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

Abstract

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.