Combinatorial Strategy to Identify Fluorescent Probes for Biothiol and Thiophenol Based on Diversified Pyrimidine Moieties and Their Biological Applications

We present a feasible paradigm of developing original fluorescent probes for target biomolecules via combinatorial chemistry. In this developmental program, pyrimidine moieties were investigated and optimized as unique recognition units for thiols for the first time through a parallel synthesis in combination with a rapid screening process. This time-efficient and cost-saving process effectively facilitated the developmental progress and provided detailed structure–reactivity relationships. As a result, Res-Biot and Flu-Pht were identified as optimal fluorescent probes for biothiol and thiophenol, respectively. Their favorable characteristics and superior applicability have been well demonstrated in both chemical and biological contexts. In particular, Res-Biot enables the direct visualization of biothiol fluctuations during oxidative stress and cell apoptosis, indicating its suitability in elucidation of a specific pathophysiological process in both living cells and living animals. Meanwhile, Flu-Pht is competent to visualize thiophenols without the interference from endogenous biothiols in living cells.