文章基本信息

标题：Sequence-Independent Traceless Method for Preparation of Peptide/Protein Thioesters Using CPaseY-Mediated Hydrazinolysis
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作者：Masahiro Ueda ; Chiaki Komiya ; Sayuki Arii 等
期刊名称：Chemical and Pharmaceutical Bulletin
印刷版ISSN：0009-2363
电子版ISSN：1347-5223
出版年度：2020
卷号：68
期号：12
页码：1226-1232
DOI：10.1248/cpb.c20-00674
出版社：The Pharmaceutical Society of Japan
摘要：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 .
关键词：carboxypeptidase;hydrazinolysis;native chemical ligation;protein semi-synthesis;protein thioester;thioesterification