An improved installation of 2-hydroxy-4-methoxybenzyl (iHmb) method for chemical protein synthesis

The 2-hydroxy-4-methoxybenzyl (Hmb) backbone modification can prevent amide bond-mediated sidereactions (e.g., aspartimide formation, peptide aggregation) by installing the removable Hmb group into a peptide bond, thus improving the synthesis of long and challenging peptides and proteins. However, its use is largely precluded by the limited Hmb's installation sites. In this report, an improved installation of Hmb (iHmb) method was developed to achieve the flexible installation and the convenient removal of Hmb. The iHmb method involves two critical steps: (1) oxidative diazotization of the readily installed 2-hydroxy-4-methoxy-5-amino-benzyl (Hmab) to give 2-hydroxy-4-methoxy-5-diazonium-benzyl (Hmdab) by combining soamyl nitrite (IAN)/HBF4, and (2) reductive elimination of Hmdab to give the desired Hmb by 1,2-ethanedithiol (EDT). The iHmb method enables the installation of Hmb at any primary amino acid including the highly sterically hindered amino acids (e.g., valine and isoleucine). The practicality and utility of the iHmb method was demonstrated by one-shot solid -phase synthesis of a challenging aspartimide-prone peptide, the mirror-image version of a hydrophobic peptide and a long-chain peptide up to 76-residue. Furthermore, the iHmb method can be utilized to facilitate chemical protein ligation, as exemplified by the synthesis of the single-spanning membrane protein sarcolipin. The iHmb method expands the toolkit for peptide synthesis and ligation and facilitates the preparation of peptides/proteins. (c) 2024 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.