Molecular diffusion in chemical reactions has been observed to deviate from the Stokes-Einstein relationship, however underlying mechanism remains elusive. The diffusion kinetics of enzymatic and small-molecule chemical reactions have shown that some reactants or catalysts exhibit enhanced translational diffusion coefficients exceeding their intrinsic values by over 20%, with minimal temperature fluctuations. While molecular motion inherently couples translational and rotational components, previous studies have primarily focused on translational diffusion, leaving rotational diffusion largely unexplored. In this study, the rotational diffusion coefficients of species involved in the Cu(I)-catalyzed 1,3-dipolar cycloaddition between 3-butynoic acid and azidoacetic acid were investigated through two independent measurements. Specifically, the changes in the translational diffusion coefficients of the substrate and product during the reaction of 3-butynoic acid and azidoacetic acid were determined by nuclear magnetic resonance (NMR) diffusion-ordered spectroscopy, and a significant increase in the substrate's diffusion coefficient was observed at the early stage of the reaction, which confirms a nonclassical diffusion phenomenon in this system. Subsequently, rotational diffusion coefficients of each species during the reaction were measured using a T₁ inversion recovery experiment, the values for both the substrate and the product showed a significant increase as the reaction progressed while the values for sodium ascorbate remained unchanged. Rotational diffusion was further measured by an independent fluorescence anisotropy/polarization experiment. The changes in fluorescence anisotropy were consistent with the NMR experiments, confirming the presence of nonclassical rotational diffusion in the reaction. The changes in rotational and translational diffusion coefficients of 3-butynoic acid and azidoacetic acid were found both strongly correlated with reaction progress and in synchronization. In summary, interpretating from two independent measurements based on NMR and fluorescence anisotropy, we propose that molecular alignment and the conversion of rotational to translational energy contribute to deviations from the Stokes-Einstein relation during reaction.

Key words: rotational diffusion, pulsed-gradient nuclear magnetic resonance, T₁ relaxation determination, fluorescence anisotropy, copper catalyzed alkyne-azide cycloaddition