Triarylmethane Fluorophores Resistant to Oxidative Photobluing.

Alexey N Butkevich,Mariano L Bossi,Gražvydas Lukinavičius,Stefan W Hell

doi:10.1021/jacs.8b11036

Abstract

Spectral stability of small-molecule fluorescent probes is required for correct interpretation and reproducibility of multicolor fluorescence imaging data, in particular under high (de)excitation light intensities of super-resolution imaging or in single-molecule applications. We propose a synthetic approach to a series of spectrally stable rhodamine fluorophores based on sequential Ru- and Cu-catalyzed transformations, evaluate their stability against photobleaching and photoconversion in the context of other fluorophores using chemometric analysis, and demonstrate chemical reactivity of fluorophore photoproducts. The substitution patterns providing the photoconversion-resistant triarylmethane fluorophores have been identified, and the applicability of nonbluing labels in live-cell STED nanoscopy is demonstrated.

Highlights

With the recent developments of fluorescence nanoscopy methods,[1] it has become possible to observe and study nanoscale structures on the cell surface[2] and within the cell interior[3] with the help of advanced optical microscopes
We propose a synthetic approach to a series of spectrally stable rhodamine fluorophores based on sequential Ru- and Cu-catalyzed transformations, evaluate their stability against photobleaching and photoconversion in the context of other fluorophores using chemometric analysis, and demonstrate chemical reactivity of fluorophore photoproducts
To evaluate the photobluing resistance of N,N′di-tert-alkylrhodamines, we had first to address the problem of their synthetic accessibility

Summary

■ INTRODUCTION

With the recent developments of fluorescence nanoscopy (super-resolution microscopy) methods,[1] it has become possible to observe and study nanoscale structures on the cell surface[2] and within the cell interior[3] with the help of advanced optical microscopes. As the ensuing amine radical cation can undergo similar N-dealkylation, the suppression of the TICT pathway, besides the improved photostability and fluorescence quantum yield, will reduce photobluing This approach was first introduced by Foley[21] and later developed by Lavis12b and Liu and Xu22 who proposed, respectively, to substitute the freely rotating N,N-dialkylamino groups in rhodamine-type fluorophores with bridged 7-azabicyclo[2.2.1]heptane, azetidine, or aziridine substituents (Figure 1b). In a recent work by Zhu and Wan,[33] an Ullmann-type amination of aryl bromides and iodides with secondary and primary amines, including 1-aminoadamantane, under exceptionally mild conditions (5 mol % of Cu catalyst, K3PO4 base in diethylene glycol, rt to 60 °C) has been reported Employing this catalytic system, upon optimization (Table S3) we were able to achieve double amination with primary N-tertalkylamines on a diverse variety of 3′,6′-dibromo- and 3′,6′diiodofluorans prepared as described above (Figure 3). The spectrally stable SiR analog 4b-Halo demonstrated similar image quality to SiR-Halo[36] in terms of target selectivity, contrast, background, and resolution in 775 nm live-cell STED nanoscopy

■ CONCLUSION

■ ACKNOWLEDGMENTS

■ REFERENCES