Heterobifunctional cross-linker with dinitroimidazole and azide modules for protein and oligonucleotide functionalization†

Abstract

Dinitroimidazole (DNIm) was recently identified as a powerful bioconjugation agent that could selectively modify thiol over amine on biomolecules at an ultrahigh speed in an aqueous buffer. However, its derivative containing a DNIm module and a terminal alkyne module failed to construct functional agents bearing a DNIm warhead via the CuAAC reaction. To solve this problem, a heterobifunctional cross-linker was designed and synthesized by linking a DNIm module with an azide module via an oxoaliphatic amido bond spacer arm. Its two modules, DNIm and azide, reacted with a thiol and cyclooctyne, respectively, in an orthogonal way. The cross-linker facilitated the preparation of various functional agents bearing a DNIm warhead via SPAAC reaction and was further applied to protein functionalization (including biotinylation and fluorescence labeling) and oligonucleotide functionalization (including PEGylation, oligonucleotide–peptide and oligonucleotide–protein conjugate). Thus, the cross-linker not only provided convenient access to those functional agents bearing a DNIm warhead but also combined DNIm chemistry with click chemistry of SPAAC to enlarge their respective application range in the bioconjugation field.