Mechanistical Insights into the Bioconjugation Reaction of Triazolinediones with Tyrosine

The bioconjugation at tyrosine residues using cyclic diazodicarboxamides, especially 4-substituted 3H-1,2,4-triazole-3,5(4H)-dione (PTAD), is a highly enabling synthetic reaction because it can be employed for orthogonal and site-selective (multi)functionalizations of native peptides and proteins. Despite its importance, the underlying mechanisms have not been thoroughly investigated. The reaction can proceed along four distinctive pathways: (i) the SEAr path, (ii) along a pericyclic group transfer pathway (a classical ene reaction), (iii) along a stepwise reaction path, or (iv) along an unusual higher order concerted pericyclic mechanism. The product mixtures obtained from reactions of PTAD with 2,4-unsubstituted phenolate support the SEAr mechanism, but it remains unclear if other mechanisms also take place. In the present work, the various mechanisms are compared using high-level quantum chemistry approaches for the model reaction of 4H,3H-1,2,4-triazole-3,5(4H)-dione (HTAD) with p-cresol and p-cresola...