis （三） 树脂 Resin 1 ． Name: 2-Chlorotrityl Chloride Resin Category: Resins 2 ． Name: Aminomethyl polystyrene Resin Category: Resins 3 ． （用于合成肽醇） Name: DHP HM Resin Category: Resins 4 ． Name: HMPA-AM Resin Category: Resins 5 ． （用于合成肽酰胺） Name: Knorr Resin Category: Resins 6 ． （用于合成肽酰胺） Name: Knorr-2-Chlorotrityl Resin Category: Resins 7 ． Name: MBHA Resin Category: Resins 8 ． Name: Merrifield Resin Category: Resins 9 ． Name: Oxime Resin Category: Resins 10 ． Name: PAM Resin Category: Resins 11 ． Name: Rink amide-AM Resin Category: Resins 12 ． Name: Rink amide-MBHA Resin Category: Resins 13 ． Name: Sieber Resin Category: Resins 14 ． Name: Wang Resin Category: Resins 15 ． （用于合成肽醛） Name: Weinreb AM Resin Category: Resins （四）保护剂 1 ． 9- 芴甲醇 Name: 9-Fluorenylmethanol Category: N-Protecting Reagents 2 ． Boc- 酸酐 Name: Boc Anhydride Category: N-Protecting Reagents 3 ． 4,4'- 二甲氧基三苯基氯甲烷 Name: DMT-Cl Category: N-Protecting Reagents 4 ． Name: N,N'-Disuccinimidyl carbonate(DSC) Category: N-Protecting Reagents 5 ． 芴甲氧羰酰氯 Name: Fmoc-Cl Category: N-Protecting Reagents 6 ． 芴甲氧羰酰琥珀酰亚胺 Name: Fmoc-OSu Category: N-Protecting Reagents 7 ． 叔丁基二甲基氯硅烷 Name: tert-Butyldimethylsilyl Chloride Category: N-Protecting Reagents 8 ． Name: Trityl Chloride Category: N-Protecting Reagents 9 ． 苯甲氧羰酰氯 (Cbz-Cl) Name: Z-Cl Category: N-Protecting Reagents 10 ． 苯甲氧羰酰琥珀酰亚胺 (Cbz-OSu) Name: Z-OSu Category: N-Protecting Reagents 11 ． Name: Z(2Cl)-OSu Category: N-Protecting Reagents 12 ． Name: Z(2-Br)-OSu Category: N-Protecting Reagents （五） 非天然氨基酸 1 ． Name: Fmoc-Dap(Boc)-OH Category: Unusual Amino Acids 2 ． Name: 3-Chloro-L-Phenylalanine Category: Unusual Amino Acids 3 ． Name: 3-Cl-Tyr-OH Category: Unusual Amino Acids 4 ． Name: 4-Amino-L-Phenylalanine Category: Unusual Amino Acids 5 ． Name: 4-Chloro-L-Phenylalanine.HCl Category: Unusual Amino Acids 16 ． Name: 3,4-Dichloro-phenylalanine Category: Unusual Amino Acids 6. Name: 4-Fluoro-L-Phenylalanine Category: Unusual Amino Acids 7. Name: Boc-4-Iodo-L-phenylalanine Category: Unusual Amino Acids 8. Name: 4-Methoxyphenylalanine Category: Unusual Amino Acids 9. Name: 3-NH2-Tyr-OH Category: Unusual Amino Acids 10. Name: 4-Nitro-L-Phenylalanine Hydrate Category: Unusual Amino Acids 11. Name: 3-(3-Pyridyl)-L-alanine.HCl Category: Unusual Amino Acids 12. Name: DL-m-Tyrosine category: Unusual Amino Acids 13. Name: Fmoc-Tyr(3-NO2)-OH Category: Unusual Amino Acids 14. Name: Boc-Homo-L-Tyrosine Category: Unusual Amino Acids 15. Name: Sarcosine tert-butyl ester.HCl Category: Unusual Amino Acids （六）标记试剂 1 ． 7- 氨基 -4- 甲基香豆素 Name: 7-Amino-4-methylcoumarin ( AMC) Category: Labeling Reagents （七）修饰及标记 1. 标记 Aminocoumarin Bodipy Lissamine Rhodaliiine NBD Fluorescein Tetramethylrhodamine 2. 修饰 Acetylation Formylation Amidation(C—terminal) Nitronation Fatty acid Phosphorylation-Serine Phosphorylation-Threonine Phosphorylation-Tyrosine Benzyloxycarbonylation Biotin Dansvlation Succinylation Dinitrobenzoylation Sulfonation 3. 蛋白载体联接 KLH(KeyhOlelimpetlleiiiocyanin) BSA(Bovine serum albUnin) 4. 复合抗原多 (MAP) Asymmetric 4 branches Asymmetric 8 branches Symmetric 4 branches Symmetric 8 branches 七、 Overview of Peptide Synthesis Introduction Proteins are present in every living cell and possess a variety of biochemical activities. They appear as enzymes, hormones, antibiotics, and receptors. They compose a major portion of muscle, hair, and skin. Consequently, scientists have been very interested in synthesizing them in the laboratory. This interest has developed into a major synthetic field known as Peptide Synthesis. The major objectives in this field are four-fold: 1. To verify the structure of naturally occurring peptides as determined by degradation techniques. 2. To study the relationship between structure and activity of biologically active protein and peptides and establish their molecular mechanisms. 3. To synthesize peptides that are of medical importance such as hormones and vaccines. 4. To develop new peptide-based immunogens. Solid Phase Peptide Synthesis (SPPS) The fundamental premise of this technique involves the incorporation of N- a -amino acids into a peptide of any desired sequence with one end of the sequence remaining attached to a solid support matrix. While the peptide is being synthesized usually by stepwise methods, all soluble reagents can be removed from the peptide-solid support matrix by filtration and washed away at the end of each coupling step. After the desired sequence of amino acids has been obtained, the peptide can be removed from the polymeric support. The general scheme for solid phase peptide synthesis is outlined in Figure 1. The solid support is a synthetic polymer that bears reactive groups such as -OH. These groups are made so that they can react easily with the carboxyl group of an N- a -protected amino acid, thereby covalently binding it to the polymer. The amino protecting group (X) can then be removed and a second N- a -protected amino acid can be coupled to the attached amino acid. These steps are repeated until the desired sequence is obtained. At the end of the synthesis, a different reagent is applied to cleave the bond between the C-terminal amino acid and the polymer support; the peptide then goes into solution and can be obtained from the solution. Fmoc Strategy in SPPS The crucial link in any polypeptide chain is the amide bond, which is formed by the condensation of an amine group of one amino acid and a carboxyl group of another. Generally, an amino acid consists of a central carbon atom (called the a -carbon) that is attached to four other groups: a hydrogen, an amino group, a carboxyl group, and a side chain group. The side chain group, designated R, defines the different structures of amino acids. Certain side chains contain functional groups that can interfere with the formation of the amide bond. Therefore, it is important to mask the functional groups of the amino acid side chain. The general scheme which outlines the strategy of Fmoc synthesis is shown in Figure 2. Initially, the first Fmoc amino acid is attached to an insoluble support resin via an acid labile linker. Deprotection of Fmoc, is accomplished by treatment of the amino acid with a base, usually piperidine. The second Fmoc amino acid is coupled utilizing a pre-activated species or in situ activation. After the desired peptide is synthesized, the resin bound peptide is deprotected and detached from the solid support via TFA cleavage. Fmoc Cleavage The removal of peptides in solid phase peptide synthesis is primarily done by acidolysis. The Fmoc chemistry employs the use of weak acids such as TFA or TMSBr. Various scavengers are included to protect the peptide from carbocations generated during cleavage which can lead to side