Regioselective rapid ene-type reaction (RRER) enables bioconjugation of histone serotonylation

Triazolinedione (TAD) derivatives have been commonly utilized as protection and labeling reagents for indole and phenol moieties <i>via</i> a reversible ene-type reaction. Previous studies showed that the TAD probes could selectively modify tyrosine and tryptophan side-chains within proteins and peptides under distinct pH conditions. Here, we report a pH-controlled regioselective rapid ene-type reaction (RRER) between TAD and 5-hydroxyindole, where the modification occurs on the C4 position rather than the C3 of inactivated indole rings. Employing this unique reaction, we have performed the selective bioconjugation of serotonylation occurring on the fifth amino acid residue, glutamine, of histone H3 (H3Q5), which does not contain any tryptophan in its protein sequence. Finally, RRER was applied to determine the H3Q5 serotonylation levels in cultured cells and tissue samples, which served as a newly developed powerful tool for <i>in vitro</i> and <i>in vivo</i> histone monoaminylation analysis. Overall, our findings in this research expanded the chemical biology toolbox for investigating histone monoaminylation and facilitated the understandings of TAD-mediated ene-type reactions.