2-Amino-6-cyanopurines as Novel Fluorescent Probes for Functional Live Imaging of Yeast Vacuoles

Fluorescent probes are vital tools for visualising biological systems. While fluorescent nucleobase analogues (FNAs) traditionally serve as nucleic acid sensors, their inherent biomimetic scaffold might offer a versatile fluorescent platform for targeted cellular visualisation by tuning their structural and spectral characteristics. Here, five 2-amino-6-cyanopurines picked from our pipeline of FNA-like molecules were tested for bioimaging applications in Saccharomyces cerevisiae and proved to be nontoxic. 2-Amino-9-cyclopentylpurine-6-carbonitrile (compound C) exhibited the highest fluorescence/concentration ratio among all tested compounds. C accumulates inside the vacuoles of living S. cerevisiae, as confirmed via colocalisation with vacuole-specific fluorescent markers. Notably, compound C showed higher photostability compared with 7-amino-4-chloromethylcoumarin, the most common blue vacuolar probe. The vacuolar labelling by C persisted in yeasts submitted to hyperosmotic stress, as well as in aerobic and anaerobic conditions, but adopted a nonspecific/diffuse pattern in fixed cells, under conditions of low energy metabolism, and after treatment with protonophores. Interestingly, the staining pattern of C remained unaffected upon inhibition of the V-ATPase activity or mutation of its V₀ subunit. This first demonstration that modified FNAs, here described as FNA-like molecules, can target intracellular organelles beyond nucleic acid chemistry opens new avenues for their use in bioimaging in living cells.