Theoretical and NMR analysis of condensation of glyoxylic acid and urea in aqueous solution with allantoin formation.

The mechanism for allantoin formation via the condensation of glyoxylic acid and urea is studied using a combined theoretical and experimental approach. Density functional theory (DFT) calculations are employed to explore the potential energy surface and identify key intermediates and transition states, while NMR spectroscopy is used to validate the proposed mechanism experimentally. The reaction proceeds through four sequential stages: (1) formation of an amino alcohol intermediate, (2) dehydration to a highly reactive imine, (3) nucleophilic addition of a second urea molecule yielding a geminal diamine, and (4) final cyclization with water elimination to form allantoin. In the absence of proton-transfer mediators, the process is kinetically hindered by high activation barriers associated with strained four-membered transition states. Participation of water or acid molecules facilitates proton transfer through six- and eight-membered transition-state structures, significantly lowering the activation barriers. The effect of protonation is systematically analyzed, revealing a combined reaction pathway in which the initial and final steps proceed via neutral species, whereas the intermediate stages are favored under protonated conditions due to stabilization of the imine intermediate. 1H, 13C, DEPT-135, HSQC, and HMBC NMR analyses confirm the presence of key intermediates, including amino alcohol and geminal diamine species, supporting the proposed mechanism. The results provide detailed insights into the interplay between protonation, solvation, and reactivity, offering a comprehensive mechanistic understanding of allantoin formation and guiding the rational design of related condensation processes.