Selective Chemical Modification of Tyrosine Residues: Development of Probes and Application in Altering Enzyme Substrate Specificity

In recent years, labeling of proteins with tyrosine (Tyr) residues has garnered significant attention owing to its selectivity and user-friendliness. Cookson’s reagent (4-phenyl-1,2,4-triazoline-3,5-dione: PTAD) is used as a protective group for the conjugated dienes because it reacts with them in a Diels–Alder reaction. Its fluorescent derivative has also been used as a derivatizing agent in liquid chromatography to quantify conjugated diene compounds. Recently, PTAD was reported to selectively react with Tyr via an ene reaction under physiological conditions. The product of the ene reaction with Tyr possesses a rotational axis at the binding site. Thus, the product obtained by modifying intrinsically fluorescent Tyr with fluorescent Cookson’s reagent may exhibit properties similar to those of thioflavin T. Thioflavin T recognizes β-sheet structures and is widely used for amyloid fibril detection and aggregate monitoring. Whereas PTAD and its derivatives have also been used for Tyr-specific modifications in various protein studies, such as for site-selective modification of antibodies and analysis of higher-order protein structures. In particular, the ene reaction at the Tyr residue in antibodies, such as trastuzumab, does not affect the antigen-binding ability of the drug. Therefore, we aimed to utilize this Tyr-selective ene reaction to visualize biomolecular functions and apply it to control enzyme functions. This review describes the characterization of the ene reaction products of Cookson-type fluorescent reagents and the effects of Tyr modification on insulin-degrading enzymes in terms of substrate specificity.