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

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 k(2) for various proposed bioorthogonal cycloadditions were estimated to be in the range from 0.9 x 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, k(2) = 1.5 x 10(-3) M-1 s(-1)) and comparable or better to that for the second generation TQ-ligation (k(2) = 2.8 x 10(-2) M-1 s(-1)). The reaction rate constants k(2) of proposed ligation reactions are in the range of the rate constants k(2) for tetrazines and norbornenes or tetrazines and cyclopropenes. These findings indicate that this chemistry is suitable for in vitro imaging experiments.

Automated summary

A new bioorthogonal cycloaddition reaction has been developed for imaging inside living cells, which is high yielding, selective, and fast in aqueous media. The proposed reaction can selectively and rapidly label cancer cells, making it suitable for in vitro imaging experiments.