reactions. These additives usually include thiol compounds, phenol, and water. The following protecting groups are compatible with TFA and TMSBr cleavage: Arg(Boc)2 Cys(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) Depending on the type of protecting groups present, certain combinations of scavengers must be used. For instance, when either Boc and t-Butyl groups are present, their carbocation counterparts (t-butyl cations and t-butyltrifluoroacetate) can react with Trp, Tyr, and Met to form their t-butyl derivatives. While EDT is a very efficient scavenger for t-butyl trifluoroacetate, it does not protect Trp from t-butylation. Therefore, water must be added in order to suppress alkylation. The indole ring of Trp and the hydroxyl group of Tyr are especially susceptible to the reactivity of the cleaved Pmc group. Again, water has been shown to be effective in suppressing this reaction. Similar occurrences can happen with the Trt and Mtr groups. Therefore, scavengers in the appropriate combination will greatly reduce the amount of side reactions. Boc Strategy in SPPS The general scheme which outlines the strategy of Boc synthesis is shown in Figure 3. Initially, the first Boc amino acid is attached to an insoluble support resin via a HF cleavable linker. Deprotection of Boc, is accomplished by treatment of the amino acid with TFA. The second Boc amino acid is coupled utilizing a pre-activated species or in situ activation. After the desired peptide is synthesized, the resin bound peptide is deprotected and detached from the solid support via HF cleavage. Boc Cleavage The Boc chemistry employs the use of strong acids such as HF, TFMSOTf, or TMSOTf. Various additives, usually thiol compounds are added to protect the peptide from the carbocations generated during cleavage. The following protecting groups are compatible with HF cleavage: Arg(Mts) Cys(4-MeOBzl) His(Z) Arg(Tos) Glu(OBzl) Lys (Cl-Z) Asp(OBzl) Glu(OcHex) Ser(Bzl) Asp(OcHex) His(Bom) Thr(Bzl) Cys(Acm) His(Dnp) Trp(CHO) Cys(4-MeBzl) His(Tos) Tyr(Br-Z) Asp(OtBu) His(Trt) The following protecting groups are compatible with TFMSOTf cleavage: Arg(Mts) His(Bom) Met(O) Asp(OBzl) His(Dnp)) Ser(Bzl) Cys(Acm) His(Tos) Thr(Bzl) Cys(4-MeBzl) His(Z) Trp(CHO) Glu(OBzl) Lys (Cl-Z) Tyr(Br-Z) The following protecting groups are compatible with TMSOTf cleavage: Arg(Mts) Glu(OcHex) Trp(CHO) Arg(Mbs) His(Bom) Trp(Mts) Asp(OBzl) Lys (Cl-Z) Tyr(Br-Z) Asp(OcHex) Met(O) Tyr(Bzl) Cys(Acm) Ser(Bzl) Tyr(Cl-Bzl) His(Bom) Thr(Bzl) General Coupling Methods in SPPS Coupling reactions in SPPS require the acylation reactions to be highly efficient to yield high-purity peptides. Coupling Methods in Fmoc SPPS The most widely used coupling method in Fmoc SPPS is the activated ester method either pre-formed (pre-activated species) or in situ (without pre-activation). Initially, the p-nitrophenyl and N-hydroxysuccinimide (ONSu) activated esters were the predominantly used forms (1-2). However, even in the presence of HOBt, the coupling reactions tended to be slow. In addition, ONSu esters of Fmoc amino acids were prone to the formation of the side product succinimido-carbonyl- b -alanine-N-hydroxysuccinimide ester (3-4). The most commonly used activated esters presently are the pentafluorophenyl (OPfp) ester and the 3-hydroxy-2,3-dihydro-4-oxo-benzo-triazone (ODhbt) ester (5-7). In the presence of HOBt, the rate of reaction is very rapid and the reaction is efficient with minimal side product formation. On the other hand, many coupling reactions can be done in situ using activating reagents such as DCC, HBTU, TBTU, BOP, or BOP-Cl. The direct addition of carbodiimide is considered to be the best choice (8-13). HBTU and TBTU would rank second, followed by BOP and finally BOP-Cl. With regards to ester coupling, the following order was found: BOP/HOBt carbodiimide/HOBt ~ carbodiimide/ODhbT DCC/OPfp (14-15). More recently, 1-hydroxy-7-azabenzotriazole (HOAt) and its corresponding uronium salt analog O-(7-azabenzotrizol-1-yl)-1,1,3,3, tetra-methyluronium hexafluorophosphate (HATU) have been developed and found to have a greater catalytic activity than their HOBt and HBTU counterparts. The use of HOAt and HATU enhances coupling yields, shortens coupling times, and reduces racemization. Consequently, these reagents are suitable for the coupling of sterically hindered amino acids, thereby ensuring greater success in the synthesis of difficult peptides (16-17). Coupling Methods in Boc SPPS The carbodiimides, primarily DCC, were the coupling reagents of choice for many years (18). The major drawbacks encountered were the precipitation of dicyclohexylurea during the activation and acylation processes and the numerous side reactions associated with its usage. Several carbodiimides which produced soluble ureas were developed, such as diisopropylcarbodiimide (DIC), t-butyl methyl- and t-butylethyl-carbodiimides (19-22), but these did not resolve the problem of side reactions. Consequently, new types of activating agents were developed. The first of these was BOP (23), PyBroP (24-25) PyBOP (26), HBTU (27), TBTU (28), and HATU (29). All of these reagents require bases for activation. All of the DCC and DCC-related derivatives discussed previously work by the formation of the symmetrical anhydride. The symmetrical anhydrides are usually very reactive and have been used extensively in SPPS, especially in Boc synthesis (30-33). Attempts at incorporating symmetrical anhydrides to Fmoc amino acids were met with some difficulties (34-36). For instance, symmetrical anhydrides prepared from N-(3-dimethylamino propyl)-N'-ethyl-carbodiimide•HCl, upon formation of the 2-alkoxy-5(4H)-oxazolone intermediate, rearranged in the presence of carbodiimides and tertiary amines (37). Also, not all of the Fmoc symmetrical anhydrides are soluble in DCM or remain insoluble regardless of the solvent used (38). An alternative to the symmetrical anhydride is the mixed anhydride which is a carboxylic-carbonate or carboxylic-phosphinic mixed anhydride. Typically, these anhydrides are prepared by reacting either isobutyl- or isopropyl-chloroformate and substituted phosphinic chlorides with the N- a -protected amino acid (39-42). The reaction is typically rapid with little or no side reactions (43-46). A type of mixed anhydride, N-carboxyanhydrides (NCA's), also known as Leuchs' anhydride have been widely used for the preparation of polyamino acids (47). This class of compounds combines N- a -protection with carboxyl group activation. Once reacted with another amino acid or peptide residue, the NCA releases carbon dioxide as its only by-product. NCA derivatives are easily prepared by treating a -amino acids with phosgene (48-51). The resulting