Novel Position-Specific ¹⁸O/¹⁶O Measurement of Carbohydrates. I. O-3 of Glucose and Confirmation of ¹⁸O/¹⁶O Heterogeneity at Natural Abundance Levels in Glucose from Starch in a C₄ Plant

The ¹⁸O/¹⁶O ratio at both molecular and positional levels in the carbohydrates of higher plants is a reliable proxy for the plant growth environment, and a potential indicator of the plant photosynthetic carbon assimilation mode, and its physiological, biochemical and metabolic status. The lack of exploitable nuclear resonance in ¹⁸O and ¹⁶O and the extremely low ¹⁷O abundance make the NMR-based PSIA (position-specific isotopic analysis) a significant challenge. In this Article, an alternative three-step wet chemistry based method for accessing the ¹⁸O/¹⁶O of glucose O-3 is presented. The O atoms (OH groups) at positions 1, 2, 5, and 6 were first protected by acetonation (converting glucose to 1,2;5,6-di-O-isopropylidene-glucofuranose). The protected glucose was then esterified at O-3 by thionoformylation. Subsequent Barton-McCombie deoxygenation quantitatively removed the O-3 from the protected sugar. Mass balance was then applied to calculate the ¹⁸O/¹⁶O of O-3 using the isotopic values of the protected sugar before and after the deoxygenation step. The method is innovative in that (i) isolation and purification of individual compounds for ¹⁸O by EA/Pyrolysis/IRMS analysis is unnecessary as the reaction mixture can be analyzed on a GC/Pyrolysis/IRMS; (ii) sample quantity is dramatically reduced; and (iii) the approach to access the O-3 isotopic signal can be easily expanded to other positions within glucose and other sugars. It was shown that O-3 is enriched by 12 mUr relative to the molecular average (O-2-O-6) for a glucose of C₄ photosynthetic origin. We highlighted the potential applications of the intramolecular O isotopic heterogeneity of glucose this method revealed.