Electrochemical Tryptophan-Selective Bioconjugation in Neutral Buffer via Cooperative <i>N</i> -Oxyl Radicals

Protein modifications targeting canonical, hydrophobic amino acid residues remain scarce under neutral, transition metal-free, and biocompatible conditions. Here, we report an electrochemical tryptophan (Trp)-selective bioconjugation that proceeds in a neutral buffer without external oxidants. The method relies on a cooperative dual N -oxyl radical manifold, employing keto-ABNO as the Trp-reactive reagent and 4-oxo-TEMPO as an electron mediator, together with NaBr, which suppresses overoxidation and radical degradation while maintaining low operating potentials. Under the optimized conditions, diverse peptides and proteins are efficiently labeled. Systematic cyclic voltammetry (CV) and UV–visible absorption spectroelectrochemistry (UV/Vis-SEC) suggest a Trp···keto-ABNO preassociation that lowers the oxidation threshold and reveal distinct roles of the two radicals, while Br – preferentially stabilizes the oxoammonium species rather than mediating redox processes. This neutral-buffer, electrocatalytic platform provides a practical route to Trp-selective labeling under physiologically compatible conditions.