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Chemical Synthesis of Proteins with Non‐Strategically Placed Cysteines Using Selenazolidine and Selective Deselenization
Author(s) -
Reddy Post Sai,
Dery Shahar,
Metanis Norman
Publication year - 2016
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201509378
Subject(s) - native chemical ligation , chemical ligation , cysteine , selenocysteine , alanine , chemistry , histidine , peptide , residue (chemistry) , ligation , active site , amino acid , biochemistry , stereochemistry , enzyme , biology , microbiology and biotechnology
Abstract Although native chemical ligation has enabled the synthesis of hundreds of proteins, not all proteins are accessible through typical ligation conditions. The challenging protein, 125‐residue human phosphohistidine phosphatase 1 (PHPT1), has three cysteines near the C‐terminus, which are not strategically placed for ligation. Herein, we report the first sequential native chemical ligation/deselenization reaction. PHPT1 was prepared from three unprotected peptide segments using two ligation reactions at cysteine and alanine junctions. Selenazolidine was utilized as a masked precursor for N‐terminal selenocysteine in the middle segment, and, following ligation, deselenization provided the native alanine residue. This approach was used to synthesize both the wild‐type PHPT1 and an analogue in which the active‐site histidine was substituted with the unnatural and isosteric amino acid β‐thienyl‐ l ‐alanine. The activity of both proteins was studied and compared, providing insights into the enzyme active site.