ESIPT-induced selective tryptophan detection: a multifunctional probe for biomacromolecule binding with theoretical and biocompatibility studies.

Abstract

A coumarin-based fluorescent ligand, 4-hydroxy-2-oxo-2H-chromene-3-carbaldehyde (HCC), was synthesized and evaluated for the selective detection of tryptophan (Trp) among the nine essential amino acids. The probe exhibited remarkable selectivity toward Trp through an excited-state intramolecular proton transfer (ESIPT) mechanism facilitated by intramolecular hydrogen transfer. Upon interaction with Trp, HCC forms an HCC-Trp complex (HCP), which undergoes a transient tautomeric transformation between enol (E) and keto (K) forms via an excited-state intramolecular proton transfer (ESIPT) process. This results in an approximately 11-fold enhancement in fluorescence intensity at 385 nm, accompanied by a bathochromic shift in both the absorption and emission spectra. The probe HCC exhibited a low detection limit of 1.53 μM and a binding constant of 1.6 × 104 M-1 towards Trp. The sensing behaviour and binding mechanism were systematically confirmed using UV-Vis and fluorescence spectroscopy, 1H NMR, mass spectrometry, Job's plot analysis, and density functional theory (DFT) calculations. Additionally, HCC exhibited notable binding affinity toward biomacromolecules such as calf thymus DNA (ct-DNA), bovine serum albumin (BSA), and pig liver esterase (PLE), supported by spectroscopic studies and molecular docking analysis. The binding constant for ct-DNA, BSA and PLE was found to be 5.7 × 104 M-1, 5.6 × 104 M-1 and 1.19 × 105 M-1, respectively. Fluorescence imaging experiments further confirmed the applicability of HCC for detecting Trp in biological samples along with in vitro anticancer studies. Thus, this study demonstrates that a simple coumarin-based small-molecule probe can selectively recognize tryptophan through an ESIPT-driven mechanism. The probe HCC exhibits high sensitivity, strong binding affinity, and compatibility with biological systems, enabling fluorescence-based detection and imaging of Trp. The findings highlight the potential of HCC as a multifunctional probe for biochemical sensing and provide a promising platform for developing small-molecule fluorophores for amino acid recognition.