Intramolecular Cyclization and a Retro-Ene Reaction Enable the Rapid Fragmentation of a Vitamin B1-derived Breslow Intermediate

In solution, analogues of the Breslow intermediate formed during catalysis by benzoylformate decarboxylase (BFDC) undergo rapid, irreversible fragmentation. The ability of BFDC to prevent this reaction and preserve its’ cofactor is a striking example of an enzyme ‘steering’ a reactive intermediate towards a productive pathway. To understand how BFDC suppresses the off-pathway reactivity of this Breslow intermediate, a clear mechanistic understanding of the fragmentation reaction is required. Here, DFT calculations reveal an unexpected mechanism for the solution-phase fragmentation that involves an intramolecular cyclization and a subsequent retro-ene reaction to release the final products. Free energy profiles demonstrate that this pathway is significantly more facile than the previously proposed mechanism that invoked Breslow intermediate enolates as intermediates. Additional computations have been performed to understand why related Breslow intermediates do not undergo analogous fragmentation reactions. Calculations performed with two closely related Breslow intermediates suggest that subtle differences in the relative values of ∆G‡ for protonation and fragmentation dictate whether a given intermediate will fragment or not. These differences and the fragmentation mechanism unveiled in this work have ramifications for the catalytic mechanism of BFDC and other thiamin-dependent enzymes and will provide general lessons related to the control of reactive intermediates by enzymes.