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

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.