Structure–Activity Relationships of Designed <i>α</i> ‐Amino Hydroxamic Acids for Affinity Labeling via In Situ Isocyanate Generation

Target identification of bioactive compounds is of significance in life sciences, ranging from molecular biology to drug development. Photoaffinity labeling (PAL), which utilizes ultraviolet (UV) light irradiation to generate a reactive species for covalent bond formation, is the gold standard method for labeling the binding target. However, requirements for UV light irradiation, which can potentially cause denaturation of biomolecules, and uncontrollable reactivity, resulting in nonproductive consumption of the active species, necessitate further improvement of the affinity labeling methodology. Here, we report our studies on the in situ generation of isocyanate from an α-amino hydroxamic acid and a sulfonyl fluoride for affinity labeling. Theoretical and experimental mechanistic studies of the reaction using various α-amino hydroxamic acid derivatives provided a design principle for efficient isocyanate formation. The best α-amino hydroxamic acid showed higher covalent bond-forming efficiency than PAL in model protein modifications.