High photostability in non-conventional coumarins with far-red/NIR emission through azetidinyl substitution

Abstract

Replacement of electron-donating N,N-dialkyl groups with three or four-membered cyclic amines (e.g., aziridine and azetidine, respectively) has been described as a promising approach to improve some of the drawbacks of conventional fluorophores, including low fluorescent quantum yields (F) in polar solvents. In this work we have explored the influence of azetidinyl substitution on non-conventional coumarin-based COUPY dyes. Two azetidine-containing scaffolds were first synthesized in four linear synthetic steps and easily transformed into far-red/NIR-emitting fluorophores through N-alkylation of the pyridine moiety. Azetidine introduction in COUPY dyes resulted in enlarged Stokes' shifts with respect the N,N-dialkylamino-containing parent dyes, but the F were not significantly modified in aqueous media, which is in contrast with previously reported observations in other fluorophores. However, azetidinyl substitution led to an unprecedented improvement in the photostability of COUPY dyes and high cell permeability was retained since the fluorophores accumulated selectively in mitochondria and nucleoli of HeLa cells. Overall, our results provide valuable insights for the design and optimization of novel fluorophores operating in the far-red/NIR region, since we have demonstrated that three important parameters (Stokes' shifts, F and photostability) cannot be always simultaneously addressed by simply replacing a N,N-dialkylamino group with azetidine, at least in non-conventional coumarin-based fluorophores.