Position-specific isotope signatures evolve during a reversible reaction

Position-specific isotope distributions capture information about reaction dynamics, metabolism, and environmental processes, encoding the formation history of organic molecules. Kinetic and equilibrium isotope effects are fundamental drivers of isotope fractionation, yet how these effects are expressed in position-specific isotope distributions remains poorly constrained. Here, we use glucose mutarotation as a model reaction for tracking the evolution of position-specific isotope ratios in a system in disequilibrium. In aqueous solution, α- and β-glucose interconvert through a low-abundance open-chain intermediate, providing an ideal framework for probing isotope effects within a single molecular pool at natural ¹³ C abundance. Using NMR spectroscopy, we simultaneously measure glucose anomer populations and position-specific ¹³ C/ ¹² C ratios, enabling direct, time-resolved observations of isotope fractionation at the anomeric (C1) carbon. Starting from α-glucose, initial β-glucose formation generates a large transient isotope disequilibrium driven by unequal forward and reverse kinetic isotope effects, producing a temporary divergence in ¹³ C abundance of more than 30‰ between anomers. Continued interconversion redistributes the isotopes and yields an equilibrium difference of ~7‰ between α- and β-glucose at the C1 position. Together, these results demonstrate how kinetic and equilibrium isotope effects acting within a reversible reaction can generate substantial, time-dependent intramolecular isotope heterogeneity, with potential implications for how position-specific isotope signatures emerge and propagate through (biogeochemical) reaction networks.