Information about plant photosynthetic carbon assimilation, physiology, and biochemistry is locked in the 18O/16O ratios of the individual positions of higher plants carbohydrates but is under-utilized due to the difficulty of making these determina-tions.
We report the extension of the wet chemistry approach we used to access the 18O/16O ratio of O-3 of glucose with a novel GC/Pyrolysis/IRMS-based method, to determine the 18O/16O ratios of O-4, O-5 and O-6. The O atoms (OH groups) at positions 1, 2, 5 and 6 of glucose were protected by acetonation (converting to 1,2;5,6-di-O-isopropylidene-glucofuranose, DAGF).
The DAGF was then converted to 6-bromo-6-deoxy-1,2;3,5-di-O-isopropylidene-glucofuranose (6-bromoDAGF) with simultaneous removal of O-6 with N-bromosuccinimide and triphenylphosphine. The DAGF was also methylated at O-3 with CH3I under the catalysis of NaH to 3-methylDAGF, which was then deacetonated to 1,2-O-isopropylidene-3-O-methyl-glucofuranose (3-methylMAGF).
O-5 and O-6 were then removed as a whole from 3-methylMAGF by I2 oxidization under the catalysis of Ph3P and imidazole.
Isotope mass balance was then applied to calculate the 18O/16O of O-5 and O-6 as a whole and O-6 respectively. Sampling at different stages of substrate conversion to product and applying a Rayleigh-type fractionation model were employed, when quantitative conversion of substrate was unachievable to calculate the 18O of the converted substrate.
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Quantitative conversion of glucose with phenylhydrazine to phenylglucosazone also allowed for the calculation of 18O2 by applying isotope mass balance between the two. A C4 starch-derived glucose intramolecular 18O profile is now determined: O-3 is relatively enriched (by 12.16mUr), O-4 relatively depleted (by 20.40-31.11mUr) and O-2 mar-ginally enriched (by 2.40mUr) against the molecular average.