Calcium chloride-promoted epimerization of N-acetyl-d-glucosamine to N-acetyl-d-mannosamine in water: NMR studies and reaction mechanism insights

A green epimerization system using only water and CaCl 2 . • NMR identifies products, by-products, and reveals the mechanism. • Isotope labeling and DFT confirm the epimerization pathway. • A direct route to ManNAc from a biomass monomer. N -acetyl- d -mannosamine (ManNAc) is a valuable precursor in the pharmaceutical and biotechnology industries. Enzymatic epimerization of N -acetyl- d -glucosamine (GlcNAc) offers mild and green conditions for ManNAc production, while its productivity is limited by poor enzyme stability and high costs. Although efficient chemical methods have been developed, they often require strict control of reaction conditions to avoid by-product formation, and their reaction mechanisms remain unclear. This study presents a green and efficient strategy for ManNAc synthesis through calcium chloride (CaCl 2 )-promoted epimerization of GlcNAc in aqueous solution. ManNAc production reached a yield of 27% under optimized conditions. The reaction mechanism was elucidated through a combination of nuclear magnetic resonance (NMR) spectroscopy and density functional theory (DFT) calculations. Using deuterium oxide (D 2 O) as the reaction medium revealed hydrogen-deuterium exchange at the C2 position of GlcNAc, providing direct experimental evidence for a reaction pathway involving an enol intermediate. This finding was further supported by subsequent 13 C isotopic labeling experiments. DFT calculations further revealed that water molecules play a critical role in mediating proton transfers, significantly reducing the energy barriers of the key steps of enol intermediate formation. CaCl 2 enhanced the yield by promoting the formation of the enol intermediate. Additionally, a preliminary investigation was conducted on the purification of ManNAc from the reaction mixture containing its structural epimers. This work not only provides a green system for ManNAc synthesis but also offers profound insights into the reaction mechanism, paving the way for developing more efficient biomass utilization strategies. Download: Download high-res image (515KB) Download: Download full-size image