Design and Photophysical Investigation of a Regioisomeric Bis[2-(2'-hydroxyphenyl)benzoxazole] Derivative for Sensing Applications.

The development of fluorescent probes with large Stokes' shifts and high sensitivity remains a critical objective in chemical sensing and bioimaging. In this study, we report the design, synthesis, and comprehensive photophysical characterization of a novel regioisomeric bis(2-(2'-hydroxyphenyl)benzoxazole) scaffold, bis(HBO) 3, expanding the limited family of ESIPT-enabled bis(HBO) systems. This newly developed probe integrates two HBO units within a distinct C2'-symmetric framework, enabling systematic investigation of regio-effects on excited-state intramolecular proton transfer (ESIPT) behavior and analyte responsiveness. Photophysical studies reveal that bis(HBO) 3 exhibits characteristic ESIPT-driven emission with a large Stokes' shift (Δλ ≈ 160-180 nm) and solvent-independent fluorescence centered around λem ≈ 530 nm. In-depth spectroscopic analysis, including low-temperature fluorescence measurements, demonstrates the presence of multiple emissive keto rotamers, providing insight into the role of conformational dynamics in modulating emission behavior. Compared with previously reported bis(HBO) analogues 1 and 2, bis(HBO) 3 displays distinct emission features due to a pronounced regio-effect. Bis(HBO) 3 exhibited selective responsiveness toward several chemical analytes. The probe shows high stability under acidic conditions but undergoes deprotonation in the presence of strong bases and certain metal ions, particularly Cu(II), Ni(II) and Fe(II), resulting in pronounced spectral changes. Notably, bis(HBO) 3 exhibits a distinct and sensitive response toward fluoride anions, characterized by the emergence of a new absorption band (~ 415 nm) and a blue-shifted emission (~ 485 nm). Quantitative analysis using the Benesi-Hildebrand method confirms a 1:1 binding stoichiometry with moderate binding affinity, distinguishing its sensing behavior from previously reported bis(HBO) systems that exhibit 1:2 binding stoichiometry. Overall, this work summarizes bis(HBO) 3 as a unique ESIPT-based fluorescent skeleton and highlights the critical role of molecular symmetry and regio-effects in governing photophysical and sensing properties.