Tunable Activated Esters Enable Lysine-Selective Protein\nLabeling and Profiling

Lysine\nresidues on protein surfaces are abundant and often found\nin enzyme active sites, making them critical targets for studying\nundruggable proteins. However, the varied microenvironment surrounding\nlysine residues results in a wide range of p<i>K</i><sub>a</sub> values, complicating site-specific covalent binding. In this\nstudy, we address the challenges posed by the diverse reactivity of\namino side chains by modulating the amide reaction activity of heteroaromatic\nactivated esters. By fine-tuning the type, position, and number of\nheteroatoms, we successfully rationalized the regulation of their\namide reaction activity, leading to the design of probes for selective\nlysine labeling within the proteome for profiling purposes. Systematic\noptimization of these esters’ reactivity and selectivity has\nyielded a series of effective probes suitable for both <i>in\nvitro</i> and cellular applications. These findings significantly\nenhance our understanding of protein functions and mechanisms, facilitated\nby the precise identification and analysis of protein labeling and\nprofiling.