Production of Anti-melanogenic Glucosides via Molecular Docking-guided Biotransformation of p-hydroxyphenethyl anisate.

p-Hydroxyphenethyl anisate (HP) has been identified as a natural compound with whitening activity in melanoma cells and zebrafish. Considering that glycosylation can enhance the solubility, stability, and bioactivity of natural products, we investigated whether HP could serve as a substrate for glycosylation enzymes. Molecular docking predicted favorable interactions between HP and amylosucrase as well as glycosyltransferases, suggesting the potential formation of α- and β-glucosides. Enzymatic reactions confirmed the production of phenethylanisate-4'-O-α-glucoside (HP-α-glc) and phenethylanisate-4'-O-β-glucoside (HP-β-glc). The structures were validated by mass spectrometry, enzymatic digestion (HP-α-glc), or nuclear magnetic resonance spectroscopy (HP-β-glc). Biological evaluation revealed that both glucosides suppressed melanogenesis in murine B16-F1 melanoma cells at non-cytotoxic concentrations. Mechanistic studies demonstrated that HP-β-glc not only inhibited tyrosinase activity but also downregulated the expression of the microphthalmia-associated transcription factor (MITF) and tyrosinase at both the protein and mRNA levels, thereby attenuating melanogenesis through transcriptional regulation. Consistently, in vivo assays demonstrated that HP-β-glc effectively inhibited pigmentation in zebrafish embryos without inducing developmental toxicity, and its effect was comparable to that of the positive control, raspberry ketone. These findings highlight the enzymatic glycosylation of HP as a viable strategy to generate bioactive derivatives with improved functional properties. In particular, HP-β-glc retains the anti-melanogenic activity of HP, providing mechanistic evidence and preclinical validation for its potential application as a safe and effective skin-whitening agent.