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二硫键广泛存在与蛋白结构中，对稳定蛋白结构具有非常重要的意义，二硫键一般是通过序列中的2个Cys的巯基，经氧化形成。
 

形成二硫键的方法很多：空气氧化法，DMSO氧化法，过氧化氢氧化法等。
 

二硫键的合成过程，  可以通过Ellman检测以及HPLC检测方法对其反应进程进行监测。  
   

如果多肽中只含有1对Cys，那二硫键的形成是简单的。多肽经固相或液相合成，然后在pH8-9的溶液中进行氧化。      
 

当需要形成2对或2对以上的二硫键时，合成过程则相对复杂。尽管二硫键的形成通常是在合成方案的最后阶段完成，但有时引入预先形成的二硫化物是有利于连合或延长肽链的。通常采用的巯基保护基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多种基团。我们分别列出两种以2-Cl树脂和Rink树脂为载体合成的多肽上多对二硫键形成路线：
 

二硫键反应条件选择    
 

 二硫键即为蛋白质或多肽分子中两个不同位点Cys的巯基（-SH）被氧化形成的S-S共价键。 一条肽链上不同位置的氨基酸之间形成的二硫键，可以将肽链折叠成特定的空间结构。多肽分 子通常分子量较大，空间结构复杂，结构中形成二硫键时要求两个半胱氨酸在空间距离上接近。 此外，多肽结构中还原态的巯基化学性质活泼，容易发生其他的副反应，而且肽链上其他侧链 也可能会发生一系列修饰，因此，肽链进行修饰所选取的氧化剂和氧化条件是反应的关键因素， 反应机理也比较复杂，既可能是自由基反应，也可能是离子反应。      

反应条件有多种选择，比如空气氧化，DMSO氧化等温和的氧化过程，也可以采用H2O2，I2， 汞盐等激烈的反应条件。
 

空气氧化法： 空气氧化法形成二硫键是多肽合成中最经典的方法，通常是将巯基处于还原态的多肽溶于水中，在近中性或弱碱性条件下（PH值6.5-10），反应24小时以上。为了降低分子之间二硫键形成的可能，该方法通常需要在低浓度条件下进行。
 

碘氧化法：将多肽溶于25%的甲醇水溶液或30%的醋酸水溶液中，逐滴滴加10-15mol/L的碘进行氧化，反应15-40min。当肽链中含有对碘比较敏感的Tyr、Trp、Met和His的残基时，氧化条件要控制的更精确，氧化完后，立即加入维生素C或硫代硫酸钠除去过量的碘。 当序列中有两对或多对二硫键需要成环时，通常有两种情况：
 

自然随机成环:       序列中的Cys之间随机成环，与一对二硫键成环条件相似；
 

定点成环：       定点成环即序列中的Cys按照设计要求形成二硫键，反应过程相对复杂。在 固相合成多肽之前，需要提前设计几对二硫键形成的顺序和方法路线，选择不同的侧链 巯基保护基，利用其性质差异，分步氧化形成两对或多对二硫键。       通常采用的巯基保护 基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多种基团。

Definition

Cocaine- and amphetamine- regulated transcript (CART) peptides, derived from proCART polypeptide in humans are neuropeptides expressed in the brain1. They mainly serve as inhibitors of food intake1.

Discovery

CART was first purified by gel filtration and sequenced in 1980 from rat hypothalamus2.

Classification

CART is a neuropeptide family member2.

Structural Characteristics

CART gene encodes a peptide of either 129 or 116 amino acid residues in rat whereas only the short form exists in humans. The predicted signal sequence is 27 amino acid residues resulting in a precursor of 102 or 89 residues3. The C-terminal end of CART, consisting of 48 amino acid residues and 3 disulphide bonds, is thought to constitute a biologically active part of the molecule.  Various CART peptides can be generated from the precursor: CARTs (55-102), (85-102), (55-76) and (62-76)3.  CART (55-102) contains three disulphide bridges that are required for its function4.                                                                    

Mode of action

The mode of action of CART peptides has not been fully established. Several studies have observed that they may function through CART receptors in turn activating down stream signaling pathways5.

Functions

CART is distributed in the central nervous system and periphery and has many physiological roles.  It elicits similar behavior as cocaine.  CART has been shown to have variety of effects on dopamine6.  It increases blood pressure, has variety of behavioral effects and influences nociception6.  In the hypothalamus CART mainly regulates energy homeostasis6.  CART is also an endogenous inhibitor of food intake6.  CART peptides are also important in anxiety, pain, arousal, startle response, regulation of calcium channels, and neuroendocrine hormone secretion5.

References

1.     Thim L, Kristensen P, Larsen PJ, Wulff BS (1998). CART, a new anorectic peptide. Int J Biochem Cell Biol, 30(12), 1281-4.

2.     Spiess J and Vale W (1980). Multiple forms of somatostatin-like activity in rat hypothalamus. Biochemistry 19, 2861–66.

3.     Dylag T, Kotlinska J, Rafalski P, Pachuta A, Silberring J (2006). The activity of CART peptide fragments. Peptides, 27(8), 1926-33.

4.     Murphy KG (2005). Dissecting the role of cocaine and amphetamine-regulated transcript (CART) in the control of appetite. Briefing in Functional Genomics and proteomic, 4 (2),  95–111.

5.     Rogge G, Jones D, Hubert GW, Lin Y & Kuhar MJ (2008). CART peptides: regulators of body weight, reward and other functions. Nature Reviews Neuroscience, 9, 747-758.

6.      Vicentic A, Jones DC (2007). The CART (cocaine- and amphetamine-regulated transcript) system in appetite and drug addiction. J Pharmacol Exp Ther., 320(2), 499-506.

