On-demand protein modification: Molecular alteration only when needed — The University of Tokyo develops new technology

A research group led by Assistant Professor Yuki Yamanashi, Graduate Student Menghan Xu, Associate Professor Shigehiro A. Kawashima, and Professor Motomu Kanai from the Graduate School of Pharmaceutical Sciences at the University of Tokyo has developed a new technology to selectively modify proteins by generating highly reactive isocyanates on demand. The results were published in the Journal of the American Chemical Society.

In biological systems, various molecules including proteins interact with each other to control diverse biological phenomena. Bioconjugation, which chemically modifies these molecules, is an important technology for analyzing molecular dynamics and controlling functions. By connecting fluorescent tags to proteins within cells or living organisms, their localization and interactions can be visualized. Additionally, by forming covalent bonds with target proteins, bioconjugation is applied as covalent drugs that more potently inhibit protein functions. Furthermore, bioconjugation plays an important role in the precise synthesis of biopharmaceuticals such as antibody-drug conjugates (ADCs).

Ideal bioconjugation requires high reactivity to complete reactions quickly, but chemical species that are too reactive pose challenges for practical application as they react non-specifically with water and cellular components.

Isocyanates are an ideal chemical species showing high reactivity with a small molecular size. However, due to their excessive reactivity that causes decomposition within tens of seconds in aqueous solution, their application to bioconjugation has rarely been achieved. The research group developed a novel reaction that generates isocyanates on demand by adding sulfonyl fluoride (activating agent) to stable hydroxamic acid-pyrrolidine compounds (isocyanate precursors).

To accelerate the reaction, they utilized reversible boronic ester formation between boronic acid and diol. This enabled efficient delivery of the activating agent near the isocyanate precursor, achieving rapid protein modification through reactions that complete within minutes in in vitro experiments using model proteins. Additionally, by using isocyanate precursors linked to ligand molecules that bind to target proteins, they demonstrated the ability to selectively label target proteins not only in test tubes but also in cell extracts and living cells.

This study demonstrated a new concept of chemical design: "to generate highly reactive isocyanates only when needed, activation is performed using stable precursors of isocyanates." This is expected to become a foundational technology for future bioconjugation methods and pharmaceutical development.

Journal Information
Publication: Journal of the American Chemical Society
Title: Induced Bioconjugation via On-Demand Isocyanate Formation
DOI: 10.1021/jacs.5c11603