NCA derivatives usually crystallize out and are ready for use under strictly defined conditions. These conditions require the pH to be carefully controlled during synthesis. At pH 10, the peptide-carbamate (produced by the reaction between the NCA and the peptide or amino acid residue) tends to lose carbon dioxide with the generation of a free a -amino end group with resulting polymerization. At pH 10.5, hydrolytic decomposition of the NCA occurs. Therefore, the reaction is performed at pH 10.2. Another required condition is that the reaction proceeds for 2 minutes at 0°C with vigorous stirring. The resulting product is free of racemization and bears a free a -amino group that can be extended by addition of another anhydride. Solution Phase Synthesis Stepwise condensation is based on the repetitive addition of single N- a -protected amino acids to a growing amino component, generally starting from the C-terminal amino acid of the chain to be synthesized. The process of coupling individual amino acids can be accomplished through employment of the carbodiimide (52-53), the mixed carbonic anhydride (54-55), or the N-carboxyanhydride methods (56-57). The carbodiimide method involves coupling N- and C- protected amino acids by using DCC as the coupling reagent. Essentially, this coupling reagent promotes dehydration between the free carboxyl group of an N-protected amino acid and the free amino group of the C-protected amino acid, resulting in the formation of an amide bond with precipitation of the by- product, N,N'-dicylcohexylurea. This method, however, is hampered by side reactions which can result in racemization (58-59) or in the presence of a strong base, the formation of 5(4H)-oxazolones (60) and N-acylureas (61). Fortunately, these side reactions can be minimized, if not altogether eliminated, by adding a coupling catalyst such as N-hydroxysuccinimide (HOSu) or 1-hydroxybenzotriazole (HOBt). In addition, this method can be employed to synthesize the active ester derivatives of N-protected amino acids (62). In turn, the resulting activated ester will react spontaneously with any other C-protected amino acid or peptide to form a new peptide. In cases where separation of the activated ester from the by-product dicyclohexylurea proves to be difficult, the mixed carbonic anhydride method can be employed. This method consists of two stages: the first stage involves activating the carboxyl group of an N- a -protected amino acid with an appropriate alkyl chlorocarbonate, such as ethylchlorocarbonate (63), or preferably isobutylchlorocarbonate (64). Activation occurs in an organic solvent in the presence of a tertiary base. The second stage involves reacting the carbonic anhydride with a free amine component of an amino acid or a peptide unit. The carbonic anhydride is usually added at a 14-fold excess over the amine component. The mixed carbonic anhydride method is noted for being highly effective at low temperatures, resulting in high yields and pure products. However, it does have its short-comings. For instance, there is a tendency for the anhydride derivative to undergo racemization as a result of the strong activation of the carbonyl group. This problem does not occur when N- a -urethane protecting groups, such as Cbz or t-Boc, are employed (65-66). Furthermore, as a result of their high reactivity, mixed carbonic anhydrides are prone to the formation of 5(4H)-oxazolones (67), urethanes (68-69), diacyimides (70-71), esters (72), and are subject to disproportion (73-74). Conditions which prompt such side reactions to occur are high temperatures, prolonged activation times (the time interval between the addition to the alkylchlorocarbonate and the amine component after the mixed anhydride is formed), steric bulk of the amine component, and incomplete formation of the mixed anhydride. Fortunately, most of these side reactions, except for oxazalone and urethane formation, can be substantially reduced by performing the reaction at low temperature (~ -15°C) and allowing for shorter activation times (~ 1-2 min). To minimize the formation of oxazolone and urethane derivatives, the following conditions must be implemented: 1) dried organic solvents such as ethyl acetate, tetrahydrofuran, t-butanol, or acetonitrile must be used (75); 2) the tertiary base, N-methylmorpholine, should be used (76); and 3) Cbz- or Boc-N- a -protected amino acids must be utilized (77). Although isobutyl- and ethylchlorocarbonate are typically used to form carbonic anhydrides, other coupling reagents do exist. For example, N-ethyloxycarbonyl-2-ethyloxy-1,2-dihydroquinoline (EEDQ) (78) and N-isobutyloxy-carbonyl-2-isobutyloxy-1,2-dihydroquinoline (IIDQ) (79) were developed to react with the carboxyl component to form the ethyl- or isobutylcarbonate derivative. Unlike the classical anhydride procedure, EEDQ and IIDQ do not require base nor low reaction temperatures. Typically, the procedure involves reacting equimolar amounts of the carboxyl and amine components in an organic solvent (a wide variety of solvents can be used) (80) at 0.1 M to 0.4 M concentrations. Then EEDQ or IIDQ is added in 5-10% excess and the mixture is allowed to stir for 15-24 hours at room temperature. After removal of the solvent, in vacuo, the residue is dissolved in ethyl acetate and washed with 1N NaHCO3, 10% citric acid, and salt water, then dried with Na2SO4 (anhydrous), and evaporated. The product can then be recrystallized or purified by chromatography. While this method circumvents the use of base, it is still subject to racemization and urethane side product formation at levels comparable to those found in the classical anhydride approach. Consequently, its only advantage may be that it is easy and convenient to use. It should be noted that a detailed comparison of the two methods has not been carried out to this date. HPLC Analysis and Purification (81-84) Analytical HPLC utilizes columns and pumping systems that can withstand and deliver very high pressures enabling the use of very fine particles (3-10 microns) as packing material. Consequently, peptides can be resolved with a high degree of resolution in a short time interval (i.e., minutes). Two common HPLC purification methods are, ion exchange and reverse phase. Ion exchange HPLC is based on direct charge interactions between the peptide and the stationary phase. The column support is derivatized with an ionic species that maintains a particular charge over a certain pH range, while the peptide or peptide mixture exhibits an opposite charge which is dependent on its amino acid composition. Separation is dependent on charge interactions. The peptide is eluted by changing the pH, the ionic strength, or both. Typically, a