N-Terminal selective modification of peptides and proteins using 2-ethynylbenzaldehydes

Jie-Ren Deng; Nathanael Chun-Him Lai; Karen Ka-Yan Kung; Bin Yang; Sai-Fung Chung; Alan Siu-Lun Leung; Man-Chung Choi; Yun-Chung Leung; Man-Kin Wong

doi:10.1038/s42004-020-0309-y

N-Terminal selective modification of peptides and proteins using 2-ethynylbenzaldehydes

Commun Chem. 2020 May 29;3(1):67. doi: 10.1038/s42004-020-0309-y.

Authors

Affiliations

¹ State Key Laboratory of Chemical Biology and Drug Discovery, and Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hum, Hong Kong, China.
² State Key Laboratory of Chemical Biology and Drug Discovery, and Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hum, Hong Kong, China. karen.kung@polyu.edu.hk.
³ Henry Cheng Research Laboratory for Drug Development and Lo Ka Chung Centre for Natural Anti-Cancer Drug Development, The Hong Kong Polytechnic University, Hung Hum, Hong Kong, China.
⁴ Henry Cheng Research Laboratory for Drug Development and Lo Ka Chung Centre for Natural Anti-Cancer Drug Development, The Hong Kong Polytechnic University, Hung Hum, Hong Kong, China. thomas.yun-chung.leung@polyu.edu.hk.
⁵ State Key Laboratory of Chemical Biology and Drug Discovery, and Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hum, Hong Kong, China. mankin.wong@polyu.edu.hk.

^# Contributed equally.

Abstract

Selective modification of the N-terminus of peptides and proteins is a promising strategy for single site modification methods. Here we report N-terminal selective modification of peptides and proteins by using 2-ethynylbenzaldehydes (2-EBA) for the production of well-defined bioconjugates. After reaction screening with a series of 2-EBA, excellent N-terminal selectivity is achieved by the reaction in slightly acidic phosphate-buffered saline using 2-EBA with electron-donating substituents. Selective modification of a library of peptides XSKFR (X = either one of 20 natural amino acids) by 2-ethynyl-4-hydroxy-5-methoxybenzaldehyde (2d) results in good-to-excellent N-terminal selectivity in peptides (up to >99:1). Lysozyme, ribonuclease A and a therapeutic recombinant Bacillus caldovelox arginase mutant (BCArg mutant) are N-terminally modified using alkyne- and fluorescein-linked 2-EBA. Alkyne-linked BCArg mutant is further modified by rhodamine azide via copper(I)-catalyzed [3 + 2] cycloaddition indicating that the reaction has high functional group compatibility. Moreover, the BCArg mutant modified by 2-ethynyl-5-methoxybenzaldehyde (2b) exhibits comparable activity in enzymatic and cytotoxic assays with the unmodified one.