Effect of biological confinement on the photophysics and dynamics of a proton-transfer phototautomer: an exploration of excitation and emission wavelength-dependent photophysics of the protein-bound drug.

The present work demonstrates the effect of biological confinement on the photophysics and dynamics of a bio-active drug molecule viz., 5-chlorosalicylic acid (5ClSA). 5ClSA is a potential candidate exhibiting Excited-State Intramolecular Proton Transfer (ESIPT) reaction and thereby generating the phototautomer (i.e. proton transferred keto form) in the excited state. Given the pK(a) of 5ClSA (around 2.64), the anionic form of the drug molecule is expected to be the interacting species with the protein under the experimental conditions (buffered solution of pH 7.40). The ESIPT photophysics of the drug (5ClSA anion) is found to be remarkably modified within the confined bio-environment of a model transport protein Bovine Serum Albumin (BSA) in terms of remarkable emission intensity enhancement coupled with a discernible red-shift of the emission maximum wavelength. Such considerable modification of the ESIPT photophysics of the 5ClSA anion has been exploited to determine the drug-protein binding strength (as characterized by the binding constant K (±10%) = 6.11 × 10(2) M(-1)). The present work also delves into evaluation of the probable binding location of the drug within the biomacromolecular assembly of the protein by a blind docking simulation technique, which reveals hydrophobic subdomain IIA to be the probable binding site of the drug. Circular dichroism (CD) spectroscopy delineates the effect of drug binding on the protein secondary structure in terms of decrease of α-helical content of BSA with increasing drug concentration. Apart from this, the excitation-emission matrix fluorescence technique is found to hint at the effect on protein tertiary structure upon binding to the drug. Chaotrope-induced protein denaturation has been explored to complement the findings on the binding interaction process. The modulated dynamics of the proton transfer phototautomer of the 5ClSA anion within the biological confinement is also investigated in this context to explore the slower rate of solvent-relaxation dynamics.