solution of low ionic strength is used; the ionic strength of the solution is then gradually or step-wise increased until the peptide is eluted from the column. One example of ion exchange separation incorporates the use of strong cation exchange columns such as sulfoethylaspartimide which separates on the basis of positive charge at an acidic pH. Reverse phase HPLC conditions are essentially the reverse of normal phase chromotography. The peptide binds on the column through hydrophobic interactions and is eluted by decreasing the ionic strength (i.e., increasing the hydrophobicity of the eluent). Generally, the column supports are composed of hydrocarbon alkane chains which are covalently attached to silica. These chains range from C4 to C18 carbon atoms in length. Since elution from the column is a function of the hydrophobicity, the longer chain hydrocarbon columns are better for small, highly charged peptides. On the other hand, large hydrophobic peptides elute better using short chain hydrocarbon supports. However, in general practice, these two types of columns can be used interchangeably with little significant differences. Other types of supports consist of aromatic hydrocarbons such as phenyl groups. A typical run usually consist of two buffers, 0.1% TFA-H2O and 80% acetonitrile/0.1% TFA-H2O, which are mixed using a linear gradient with a flow rate which will give a 0.5% to 1.0% change per minute. Typical columns for analytical and purification runs are 4.6 x 250 mm (3-10 microns) and 22 x 250 mm (10 microns), respectively. If radial packed columns are used, then column sizes are 8 x 100 mm (3-10 microns) and 25 x 100 mm (10 microns), respectively. A variety of other buffers can contain many different types of reagents such as 0.1% heptafluorobutyric acid, 0.1% phosphoric acid, dilute HCl, formic acid (5-60%, pH 2-4), 10-100 mM NH4HCO3, sodium/ammonium acetate, TFA/TEA, sodium or potassium phosphate, or triethylammonium phosphate (pH 4-8). In addition, water miscible eluents can also be added such as methanol, propanol, and isopropanol. Therefore, many combinations of solvents and additives for a buffer are possible. It should be noted that silica-based reverse phase column packing must not be exposed to high pH's or even slightly basic pH's for extended periods of time because the column can be destroyed at those pH levels. The crude peptide obtained from SPPS will contain many by-products which are a result of deletion or truncated peptides as well as side products stemming from cleaved side chains or oxidation during the cleavage and deprotection process. Earlier purification methods included ion exchange, partition, and counter current chromatography. Recent purification methods include reverse phase HPLC which is generally successful with peptides containing 60 residues or less. In conjunction, ion exchange HPLC can be used in cases where reverse phase HPLC does not work. Typically, analytical HPLC results are used to determine the purification conditions. For example, if a peptide elutes out at 30% (0.1% TFA) aqueous acetonitrile (determined by analytical HPLC analysis), a buffer containing a lower concentration of acetonitrile is chosen such that the peptide peak will come out 4-5 minutes after the solvent peak under isocratic conditions (e.g., 28% (0.1% TFA) aqueous acetonitrile). The purification conditions will entail using a linear gradient of 16-35% (0.1% TFA) aqueous acetonitrile over one or two hours depending on the type of column chosen. The collected fractions will then be checked by analytical HPLC, using the buffer chosen for isocractic conditions. Handling and Storage of Peptides Peptides have widely varying solubility properties. The main problem associated with the dissolution of a peptide is secondary structure formation. This formation is likely to occur with all but the shortest of peptides and is even more pronounced in peptides containing multiple hydrophobic amino acid residues. Secondary structure formation can be promoted by salts. It is recommended first to dissolve the peptide in sterile distilled or deionized water. Sonication can be applied if necessary to increase the rate of dissolution. If the peptide is still insoluble, addition of a small amount of dilute (approximately 10%) acetic acid (for basic peptides) or aqueous ammonia (for acidic peptides) can facilitate dissolution of the peptide. For long-term storage of peptides, lyophilization is highly recommended. Lyophilized peptides can be stored for years at temperatures of -20°C or lower with little or no degradation. Peptides in solution are much less stable. Peptides are susceptible to degradation by bacteria so they should be dissolved in sterile, purified water. Peptides containing methionine, cysteine, or tryptophan residues can have limited storage time in solution due to oxidation. These peptides should be dissolved in oxygen-free solvents. To prevent the damage caused by repeated freezing and thawing of peptides, dissolving the amount needed for the immediate experiment and storing the remaining peptide in solid form is recommended. 八、多肽大规模生产工艺 Peptide Manufacturing Method Chemical Synthesis Solution Phase: Convergent method in which the desired sequence is constructed by fragment condensation. These peptides usually contain 3 to 5 amino acids or less than 10 amino acids. This method is easy for scale up to hundreds of kilograms of product but limited in number of amino acids and costly development. Solid Phase: Stepwise method, in which a peptide is constructed by the addition of the protected amino acids constituting its sequence to an insoluble polymeric support. This method is very fast to have product but the scale up may be costly. Solid Phase Peptide Synthesis (SPPS) Boc Synthesis tert-Butyloxycarbonyl (Boc) Strategy: The Boc method has been exclusively used during the first 15 years of SPPS. The Boc protecting group on the alpha amino of the amino acid is removed by Trifluoroacetic acid (TFA) and the final cleavage of the peptide from the resin along with the removal of the amino acid side chain protecting groups requires strong acid, such as hydrogen fluoride (HF) or trifluoromethanesulfonic acid (TFMSA). Dichloromethane (DCM) is the primary solvent used for resin deprotection, coupling, and washing. In peptide synthesis, t-Butyloxycarbonyl (Boc) protected amino acid residues (in excess millimole quantities) are sequentially bound to a resin support. These residues are assigned individual coupling cycles in accordance to their respective positions within the given peptide chain. Once a residue has been successfully coupled as evidenced by the Ninhydrin test, the compound is deprotected and neutralized for