Fig. 1: Background information of bioorthogonal reaction and development of MAAD reaction.

a Typical bioorthogonal reactions: Staudinger ligation, copper-catalyzed azide-alkyne cycloaddition, strain-promoted [3 + 2] cycloaddition, and tetrazine ligation. b MAAD reaction reported in this article which was applied to nucleic acid, proteins, and cells. c The proposed mechanism of the MAAD reaction shows distinct reactivity through a concerted transition state. d The efficiency of MAAD reaction in a variety of solvents highlights its fast rate in aqueous solvent. The online infrared experiments (FTIR) confirmed that the reaction completes within 90 seconds under standard conditions (THF, 25 °C, 0.1 M). e The substrate scope of MAAD reaction shows its efficiency and compatibility toward functional groups. The structure of product P3 was determined by X-ray analysis. f Reaction profiles under varying concentrations of M1 and A1. The reaction progress was monitored by in situ FT-IR based on the formation of P1 (1752 cm^-1). Left: concentrations of M1 (0.085–0.11 M). Right: concentrations of A1 (0.09–0.11 M). Increasing the concentrations of either M1 or A1 resulted in faster product formation, consistent with second-order kinetics. g Control experiments assessing the impact of biologically relevant additives on the MAAD reaction. M1 (100 μM) and A1 (100 μM) were reacted in PBS/DMSO (20: 1, v/v) at 25 °C with or without additives. For all examined conditions, the yields of P1 were >99%, indicating the tolerance of the MAAD reaction to ionic and biomolecular components. FT-IR Fourier transform infrared spectroscopy, Bn benzyl, ⁱPr iso-propyl, ^tBu tert-butyl.