Temperature‐dependent thermodynamic and kinetic analysis of ketohexose tautomerism by quantitative <scp> ¹ H NMR </scp> : A comparative study of D‐fructose and D‐allulose

Abstract A quantitative thermodynamic and kinetic investigation of ketohexose tautomerism was performed using temperature‐resolved 1 H 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 1 H and 13 C 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 13 C NMR measurements confirmed the quantitative reliability of the 1 H qNMR approach. Time‐resolved in situ 1 H 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 1 H NMR as an efficient platform for simultaneous equilibrium and pathway‐resolved kinetic characterization of carbohydrate systems.