the next coupling cycle. More or less peptide can be synthesized by varying quantities of reactants and solvents proportionately. Additionally, coupling intervals may be varied from 30 minutes to 72 hours; peptide resin washing intervals may range from 1 to 30 minutes. Resin: Modified polystyrene resins are used for Boc peptide synthesis. In usually, the small particle sized resin of low cross-linking is favored. The cross-linked with 1% divinylbenzene (DVB) is most common used: A higher level of cross-linking would reduce the swelling. The old most popular resin is 200-400 mesh (38-75 m m) these resins allow for rapid diffusion of reagents inside the beads. The 100-200 mesh (75-150 m m) resins are very popular now these resins allow for fast draining the reagents. The substitution of resins should approximately 0.5-0.6 meq/gm. The higher substitution resins are available for short and large quality of peptide. The Chloromethyl polystyrene resin (Merrifield resin) is most common for acid peptide. The phenylacetamidomethyl resin (Pam resin) is more stable to TFA deprotection than the benzyl ester linker but HF cleavage may give yields as low as 70%. The 4-methylbenzhydrylamine resin (MBHA resin) is most common for amide peptide. The resins with 100-200 mesh, 1%DVB, 0.5-0.6 meq/gm are most common used at our lab. Amino Acids A: Ala C: Cys(4-Me-Bzl), Cys(4-Me-OBzl), Cys(Acm) D: Asp(OBzl), Asp(OcHex), Asp(Fmoc) E: Glu(OBzl), Glu(OcHex) F: Phe G: Gly H: His(Tos), His(Bom), His(Dnp) I: Ile(1/2H2O) K: Lys(2-Cl-Z), Lys(Fmoc), Lys(Alloc) L: Leu(H2O) M: Met N: Asn * , Asn(Xan) P: Pro Q: Gln * , Gln(Xan) R: Arg(Tos) S: Ser(Bzl) T: Thr(Bzl) V: Val W: Trp, Trp(Formyl) Y: Tyr(2-Br-Z), Tyr(2,6 Di-Cl-Bzl) Note: 1. Asn Gln, * : need HOBT. Asn can not add with HBTU or BOP. 2. Asp: Asp-Gly, Asp-Asn, Asp-Ala, Asp-Ser, and Asp-Leu, use the cyclohexyl ester Asp(OcHex) instead the Asp(OBzl) to prevent to lost water during HF cleavage. His(Tos): 1 hour deprotection after His. No HOBT should add after His for 2 to 3 amino acids. Coupling Reagents The most commonly reagents are DCC, DIC, HBTU, BOP. BOP, HBUT, TBTU, PyBOP, and HATU, are in situ activating reagents. The coupling reaction is very fast. To avoid peptide termination, the ratio of amino acid to coupling reagent should be 1 to 0.9 for HBTU, TBTU or HATU. DCC and DIC are traditionally Equipment and Materials Automated C S Bio peptide synthesizer Test tube heating block Dichloromethane (DCM) N-Dimethylformamide (DMF) Methylsulfoxide (DMSO) 1-Methyl-2-Pyrrolidione (NMP) Ethanol (ETOH) Diisopropylethylamine (DIEA) Triethylamine (TEA) Trifluoroacetic acid (TFA) Indole Dicyclohexylcarbodiimide (DCC) Diisopropylcarbodiimide (DIC) Benzotriazol-1-yl-Oxy-Tris-(Dimethylamino) phosphonium Hexafluorophosphate (BOP) 2-(1H-Benzotriazol-1-yl)-1,1,3,3-Tetramethyluronium Hexafluorophosphate (HBTU) 1-Hydroxybenzotriazole monohydrate (HOBT) Boc Amino Acids Acetic anhydride Ninhydrin Phenol Pyridine (Pyr) Air 60 psi (4.14 bar or 413.8 kPa) Nitrogen 20 psi (1.38 bar or 137.9 kPa) Synthesis Protocols t-Boc + DCC / DIC: Cycle No Solvent Time (min.) 1 2 – 3 DCM 1 - 3 2 1 40% TFA in DCM 1 - 3 3 1 40% TFA in DCM 25 - 30 4 1 – 2 DCM 1 - 3 5 1 – 2 ETOH or DMF 1 - 3 6 1 – 2 DCM 1 - 3 7 2 10% DIEA in DCM 3 - 5 8 2 – 3 DCM 1 - 3 9 1 Coupling 60 - 999 10 1 – 2 DCM 1 - 3 11 1 – 2 10% DIEA in DCM 1 - 3 12 1 – 2 DCM 1 – 3 Re-coupling: t-Boc and DCC / DIC: Cycle No Solvent Time (min.) 1 1 – 2 DCM 1 - 3 2 2 10% DIEA in DCM 3 - 5 3 2 – 3 DCM 1 - 3 4 1 Coupling 60 - 999 5 1 – 2 DCM 1 - 3 6 1 – 2 10% DIEA in DCM 1 - 3 7 1 – 2 DCM 1 - 3 t-Boc + BOP / HBTU / TBTU: Cycle No Solvent Time (min.) 1 2 – 3 DCM 1 - 3 2 1 40% TFA in DCM 1 - 3 3 1 40% TFA in DCM 25 - 30 4 1 – 2 DCM 1 - 3 5 1 – 2 ETOH or DMF 1 - 3 6 1 – 2 DCM 1 - 3 7 1 10% DIEA in DCM 1 - 3 9 1 Coupling 60 - 999 10 1 – 2 DCM 1 - 3 11 1 – 2 10% DIEA in DCM 1 - 3 12 1 – 2 DCM 1 - 3 Re-coupling: t-Boc and BOP / HBTU / TBTU: Cycle No Solvent Time (min.) 1 1 - 2 DCM 1 – 3 2 1 10% DIEA in DCM 1 – 3 3 1 Coupling 60 – 999 4 1 - 2 DCM 1 – 3 5 1 - 2 10% DIEA in DCM 1 – 3 6 1 - 2 DCM 1 – 3 Aceylation protocol. Cycle No Solvent Time (min.) 1 1 - 2 DCM 1 - 3 2 1 10% DIEA AcOH in DCM 5 - 20 3 2 - 3 DCM 1 – 3 Note: 1. DIEA can be substituted with TEA. 2. Net TFA for 2 to 5 minutes deprotection with DMF flow wash by Kent’s method. Ninhydrin Test A small sample of resin (Approx. 3-10 mg) is removed from the synthesizer’s reaction vessel and placed in a test tube. The resin is washed with 3-4 mL 10% DIEA/DCM solution one time and 2 times with 3-4 ml ETOH. Washing is accomplished by addition of solution, mixing, and decanted. One drop from each of the following reagents are added to each tube: Ninhydrin Solution (5.0 gm Ninhydrin in 100 mL ETOH). Phenol Solution (80 gm Phenol in 20 mL ETOH). Pyridine. The contents of each tube are then mixed thoroughly. All tubes are placed in a heating block and heated at 120 ° C for 3 min. The tubes are then promptly removed. Holding each tube against a white background and evaluating the solution color interpret test results. Results for the sample-containing tubes are compared with that of the standard. A blue, purple, or red color is indicative of free amine (positive result). The absence of color - as exhibited by the clear polystyrene blank solution - is indicative of no free amine (negative result). Samples are re-tested when variable results are obtained. A negative test results means that the synthesis can continue to the next cycle. A positive test results means a determination will be made by the chemist to either re-couple the same amino acid using a double couple program or that the resin will be acylated using the acylation program. Hydrogen Fluoride Cleavage The resin bound peptides or side chain protected peptides are treated with liquid hydrogen fluoride. This cleaves the peptide from the resin and protecting groups from the constituent amino acid moieties. An HF cleavage apparatus consists of Kel-F or Teflon reaction vessels, valves and tubing. A high vacuum pump is employed in this process. The peptide resin is weighed and transferred to the HF reaction vessels. Anisole or P-Cresol is added to the resin, 1 mL per gram of peptide resin. If Cysteine, Methionine or Tryptophan is present in the peptide, DMS or 1.2 Ethanedithiol is also added, 0.25 mL per gram resin. The HF reaction vessel is attached to the HF apparatus and placed in a dry ice/acetone batch for approximately 5 minutes. During this time turn on vacuum pump and check the HF apparatus for any vacuum leaks. (IF THERE IS A LEAK DO NOT PROCEED UNTIL THIS IS FIXED) The pump valve is then turned off creating a closed system between the HF cylinder and reaction vessel, which is regulated