A ¹³C NMR spectrometric method for the determination of intramolecular δ¹³C values in fructose from plant sucrose samples

Recent developments in ¹³C NMR spectrometry have allowed the determination of intramolecular ¹³C/¹²C ratios with high precision. However, the analysis of carbohydrates requires their derivatization to constrain the anomeric carbon. Fructose has proved to be particularly problematic because of a byproduct occurring during derivatization and the complexity of the NMR spectrum of the derivative. Here, we describe a method to determine the intramolecular ¹³C/¹²C ratios in fructose by ¹³C NMR analysis of the acetyl-isopropylidene derivative. We have applied this method to measure the intramolecular ¹³C/¹²C distribution in the fructosyl moiety of sucrose and have compared this with that in the glucosyl moiety. Three prominent features stand out. First, in sucrose from both C₃ and C₄ plants, the C-1 and C-2 positions of the glucosyl and fructosyl moieties are markedly different. Second, these positions in C₃ and C₄ plants show a similar profile. Third, the glucosyl and fructosyl moieties of sucrose from Crassulacean acid metabolism (CAM) metabolism have a different profile. These contrasting values can be interpreted as a result of the isotopic selectivity of enzymes that break or make covalent bonds in glucose metabolism, whereas the distinctive ¹³C pattern in CAM sucrose probably indicates a substantial contribution of gluconeogenesis to glucose synthesis.