Application of bioorthogonal hetero-Diels–Alder cycloaddition of 5-arylidene derivatives of 1,3-dimethylbarbituric acid and vinyl thioether for imaging inside living cells

Abstract

New bioorthogonal cycloaddition of 5-arylidene derivatives of 1,3-dimethylbarbituric acid as 1-oxa-1,3-butadienes and vinyl thioether as a dienophile has been applied to imaging inside living cells. The reaction is high yielding, selective, and fast in aqueous media. The proposed 1-oxa-1,3-butadiene derivative conjugated to a FITC fluorochrome selectively and rapidly labels the cancer cells pretreated with the dienophile-taxol. The second order rate constants k₂ for various proposed bioorthogonal cycloadditions were estimated to be in the range from 0.9 × 10⁻² M⁻¹ s⁻¹ to 1.4 M⁻¹ s⁻¹, which is much better than in the case of the first generation TQ-ligation (o-quinolinone quinone methide and vinyl thioether ligation, k₂ = 1.5 × 10⁻³ M⁻¹ s⁻¹) and comparable or better to that for the second generation TQ-ligation (k₂ = 2.8 × 10⁻² M⁻¹ s⁻¹). The reaction rate constants k₂ of proposed ligation reactions are in the range of the rate constants k₂ for tetrazines and norbornenes or tetrazines and cyclopropenes. These findings indicate that this chemistry is suitable for in vitro imaging experiments.