by the HF cylinder valve. This valve is then opened permitting approximately 5 - 10 mL of HF per gram of peptide resin to accumulate in the reaction vessel. When adequate HF has collected, the HF cylinder valve is closed, and the reaction vessel is warmed to 0 ° C in an ice bath. The reaction vessel contents are then magnetically stirred at this temperature for 45 - 90 minutes completing the reaction. HF is evacuated from the reaction vessel into a trap containing calcium oxide; HF is absorbed as calcium fluoride. While stirring at 0 ° C, evacuation continues until all HF has been removed. After evacuation, the reaction vessel is removed from the HF apparatus. Approximately 50 - 100 mL of anhydrous ether per gram of resin are added and the mixture is agitated. When dispersed, the resin and cleaved peptide are collected by filtration using a sintered glass funnel. This washing procedure is repeated twice before extracting the peptide with acetic acid, Buffer B of purification, or TFA and water. The resulting filtrate contains the crude peptide then lyophilize. Equipment and Materials HF apparatus High vacuum pump Freeze Dryers (Lyophilizer) Anisole Dimethyl sulfide (DMS) 1, 2 Ethanedithiol (EDT) P-Cresol Ether, anhydrous Acetic acid (AcOH) Acetonitrile (CH3CN) Trifluoroacetic acid (TFA) Water - Distilled water and deionized water Ice Dry Ice Acetone Filter funnel Filtering flask ---------------------------------------------------------------------------------------- Fmoc Synthesis 9-Fluorenylmethyloxycarbonyl (Fmoc) Strategy: The Fmoc protecting group is deprotected by mild base treatment of 20% piperidine in N-Dimethylformamide (DMF), 2% DBU, 2% piperidine in DMF and the final cleavage of the peptidyl resin and side chain groups deprotection by Trifluoroacetic acid (TFA). N-Dimethylformamide (DMF) is the primary solvent used for resin deprotection, coupling, and washing. N,N-Dimethylacetamide (DMA) or 1-Methyl-2-Pyrrolidione (NMP) may also be used. In peptide synthesis, Fmoc protected amino acid residues (in excess millimole quantities) are sequentially bound to a resin support. These residues are assigned individual coupling cycles in accordance to their respective positions within the given peptide chain. Once a residue has been successfully coupled as evidenced by the Ninhydrin test, the compound is deprotected for the next coupling cycle. By the methods employed, material of equivalent quality in larger or smaller amounts can be obtained while varying quantities of reactants and solvents proportionately. Additionally, coupling intervals may be varied from 30 minutes to 72 hours; peptide resin washing intervals may range from 1 to 30 minutes. Resin: The hydroxymethyl-based resins (Wang, HMPA, HMPB resins) are the most common used except the Cys, His and Pro residues at the C-terminus, in this case Trityl-based resins such as 2-chlorotrityl resin should be used. The Rink amide MBHA resin consist 4-methylbenzydrylamine (100-200 mesh, 1% DVB) polystyrene, derivatized sequentially with norleucine and the Fmoc protected modified form of the Rink amide linker, which incorporates an acetic acid spacer, is not degraded by TFA, and is therefore compatible with the standard 95% TFA cleavage reaction. The resins with 100-200 mesh, 1%DVB, 0.5-0.6 meq/gm are most common used at our lab. Amino Acids A: Ala C: Cys(Trt), Cys(Mmt), Cys(Acm) D: Asp(OtBu) E: Glu(OtBu) F: Phe G: Gly H: His(Trt) I: Ile K: Lys(Boc), Lys(Fmoc), Lys(Alloc) L: Leu M: Met N: Asn * , Asn(Trt) P: Pro Q: Gln * , Gln(Trt) R: Arg(Pbf), Arg(Pmc) S: Ser(tBu), Ser(Trt) T: Thr(tBu), Thr(Trt) V: Val W: Trp, Trp(Boc) Y: Tyr(tBu) Note: Asn and Gln: need HOBT. Coupling Reagents The most commonly reagents are DCC, DIC, HBTU, BOP. BOP, HBUT, TBTU, PyBOP, and HATU, are in situ activating reagents. The coupling reaction is very fast. To avoid terminus the peptide, the ratio of amino acid to coupling reagent should be 1 to 0.9 for HBTU, TBTU, or HATU. DCC and DIC are traditional. Equipment and Materials Automated C S Bio peptide synthesizer Test tube heating block Dichloromethane (DCM) N-Dimethylformamide (DMF) 1-Methyl-2-Pyrrolidione (NMP) Methylsulfoxide (DMSO) Piperidine N,N-Dimethylacetamide (DMA) Ethanol (ETOH) Diisopropylethylamine (DIEA) Trifluoroacetic acid (TFA) Dicyclohexylcarbodiimide (DCC) Diisopropylcarbodiimide (DIC) Benzotriazol-1-yl-Oxy-Tris-(Dimethylamino) phosphonium Hexafluorophosphate (BOP) 2-(1H-Benzotriazol-1-yl)-1,1,3,3-Tetramethyluronium Hexafluorophosphate (HBTU) 1-Hydroxybenzotriazole monohydrate (HOBT) Fmoc Amino Acids Acetic anhydride (AcOH) Ninhydrin Phenol Pyridine (Pyr) Air 60 psi Nitrogen 20 psi Synthesis Protocols Fmoc amino acid and DCC or DIC: Cycle No Solvent Time (min.) 1 2 – 3 DMF 1 - 3 2 1 20% Piperidine in DMF 5 - 10 3 1 20% Piperidine in DMF 5 - 20 4 2 – 3 DMF 1 - 3 5 1 – 2 DCM 1 - 3 6 2 DMF 1 - 3 7 1 Coupling 60 - 999 8 2 – 3 DMF 1 - 3 Doubling coupling: Fmoc + DCC or DIC: Cycle No Solvent Time (min.) 1 2 - 3 DMF 1 - 3 2 1 Coupling 60 - 999 3 2 – 3 DMF 1 - 3 Fmoc + BOP or HBTU or TBTU: Cycle No Solvent Time (min.) 1 2 - 3 DMF 1 - 3 2 1 20% Piperidine in DMF 5 - 10 3 1 20% Piperidine in DMF 5 - 20 4 2 - 3 DMF 1 - 3 5 1 - 2 DCM 1 - 3 6 2 DMF 1 - 3 7 1 Coupling 60 - 999 8 2 – 3 DMF 1 - 3 Double coupling: Fmoc + BOP or HBTU or TBTU: Cycle No Solvent Time (min.) 1 2 - 3 DMF 1 – 3 2 1 Coupling 60 – 999 3 2 – 3 DMF 1 – 3 Aceylation protocol. Cycle No Solvent Time (min.) 1 1 - 2 DMF 1 - 3 2 1 10% DIEA AcOH in DMF 5 - 30 3 2 - 3 DMF 1 - 3 Note: 2% DBU in DMF may substitute the 20% Piperidine in DMF. TFA Cleavage for Fmoc Synthesis Before acid cleavage of the peptidyl resin can be performed, the N-terminal Fmoc group must be removed using piperidine. Having successfully synthesized a protected peptide, one is confronted with a difficult task of having to simultaneously detach the peptide from the resin support and remove all the side chain protecting groups of the amino acid residues to yield the desired peptide. In Fmoc SPPS, this step is normally carried out by treating the peptidyl resin with TFA. The peptide resin is weighed and transferred to the reaction vessels. If Cysteine, Methionine and Tryptophan are present in the peptide, Reagent K (TFA / water / phenol / thioanisole / EDT = 82.5 : 5 : 5 : 5: 2.5) will be applied. Otherwise TFA / water / TIS = 95 : 2.5 : 2.5 will be used. Place dry resin in a flask and add TFA solution containing appropriate scavengers (10 - 25 ml/gm) resin. Stopper the flask and leave to stand at room temperature with occasional swirling. React for 2-4 hours. Remove the resin by filtration under pressure. Wash the resin twice with TFA. Combine filtrates, and add (drop-wise) an 8 -10-fold volume of cold ether. Sometimes