Proteins incorporating artificial moieties such as fluorophores and drugs have enjoyed increasing use in chemical biology and drug development research. Preparation of such artificial protein derivatives has relied mainly on native chemical ligation in which peptide/protein thioesters chemoselectively react with N-terminal cysteine (Cys) peptides to afford protein molecules. The protein thioesters derived from expressed proteins represent thioesters that are very useful for the preparation of artificial proteins by native chemical ligation with synthetic peptides with N-terminal Cys. We recently have developed a traceless thioester-producing protocol using carboxypeptidase Y (CPaseY) which is compatible with an expressed protein. The traceless character is ensured by CPaseY-mediated hydrazinolysis of C-terminal Xaa (X)-Cys-proline (Pro)-leucine (Leu)-OH sequence followed by an auto-processing of the Cys-Pro (CP) dipeptide unit, affording the corresponding X-thioester (X-SR). However, hydrazinolysis of the amide bond in the prolyl leucine junction depends significantly on the nature of X. In the case of hydrophobic X residues, the hydrazinolysis overreacts to give several hydrazides while the reaction of hydrophilic X residues proceeds slowly. In this research, we attempted to develop an X-independent CPaseY-mediated protocol and found that the incorporation of a triple CP sequence into the C-terminal end (X-(CP)₃-Leu-OH) allows for efficient X-SR formation in a manner that is independent of X.

结合了人工部分（例如荧光团和药物）的蛋白质在化学生物学和药物开发研究中的使用越来越多。这种人工蛋白质衍生物的制备主要依赖于天然化学连接，其中肽/蛋白质硫酯与N-末端半胱氨酸（Cys）肽化学选择性反应以提供蛋白质分子。衍生自表达蛋白质的蛋白质硫酯代表硫酯，通过与具有N端Cys的合成肽进行天然化学连接，对于制备人工蛋白质非常有用。我们最近开发了一种使用羧肽酶Y（CPaseY）的无痕硫代酯生产协议，该协议可与表达的蛋白质相容。CPaseY介导的C末端Xaa（X）-Cys-脯氨酸（Pro）-亮氨酸（Leu）-OH序列，然后自动处理Cys-Pro（CP）二肽单元，得到相应的X-硫代酯（X- SR ）。然而，脯氨酰亮氨酸连接中酰胺键的肼解作用很大程度上取决于X的性质。在疏水性X残基的情况下，肼解反应过度反应，得到几种酰肼，而亲水性X残基的反应进行缓慢。在这项研究中，我们尝试开发一种独立于X的CPaseY介导的协议，并发现将三重CP序列并入C末端（X-（CP）₃ -Leu-OH）允许以独立于X的方式有效地形成X -SR 。