Tandem ligation of unprotected peptides through thiaprolyl and cysteinyl bonds in water.
Tandem ligation of unprotected peptides through thiaprolyl and cysteinyl bonds in water.
复制标题
在水中通过硫杂丙烯酰基和半胱氨酰键串联连接未受保护的肽。
DOI：
10.1021/ja0035654
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发表时间：
2001
影响因子：
15
通讯作者：
Yang,JL
中科院分区：
文献类型：
--
作者：
Tam,JP;Yu,Q;Yang,JL
用于蛋白质合成和修饰的串联连接需要在没有保护基团方案的情况下形成多个游离肽段的两个或更多个区域特异性酰胺键。我们在这里描述了一个半正交的策略连接三个未受保护的肽段，其中两个含有N-末端（NT）半胱氨酸，形成串联两个酰胺键，一个Xaa-SPro（硫杂脯氨酸），然后Xaa-Cys。该策略利用了NT-半胱氨酰肽在酸性条件下选择性地经历SPro-亚胺连接而不是Cys-硫酯连接的强烈偏好。在操作上，其在N → C方向上进行，首先通过在pH < 3下的亚胺连接以在含有羧基末端（CT）-乙醛酯的肽与含有NT-Cys和CT-硫酯两者的第二肽之间提供Xaa-噻唑烷酯键。然后在pH > 7下通过硫酯连接将新产生的具有CT-硫酯的O-酯连接片段连接到另一个NT-半胱氨酰肽以形成Xaa-Cys键。同时，这种碱性条件也催化Xaa-噻唑烷酯的O，N-酰基迁移到第一个连接位点的Xaa-SPro键上，以完成串联的三段连接。两个连接反应均在水性缓冲溶剂中进行。这种三段连接策略的有效性在19至70个氨基酸的六种肽中进行了测试，包括生长抑素的硫脯氨酸→脯氨酸类似物和两种CC-趋化因子。在这些肽和蛋白质中的硫代脯氨酸替换没有导致改变的生物活性。通过消除保护基方案和偶联试剂，串联连接的多个游离肽段在水溶液中提高了蛋白质合成的范围，并可能提供一个有用的方法组合段合成。
Tandem ligation for the synthesis and modification of proteins entails forming two or more regiospecific amide bonds of multiple free peptide segments without a protecting-group scheme. We here describe a semi-orthogonal strategy for ligating three unprotected peptide segments, two of which contain N-terminal (NT) cysteine, to form in tandem two amide bonds, an Xaa-SPro (thiaproline), and then an Xaa-Cys. This strategy exploits the strong preference of an NT-cysteinyl peptide under acidic conditions to undergo selectively an SPro-imine ligation rather than a Cys-thioester ligation. Operationally, it was performed in the N → C direction, first by an imine ligation at pH < 3 to afford an Xaa-thiazolidine ester bond between a peptide containing a carboxyl terminal (CT)-glycoaldehyde ester and a second peptide containing both an NT-Cys and a CT-thioester. The newly createdO-ester-linked segment with a CT-thioester was then ligated to another NT-cysteinyl peptide through thioester ligation at pH > 7 to form an Xaa-Cys bond. Concurrently, this basic condition also catalyzed theO,N-acyl migration of an Xaa-thiazolidine ester to the Xaa-SPro bond at the first ligation site to complete the tandem three-segment ligation. Both ligation reactions were performed in aqueous buffered solvents. The effectiveness of this three-segment ligation strategy was tested in six peptides ranging from 19 to 70 amino acids, including thiaproline → proline analogues of somatostatins and two CC-chemokines. The thiaproline replacements in these peptides and proteins did not result in altered biological activity. By eliminating the protecting-group scheme and coupling reagents, tandem ligation of multiple free peptide segments in aqueous solutions enhances the scope of protein synthesis and may provide a useful approach for combinatorial segment synthesis.