A cysteine selenosulfide redox switch for protein chemical synthesis

Bérénice Leclercq,Nathalie Ollivier,Vincent Diemer,Hervé Drobecq,Oleg Melnyk,Jérôme Vicogne,Vangelis Agouridas

doi:10.1038/s41467-020-16359-6

Abstract

The control of cysteine reactivity is of paramount importance for the synthesis of proteins using the native chemical ligation (NCL) reaction. We report that this goal can be achieved in a traceless manner during ligation by appending a simple N-selenoethyl group to cysteine. While in synthetic organic chemistry the cleavage of carbon-nitrogen bonds is notoriously difficult, we describe that N-selenoethyl cysteine (SetCys) loses its selenoethyl arm in water under mild conditions upon reduction of its selenosulfide bond. Detailed mechanistic investigations show that the cleavage of the selenoethyl arm proceeds through an anionic mechanism with assistance of the cysteine thiol group. The implementation of the SetCys unit in a process enabling the modular and straightforward assembly of linear or backbone cyclized polypeptides is illustrated by the synthesis of biologically active cyclic hepatocyte growth factor variants.

Highlights

The control of cysteine reactivity is of paramount importance for the synthesis of proteins using the native chemical ligation (NCL) reaction
The NCL reaction is classically performed in the presence of an aryl thiol catalyts[22], of which 4-mercaptophenylacetic acid (MPAA) is considered as the gold standard[23]
The reactivity of the selenoethyl cysteine (SetCys) residue is peculiar in that the loss of the 2selenoethyl arm through carbon–nitrogen bond cleavage proceeds under very mild conditions in water (Fig. 2a)

Summary

Introduction

The control of cysteine reactivity is of paramount importance for the synthesis of proteins using the native chemical ligation (NCL) reaction. SetCys spontaneously loses its selenoethyl arm in water at neutral pH in the presence of popular disulfide bond reductants such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) This chemical behavior contrasts with the known difficulty in breaking carbon–nitrogen bonds, a process that usually requires harsch conditions[17,18], metal catalysis[19], or radical reactions[20,21]. The detailed mechanistic investigations reported here point toward an anionic mechanism that depends on the ionization state of SetCys in its ring-opened and reduced form In this respect, SetCys uncovers an unusual mode of reactivity for Cys and provides a useful means for accessing complex protein scaffolds as illustrated by the total one-pot synthesis of biologically active backbone-cyclized variants of the hepatocyte growth factor (HGF) kringle 1 (K1) domain

Methods

Results

Conclusion