A Multidisciplinary Experiment for Undergraduate Students: 5-Formylcytosine Labeling through Aqueous Wittig Reaction

Increasingly, many biological problems require molecular and quantitative answers that can be provided only by chemical tools. This laboratory experiment, designed for undergraduate education, introduces students to fluorescence-based labeling as a powerful method for epigenetic research. Commercial (tert-butoxycarbonylmethyl)triphenylphosphonium bromide (tBuA+) was employed to selectively convert 5-formylcytosine (5fC), a key epigenetic modification, into its cyclic derivative (cycle-C). This transformation was achieved through a mild cascade reaction comprising a Wittig reaction followed by intramolecular cyclization. cycle-C emits strong blue fluorescence, thus enabling fluorescence labeling and turn-on detection of 5fC. The three-phase lab emphasizes (1) collaborative optimization of reaction parameters, (2) specificity verification through control experiments, and (3) practical applications in fluorescence detection and nucleic acid labeling. Pedagogically, this multidisciplinary experiment bridges synthetic chemistry with biological applications by demonstrating tailored chemical reactions for biomolecular labeling. Through hypothesis-driven experimentation and data-driven optimization, it trains students in experimental design and critical decision-making while exposing them to epigenetic research frontiers. The modular design enables adaptation to diverse teaching environments through core-principle-focused implementation.