多肽H2N-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu-COOH的合成步骤：

1、合成CTC树脂：称取2.85g CTC Resin（如初始取代度约为1.15mmol/g）和3.93mmol Fmoc-Leu-OH于反应器中，加入适量DCM溶解氨基酸（需要注意，此时CTC树脂体积会增大好几倍，避免DCM溶液过少），再加入9.83mmol DIPEA（Mw：129.1,d：0.740g/ml），反应2-3小时后，可不抽滤溶液，直接加入1ml的HPLC级甲醇，封端半小时。依次用DMF洗涤2次，甲醇洗涤1次，DCM洗涤一次，甲醇洗涤一次，DCM洗涤一次，DMF洗涤2次（这里使用甲醇和DCM交替洗涤，是为了更好地去除其他溶质，有利于后续反应）。得到  Fmoc-Leu-CTC Resin。结构图如下：

2、脱Fmoc：加3倍树脂体积的20%Pip/DMF溶液，鼓氮气30分钟，然后2倍树脂体积的DMF 洗涤5次。得到 H2N-Leu-CTC Resin 。（此步骤脱除Fmoc基团，茚三酮检测为蓝色，Pip为哌啶）。结构图如下：

3、缩合：取9.83mmol Fmoc-Cys(Trt)-OH 氨基酸，加入到上述树脂里，加适当DMF溶解氨基酸，再依次加入19.66mmol DIPEA，9.34mmol HBTU。反应30分钟后，取小样洗涤，茚三酮检测为无色。用2倍树脂体积的DMF 洗涤3次树脂。（洗涤树脂，去掉残留溶剂，为下一步反应做准备）。得到Fmoc-Cys(Trt)-Leu-CTC Resin。氨基酸：DIPEA：HBTU：树脂=3：6：2.85：1（摩尔比）。结构图如下：

4、依次循环步骤二、步骤三，依次得到

H2N-Cys(Trt)-Leu-CTC Resin

Fmoc-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Ala-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Ala-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Cys(Trt)-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Cys(Trt)-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Met-Cys(Trt)-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Met-Cys(Trt)-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Pro-Met-Cys(Trt)-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Pro-Met-Cys(Trt)-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Val-Pro-Met-Cys(Trt)-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Val-Pro-Met-Cys(Trt)-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Gln(Trt)-Val-Pro-Met-Cys(Trt)-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Gln(Trt)-Val-Pro-Met-Cys(Trt)-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Gly-Gln(Trt)-Val-Pro-Met-Cys(Trt)-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

H2N-Gly-Gln(Trt)-Val-Pro-Met-Cys(Trt)-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

Fmoc-Tyr(tBu)-Gly-Gln(Trt)-Val-Pro-Met-Cys(Trt)-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin

以上中间结构，均可在专肽生物多肽计算器-多肽结构计算器中，一键画出。

最后再经过步骤二得到 H2N-Tyr(tBu)-Gly-Gln(Trt)-Val-Pro-Met-Cys(Trt)-Asp(OtBu)-Ala-Gly-Glu(OtBu)-Gln(Trt)-Cys(Trt)-Ala-Val-Arg(Pbf)-Lys(Boc)-Gly-Ala-Arg(Pbf)-Ile-Gly-Lys(Boc)-Leu-Cys(Trt)-Asp(OtBu)-Cys(Trt)-Pro-Arg(Pbf)-Gly-Thr(tBu)-Ser(tBu)-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Leu-Leu-Lys(Boc)-Cys(Trt)-Leu-CTC Resin，结构如下：

5、切割：6倍树脂体积的切割液（或每1g树脂加8ml左右的切割液），摇床摇晃 2小时，过滤掉树脂，用冰无水乙醚沉淀滤液，并用冰无水乙醚洗涤沉淀物3次，最后将沉淀物放真空干燥釜中，常温干燥24小试，得到粗品H2N-Tyr-Gly-Gln-Val-Pro-Met-Cys-Asp-Ala-Gly-Glu-Gln-Cys-Ala-Val-Arg-Lys-Gly-Ala-Arg-Ile-Gly-Lys-Leu-Cys-Asp-Cys-Pro-Arg-Gly-Thr-Ser-Cys-Asn-Ser-Phe-Leu-Leu-Lys-Cys-Leu-COOH。结构图见产品结构图。

切割液选择：1）TFA：H2O=95%：5%

2）TFA：H2O：TIS=95%：2.5%：2.5%

3）三氟乙酸：茴香硫醚：1，2-乙二硫醇：苯酚：水=87.5%：5%：2.5%：2.5%：2.5%

（前两种适合没有容易氧化的氨基酸，例如Trp、Cys、Met。第三种适合几乎所有的序列。）

6、纯化冻干：使用液相色谱纯化，收集目标峰液体，进行冻干，获得蓬松的粉末状固体多肽。不过这时要取小样复测下纯度 是否目标纯度。

7、最后总结：

杭州专肽生物技术有限公司（ALLPEPTIDE https://www.allpeptide.com）主营定制多肽合成业务，提供各类长肽，短肽，环肽，提供各类修饰肽，如：荧光标记修饰（CY3、CY5、CY5.5、CY7、FAM、FITC、Rhodamine B、TAMRA等），功能基团修饰肽（叠氮、炔基、DBCO、DOTA、NOTA等），同位素标记肽（N15、C13），订书肽（Stapled Peptide）,脂肪酸修饰肽（Pal、Myr、Ste），磷酸化修饰肽（P-Ser、P-Thr、P-Tyr），环肽（酰胺键环肽、一对或者多对二硫键环），生物素标记肽，PEG修饰肽，甲基化修饰肽等。

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