Chemical formation of 4-hydroxy-2,5-dimethyl-3[2H]-furanone from d-fructose 1,6-diphosphate Original Research Article

The selective chemical formation of 4-hydroxy-2,5-dimethyl-3[2H]-furanone (HDF) from d-fructose 1,6-diphosphate in the presence of reduced nicotinamide-adenine-dinucleotides (NAD(P)H) was investigated by means of HPLC–DAD and HPLC–UV–MS/MS. The temperature optimum for HDF formation was 30 °C, whereas the pH value (pH 3–10) and chemical nature of the buffer had no significant influence. A linear correlation of reaction time and d-fructose 1,6-diphosphate concentration with the obtained HDF yield was observed. Proteins appeared to have a stabilizing effect. The NAD(P)H were mandatory, even in the presence of protein, implying a non-enzymatic hydride-transfer to an unknown intermediate which finally leads to the selective formation of HDF. The hydride-transfer was confirmed by the application of selectively pro-4R or pro-4S deuterium labeled NADH resulting in each case in the formation of HDF exhibiting a deuterium labeling of approx 30% and employment of [4R,S-2H2]-NADH led to a deuterium labeling of approx 66%. The incubation of [1-13C]-d-fructose 1,6-diphosphate with [4R,S-2H2]-NADH revealed that the hydride is transferred to C-5 or C-6 of the d-fructose 1,6-diphosphate skeleton. Thus, a chemical HDF formation from d-fructose 1,6-diphosphate under physiological reaction conditions was shown and for the first time to our knowledge a non-enzymatic hydride-transfer from NADH to a carbohydrate structure was demonstrated.