it is necessary to evaporate most of the TFA to achieve a good precipitation of the crude peptide. The Ether can be cooled with ice to further assist precipitation. Filter the precipitated peptide through hardened filter paper in a Hirsch funnel or filter funnel under a light vacuum. Wash the precipitate further with cold ether, dissolve the peptide in a suitable aqueous buffer and lyophilize. Equipment and Materials Trifluoroacetic acid (TFA) Phenol Thioanisole Triisopropylsilane (TIS) 1, 2 Ethanedithiol (EDT) Ether, anhydrous Ice Dry Ice Acetone Acetic acid (AcOH). Rotary evaporator Centrifuge Freeze Dryers (Lyophilizer) Filter funnel Stirrer or stir bar Filtering flask Round bottom flask Cyclization (Oxidation) Cyclization is accomplished by oxidizing free sulfhydril groups among constituent amino acids within the peptide chain. To detect the presence of sulfhydril groups, a sample of the uncyclized peptide is evaluated with Ellman’s test. Ellman’s test Reagent 1: Ellman reagent - 40.0 mg of 5,5’-dithio-bis (2-nitrobenzoic acid), DTNB, are dissolved in 10 mL of pH 8 phosphate buffer. Make up fresh before use. Reagent 2: 0.1 N Sodium phosphate Na2PO4 buffer, pH 8. Make 4.60 gm sodium phosphate monohydrate NaH2PO4-H2O and 307.4 mL of 0.1 N NaOH to 1 L distilled water. Standard sulfhydril compound: 0.24 mg of Cysteine is dissolved in 10 mL of distilled water. Peptide sample: 0.24 mg of peptide is dissolved in 10 mL of distilled water. Test solutions are prepared in glass tubes by combining the following: 0.1 mL of standard or peptide solution 0.1 mL of reagent 1 5 mL of reagent 2 A blank solution of 0.1 ml reagent 1 and 5 ml reagent 2 is compared to the test solution. More yellow is the test solution is indicative of free S-H. Method 1: Disulfide bridge formation with potassium ferricyanide. The bulk cleaved peptide is transferred to an appropriate size container and dissolved in water to a concentration of 1 L – 5 L per gram of peptide. After thoroughly mixing the solution, the pH is adjusted to 7.4 - 7.6 with ammonium hydroxide. From a separatory funnel, 0.01 M K3Fe(CN)6 is added to the peptide solution at a rate of approximately 5 drops per minute. Upon addition of the oxidizing agent, the reaction mixture will exhibit a yellow color that dissipates. The reaction is complete when this color persists for 30 minutes. The pH of the reaction mixture is adjusted to 4.5 with acetic acid. If Lysine or Arginine residues are present in the peptide Bio-Rex 70 resin may be used. Add the Bio-Rex 70 resin (80 - 150 gm/m mole peptide) into solution and stir overnight. Or pass the solution through C-18 column. Extract the peptide from Bio-Rex 70 resin or C-18 column. Method 2: Disulfide bridge formation with air pump or without air pump: The bulk cleaved peptide is transferred to an appropriate size container and dissolved in water to a concentration 1 L - 5 L per gram of peptide. After thoroughly mixing the solution, the pH is adjusted to 8.0 - 8.3 with ammonium hydroxide. By using the air pump, pump the air into the solution for 20 to 60 hours. The pH of the reaction mixture is adjusted to 4.5 with acetic acid. If Lysine or Arginine residues are present in the peptide Bio-Rex 70 resin may be used. Add the Bio-Rex 70 resin (80 - 150 gm/m mole peptide) into solution and stir overnight. Or pass the solution through C-18 column. Extract the peptide from Bio-Rex 70 resin or C-18 column. Method 3: Disulfide bridge formation with Iodine: The bulk cleaved peptide is transferred to an appropriate size container and dissolved in water to a concentration of 1 L – 5 L per gram of peptide. After thoroughly mixing the solution, drop a drop of the solution of 10 gm Iodine in 100 ml methanol slowly. Upon addition of the oxidizing agent, the reaction mixture will exhibit a yellow color that dissipates. The reaction is complete when this color persists for 30 minutes then adds the absorbic acid until the solution color is clear. If Lysine or Arginine residues are present in the peptide Bio-Rex 70 resin may be used. Add the Bio-Rex 70 resin (80 - 150 gm/m mole peptide) into solution and stir overnight. Or pass the solution through C-18 column. Extract the peptide from Bio-Rex 70 resin or C-18 column. Equipment and materials Container (2 L to 100 L) Stirrer Stirrer bar pH meter or pH paper Glass filter funnel (1 L to 3L) Vacuum filtration flask Air pump Separator funnel Ammonium hydroxide (NH4OH) Acetic acid (AcOH) Trifluoroacetic acid (TFA) Potassium ferricyanide (K3Fe(CN)6) Iodine Methanol Bio Rex 70 resin Reverse phase resin: C-18 Water – Distilled water / deionized water ------------------------------------------------------------------------------------------------------------------ Purification A revolution in purification techniques for many peptides and proteins is high performance liquid chromatography (HPLC). It is highly cost effective in that it is able to replace multiple steps in conventional purification processes, thus eliminating handling, labors costs, and also able to perform purifications which can not be carried out by any other process. Method 1. 1: Ion Exchange Chromatography (CM-52 or DE-52). Cation exchanger CM-52 or anion exchanger DE-52 as appropriate may treat the crude peptide. The mobile phase of cation exchange chromatography consists of: Buffer A: 0.05 M - 0.1 M Ammonium acetate. Buffer B: 0.1 M - 1 M ammonium acetate. The mobile phase of anion exchange chromatography consists of: Buffer A: 0.05 M - 0.1 M Ammonium bicarbonate. Buffer B: 0.1 M - 1 M ammonium bicarbonate. In either, the buffer molarity may be varied to accommodate the particular peptide in maximizing yield and purity. Also 0 - 6 M urea can be used in buffer A or Buffer B depending on the solubility of the peptide. Stir and pour ion exchanger into Buffer A. Stir the slurry with a magnetic stirrer in a stopper flask connected to a pump until no more bubbles appear. Set up the glass column vertically containing about 3 - 4 cm Buffer A at the bottom. The resin slurry in buffer A is then poured into the column to a depth of 20 cm. Additional buffer A is passed through the column and then elulant is monitored until its pH is comparable with that of the buffer. The peptide in buffer A solution is then applied to the column and eluted with a gradient of 0 - 100 percent buffer B by gravity. Fractions of the sample are collected in appropriately sized tubes. TLC or analytical HPLC analyzes these fractions for purity. Pure fractions are lyophilized and forwarded to QC; impure fractions are subjected to further purification steps. Method 1.2 : Ion Exchange Chromatography (Bio-Rex 70). The Bio Rex 70 resin used in the extraction must be in the sodium free form. 1 kg