Small molecule fluorophores are critical for fluorescence spectroscopy, and enable the measurement of a wide array of biological processes and dynamics. For many biological sensing applications, the overall efficacy of the fluorophore is closely correlated to the strength of the fluorophore’s excited state dipole. Structurally, this is achieved by designing electronically asymmetric dyes having a donor-π-acceptor (D- π -A) molecular architecture. The electron donating and accepting substituents are chosen to maximize mesomeric effect differences, thus facilitating intramolecular charge-transfer (ICT), while also balancing application-specific requirements. In biosensing for example, donor and acceptor substituents may also be chosen based on water-solubility, toxicity, and specificity considerations. Also important, is the planarity, rigidity, and stability conferred by the π-conjugated bridge. These structural features of the π-bridge ensure strong π-π overlap, inhibited rotation, and minimal degradation, thus enabling strong absorbance, large quantum yields, and enhanced photostability – key requirements for sensing applications. One such class of π-bridge moieties demonstrating structure-function characteristics ideal for fluorophores is a class of heterocycles known as thiazolo[5,4-d]thiazoles (TTz). TTzs are fused, bicyclic heteroaromatic compounds whose unique molecular structure ensures planarity, rigidity, and stability. As such, reported is a novel mixed-pot synthetic approach for accessing D- π -A TTz fluorophores in a single step. The mixed-pot synthesis is modified from the classic literature approach by incorporating additional aromatic aldehydes, resulting in a product distribution of two symmetric TTzs and one asymmetric TTz (a-TTz). A first generation of TTz fluorophores were synthesized, photophysically characterized, and assessed for their solvent, temperature, pH, and membrane voltage (VM) sensing capabilities. These first-generation a-TTzs showed promising VM sensitivity, good membrane localization, negligible cytotoxicity, and excellent photostability compared to current-state-of-the art voltage sensitive dyes (VSDs). Given the promising results of the first-generation dyes, a second generation of a-TTzs VSDs were designed specifically to increase voltage sensitivity, water-solubility, and specificity. Additionally reported, are a series of computational and analytical studies designed to probe mechanistic and thermochemical understandings of the novel mixed-pot TTz reaction. These studies provide compelling evidence for a new mechanism previously unconsidered within the literature. Furthermore, kinetic and thermodynamic differences between products and probable intermediates are elucidated in a detailed reaction coordinate (RC) diagram. Combined with reaction studies under both aerobic and anaerobic conditions, the RC diagram provides insight into how thermodynamic control can be achieved so that the mixed-pot product distribution trends more favorably towards high a-TTz yields.
