Expanding the Versatility of Microbial Transglutaminase Using α‐Effect Nucleophiles as Noncanonical Substrates

Abstract

The substrate promiscuity of microbial transglutaminase (mTG) has been exploited in various applications in biotechnology,inparticular for the attachment of alkylamines to glutamine-containing peptides and proteins.H ere,w e expand the substrate repertoire to include hydrazines,h ydrazides,a nd alkoxyamines,resulting in the formation of isopeptide bonds with varied susceptibilities to hydrolysis or exchange by mTG.F urthermore,w ed emonstrate that simple unsubstituted hydrazine and dihydrazides can be used to install reactive hydrazide handles onto the side chain of internal glutamine residues.The distinct hydrazide handles can be further coupled with carbonyls,i ncluding ortho-carbonylphenylboronic acids, to form site-specific and functional bioconjugates with tunable hydrolytic stability.T he extension of the substrate scope of mTG beyond canonical amines thus substantially broadens the versatility of the enzyme,providing anew approach to facilitate novel applications. Scheme 1. Transamidation reaction catalyzed by microbial transglutaminase (mTG) using an amine or an a-effect nucleophile a) hydrazine, b) hydrazide, or c) alkoxyaminea sthe acyl acceptor substrate.