Temperature-dependent thermodynamic and kinetic analysis of ketohexose tautomerism by quantitative 1H NMR: A comparative study of D-fructose and D-allulose

초록

A quantitative thermodynamic and kinetic investigation of ketohexose tautomerism was performed using temperature-resolved 1H quantitative nuclear magnetic resonance (qNMR) spectroscopy. The tautomeric equilibria and mutarotation kinetics of D-fructose and its C3 epimer, D-allulose, were systematically examined in aqueous solution. Complete 1H and 13C resonance assignments enabled reliable quantification of all detectable cyclic and acyclic species, including minor open-chain forms. Temperature-dependent equilibrium analysis revealed fundamentally distinct thermodynamic landscapes: D-fructose was strongly biased toward β-pyranose, whereas D-allulose preferentially populated furanose forms, particularly α-furanose. Increasing the temperature shifted both systems toward furanose and keto species, consistent with entropically favored populations of less conformationally constrained structures. Independent 13C NMR measurements confirmed the quantitative reliability of the 1H qNMR approach. Time-resolved in situ 1H NMR measurements enabled the determination of apparent ring-opening rate constants using a reversible first-order kinetic framework. Activation parameters derived from Arrhenius and Eyring analyses revealed that the most thermally responsive pathways differ between the epimers: D-fructose exhibited activation energies of 59–64 kJ·mol−1 for furanose ring opening, whereas D-allulose showed substantially higher barriers of 77–83 kJ·mol−1 for pyranose ring-opening processes. These findings demonstrate that minimal stereochemical variation reorganizes not only equilibrium distributions but also pathway-specific kinetic responses in ketohexose tautomerism. The study establishes temperature-dependent quantitative 1H NMR as an efficient platform for simultaneous equilibrium and pathway-resolved kinetic characterization of carbohydrate systems.

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