of resin first washed with 1 L of 1 N HCl then washed D.I. water until pH of washing solution equal to D.I. water pH should be used. The crude peptide (after cyclization contains Lys, Arg, Orn, or positive charged group) may be absorbed by chloride form Bio-Rex 70 after stirring over night. The resin is filtered and packed into a column. Elute with 70% AcOH isocratically by gravity. It takes approximately 10 times of column volume until the peptide is completely eluted out from the column. Fractions of the sample are collected in appropriately sized tubes. These fractions are analyzed for purity by analytical HPLC. Pure fractions are lyophilized and forwarded to QC; impure fractions are subjected to further purification steps. Method 1.3: Ion Exchange Chromatography (AG1-X8). An anion exchange resin AG1-X8 is used for the conversion of Trifluoroacetic or fluoride to acetate as the counter anion for peptides. Pack a column with AG1-X8 (size of column is dependent on the quantity of peptide. Peptide : AG1X8 = 1 gm : 25 gm). Solubilize peptide in elution buffer (10% to 70% AcOH) and load onto column. Elute isocratically by gravity with the same buffer. It takes approximately 10 times of column volume until the peptide is completely eluted out from the column. Fractions of the sample are collected in appropriately sized tubes. These fractions are analyzed for purity by analytical HPLC. Pure fractions are lyophilized and forwarded to QC; impure fractions are subjected to further purification steps. Method 2 : Gel Filtration Chromatography. Bio-gel P series resin P-2, P-4, P-6 and Sephadex G-series - G-25, G-50 serve as matrices in this technique. The mobile phase consists of a 5 - 70% acetic acid buffer. Prior to use, this buffer is vacuum filtered through a membrane of 0.45 micron pore size and degassed. The column matrix is prepared by adding the resin to the acetic acid buffer and allowing it to stand 20 - 30 minutes. A glass column (size of column is dependent on the quantity of peptide) is then primed with additional buffer before filling 50 - 70% of glass column capacity with the swollen resin. Once packed, the column is equilibrated with acetic acid buffer (about 5 times of column volume). The crude or partial pure peptide, in solution, is then applied to the column and eluted isocratically by gravity. Fractions of the sample are collected in appropriately sized tubes. These fractions are analyzed for purity by analytical HPLC. Pure fractions are lyophilized and forwarded to QC; impure fractions are subjected to further purification steps. Method 3.1 : Reverse phase Chromatography (Low pressure). The silane or polystyrene reverse phase (C-18, C-8, or C-4) resin (60 Å to 300 Å) is used as matrices in this procedure. The resin is slurry with MeOH and packed into glass column (size of column is dependent on the quantity of peptide. Peptide : resin = 1 gm : 50 gm). Typically, the mobile phase may consist of buffer A and B as follows: Buffer A1 - 0.1% TFA in water. Buffer A2 - 0.05 M NH4OAc in water. Buffer A3 - 1% AcOH in water. Buffer A4 - 0.05 M NaH2PO4 (Sodium Phosphate monobasic) pH 4.5 in water. Buffer A5 - 1% H3PO4 (Phosphoric acid) Buffer A6 - TEAP pH 2.5 or pH 4.5 or pH 6.5 Buffer A7 - 0.1% HCl in water Buffer B - 5% to 99% CH3CN with same ion pairing of Buffer A The ion pairing concentration and pH changes reversed phase selectivity for peptide purification. The buffer molarity may be varied to accommodate the particular peptide in maximizing yield purity. The column is equilibrated with buffer A. The peptide is dissolved in buffer A, and if necessary small quantities of buffer B, AcOH or TFA can be added. The clear peptide solution is loaded onto the column and eluted linearly on a gradient from 0 -100% buffer B. Fractions of the sample are collected in appropriately sized tubes. These fractions are analyzed for purity by analytical HPLC. Pure fractions are lyophilized and forwarded to QC; impure fractions are subjected to further purification steps. Method 3.2 : Reverse phase Chromatography (Preparative). The silane or polystyrene reverse phase (C-18, C-8, or C-4) resin (60Å to 300Å) is used as matrices in this procedure. The resin is slurry with MeOH, IPA or other suitable solvents and packed into steel column (size of column is 1” to 8” diameter with 10” to 40” long). Typically, the mobile phase may consist of buffer A and B as follows: Buffer A1 - 0.1% TFA in water. Buffer A2 - 0.05 M NH4OAc in water. Buffer A3 - 1% AcOH in water. Buffer A4 - 0.05 M NaH2PO4 (Sodium Phosphate monobasic) pH 4.5 in water. Buffer A5 - 1% H3PO4 (Phosphoric acid) Buffer A6 - TEAP pH 2.5 or pH 4.5 or pH 6.5 Buffer A7 - 0.1% HCl in water Buffer B – 0% to 100% CH3CN in buffer Ax (x = 1 or 2 or 3). The buffers are vacuum filtered through a membrane of 0.45 micron pore size or degassed prior to use. The buffer molarity may be varied to accommodate the particular peptide in maximizing yield purity. The column of choice is configured to an HPLC instrument having dual pumps and equilibrated with buffer A. The peptide is dissolved in buffer A, and if necessary small quantities of buffer B, ion can be added. The clear peptide solution is loaded onto the column and eluted linearly using on a gradient that is varied to accommodate to particular peptide. Fractions of the sample are collected in appropriately sized tubes. These fractions are analyzed for purity by analytical HPLC. Pure fractions are lyophilized and forwarded to QC; impure fractions are subjected to further purification steps. Equipment and Materials Glass columns with coarse (60 u) filters. The selection of the column and filter size is based on the quantity of the peptide to be purified. Preparative HPLC columns Solvent reservoirs Peristaltic pumps for delivering the solvents HPLC system Magnetic stirrer stir bar Lab jack Fraction collector Rotary evaporator Freeze Dryers (Lyophilizer) Beakers flask Ammonium acetate (NH 4 OAc) Ammonium bicarbonate (NH4HCO3) Acetic acid (AcOH) Hydrogen chloride (HCl) Acetonitrile (CH3CN) Ethanol (ETOH) Trifluoroacetic acid (TFA) Methanol (MeOH) Hydroxymethyl Aminomethane - Tris Phosphoric Acid (H3PO4) Triethylamine (TEA) Ammonium acetate (NH4Ac) Ammonium bicarbonate (NH4HC3) Water: Deionized water (D. I. water) / Distilled water Whatman diethylmethyl cellulose DE-52 Whatman carboxymethyl cellulose CM-52 Bio-Rad 70 AG1-X8 Bio-gel, P-series (P-2, P-4, P-6) Sephadex G-series (G-25, G-50) Silica gel with octadecyl boned phase resin - C-18 (from 5 m m to 40 m m): 60 Å to 300 Å Silica gel with octyl boned phase resin - C-8 (from 5 m m to 40 m m): 60 Å to 300 Å Silica gel with butyl boned phase resin - C-4 (from 5 m m to 40 m m): 60 Å to 300 Å
标签: 多肽合成
