Ethoxy Meso-Modified Heptamethine Cyanine Fluorophores: Synthesis, Photophysical Properties and BSA Sensing Study

Heptamethine cyanine fluorophores are widely used in NIR imaging, due to their excellent photophysical properties. In the present work, new meso-ethoxy substituted heptamethine carbocyanine fluorophores 6a-c were prepared by replacing the meso-chlorine atom on a cyclohexenyl-bridged scaffold through a base-driven S_RN1 substitution reaction with an ethoxy group. Once isolated, the compounds were examined in several solvents to study how strongly the medium influenced their absorption, emission, and brightness. The fluorophores absorbed in the 755-770 nm range and showed their strongest fluorescence in ethanol, whereas their emissions were heavily quenched in buffer solutions. Calculations of the HOMO and LUMO energies supported the observed data. A comparison with indocyanine green (ICG) under continuous light exposure showed that fluorophores 6a-c degraded more slowly and maintained their absorbance for a considerably longer period. Having long hydrophobic alkyl chains raised the hypothesis that they could act as sensors for bovine serum albumin (BSA), which is known for its hydrophobic pockets. Therefore, they were tested for BSA binding both in silico and in cuvette. Fluorophore 6b produced a clear optical response when BSA was added, giving both a red shift and an increase in absorbance and fluorescence. Quantitative analysis of the fluorophore 6b-BSA interaction revealed a dissociation constant (K_d) of 0.75 μM and an apparent binding stoichiometry of approximately 1:1 (Hill coefficient n = 1.25), confirming high-affinity, single-site binding. Using the linear range of the fluorescence titration, the limit of detection (LOD) and limit of quantitation (LOQ) for BSA were determined to be 1.9 and 6.0 μM, respectively. Selectivity experiments demonstrated that fluorophore 6b does not exhibit fluorescence enhancement in the presence of collagen or human parvalbumin, confirming its selectivity toward BSA. These observations suggest that introducing an alkoxy group at the meso-position can improve carbocyanine fluorophores properties and achieve high photostability, with significant potential for various biomedical applications.