Vitamin B2 Operates by Dual Thermodynamic and Kinetic Mechanisms to Selectively Tailor Urate Crystallization

Here we demonstrate how a biologically relevant molecule, riboflavin (vitamin B2), operates by a dual mode of action to effectively control crystallization of ammonium urate (NH4HU), which is associated with cetacean kidney stones. In situ microfluidics and atomic force microscopy experiments confirm a strong interaction between riboflavin and NH4HU crystal surfaces that substantially inhibits layer nucleation and spreading by kinetic mechanisms of step pinning and kink blocking. Riboflavin does not alter the distribution of tautomeric urate isomers, but its adsorption on NH4HU crystal surfaces does interfere with the effects of minor urate tautomer by limiting its ability to induce NH4HU crystal defects while also suppressing NH4HU nucleation and inhibiting crystal growth by 80% at an uncharacteristically low modifier concentration. Time-resolved spectroscopy measurements, ab initio calculations, and molecular dynamics simulations confirm that each riboflavin molecule forms a complex with six or more urate molecules to lower supersaturation, thereby reducing the rate of NH4HU crystallization by a thermodynamically driven mechanism. The degree of complexation observed for riboflavin far exceeds that of common chelating agents, and results in crystal dissolution when the free urate concentration falls below NH4HU solubility. The synergism that is created by riboflavin's dual kinetic and thermodynamic mechanisms is rarely achieved by more conventional crystal growth inhibitors. These insights offer new approaches that could be influential for the design of molecular modifiers in crystal engineering applications, the development of therapeutics for pathological conditions, and establishing broader understanding of the roles played by foreign agents in natural and biological crystallization.