Microbial transglutaminase (mTG) is known to accept a wide range of primary amine substrates. This substrate promiscuity underlies the utility of mTG as a tool to equip proteins with diverse functional moieties. In one prominent example, the enzyme has been employed for the preparation of site-specific antibody-drug conjugates for pharmaceutical application. Here, using an engineered trastuzumab antibody as our protein for demonstration, we show that in addition to amine-functionalized payloads, mTG can also recognize hydrazine, hydrazide and alkoxyamine as substrates, resulting in products with varied susceptibility to enzyme-mediated hydrolysis or exchange. Furthermore, we show that simple unsubstituted hydrazine and dihydrazides can be used to install distinct hydrazide functionalities onto the protein. The introduced reactive handle can subsequently couple with carbonyls, including the fast variants based on <i>ortho</i>-carbonylphenylboronic acids, to form site-specific and functional bioconjugates with tunable hydrolytic stability. The extension of the substrate scope of mTG beyond the canonical amine thus broadens the versatility of the enzyme and enables it to partake in novel opportunities in biotechnology.

α-Effect nucleophiles as noncanonical substrates for microbial transglutaminase

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.