Facile Synthesis of H‐L‐Photo‐Lysine and Its Genetic Incorporation Into Proteins

Photocrosslinking has emerged as a powerful tool for studying protein interactions in physiologically relevant conditions. Among various photoreactive groups, diazirine-based noncanonical amino acids (ncAAs) are widely used due to their small size, stability, and high reactivity upon UV irradiation. Here, we report a convenient synthesis of H-L-photo-lysine (photoK), a diazirine-containing ncAA. The method offers improved yields, milder reaction conditions, and simplified purification. The synthetic photoK was successfully incorporated into a GFP variant via genetic code expansion, demonstrating its utility in protein labeling. Additionally, photoK was applied to study SIRT6, a human sirtuin with critical roles in genome stability, metabolism, and disease, yet with limited known substrates. Using a photoactivatable SIRT6K15photoK mutant, we confirmed its UV-dependent crosslinking with histone H3, a known substrate. Application of SIRT6K15photoK in HEK293 cell lysates enabled the capture of potential physiological SIRT6 interaction partners. Photocrosslinking was also performed in Escherichia coli expressing SIRT6K15photoK. UV irradiation induced formation of protein conjugates, indicative of specific interactions with cellular binding partners. Importantly, pretreatment with a SIRT6-selective inhibitor substantially reduced crosslinking, confirming interaction specificity. This approach provides a robust strategy for unbiased substrate profiling of SIRT6 and highlights the application of diazirine-containing photocrosslinking in expanding the interactome of under-studied enzymes.