Methyl 3-(2-formyl-5-methoxynaphthalen-1-yl)propanoate as a fluorescent probe for folding and binding studies of human serum albumin

Abstract

Methyl 3-(2-formyl-5-methoxynaphthalen-1-yl) propanoate (MFMNP) is synthesized and selected as the most promising fluorescent probe, exhibiting superior fluorescence intensity and the highest Stokes shift among its other derivatives. In aqueous media, the dual emission behavior of MFMNP is attributed to the equilibrium between the aldehyde and its corresponding geminal diol form. Comprehensive spectroscopic analyses, including both steady-state and time-resolved measurements, were performed to decipher the photophysical behavior of MFMNP. The interactions of MFMNP with both native and denatured forms of human serum albumin (HSA) were comprehensively examined, along with its binding affinity toward bovine serum albumin (BSA). The denaturing effect of sodium dodecyl sulfate (SDS) as well as the renaturing effect of SDS in urea-denatured HSA are demonstrated by steady-state and time-resolved fluorescent spectroscopy. Warfarin and ibuprofen, established as site I and site II markers, respectively, are employed in competitive binding assays to assess the site-specific interaction of MFMNP with HSA. Förster resonance energy transfer (FRET) from HSA to MFMNP was carried out, demonstrating the remarkable quenching efficiency of MFMNP. Molecular docking and molecular dynamics (MD) simulations were employed to further elucidate the binding mechanism between MFMNP and HSA, revealing key amino acid residues involved in the interaction. The binding and fluorescence quenching constants for the MFMNP–HSA complex were experimentally determined. The theoretically determined binding constant was found to be in good agreement with computational predictions.