Theoretical Exploration of the Mechanism of Riboflavin Formation from 6,7-Dimethyl-8-ribityllumazine: Nucleophilic Catalysis, Hydride Transfer, Hydrogen Atom Transfer, or Nucleophilic Addition?

Abstract

The cofactor riboflavin is biochemically synthesized by a constitutionally intricate process in which two molecules of 6,7-dimethyl-8-ribityllumazine react with each other to form one molecule of the cofactor and one molecule of 5-amino-6-(ribitylamino)uracil. Remarkably, this complex molecular transformation also proceeds non-enzymatically in boiling aqueous solution at pH 7.3. Four different mechanistic pathways for this transformation (nucleophilic catalysis, hydride transfer, hydrogen atom transfer, and a nucleophilic addition mechanism) have now been analyzed by density functional theory [M06-2X/def2-TZVPP/CPCM//M06-2X/6-31+G(d,p)/IEFPCM]. On the basis of these computational results, a so far unpublished nucleophilic addition mechanism is the lowest energy pathway yielding riboflavin. The previously proposed mechanism involving nucleophilic catalysis is higher in energy but is still a viable alternative for an enzyme-catalyzed process assisted by suitably positioned catalytic groups. Pathways involving the transfer of a hydride ion or of a hydrogen atom are predicted to proceed through higher energy transition states and intermediates.

Natural [4 + 2]-Cyclases

“…Furthermore, this pathway was not only lower in energy than that involving the hydride transfer and Diels-Alder reactions but also the pathway involving activation by an exogenous nucleophile. 60 …”

Natural Diels–Alderases: Elusive and Irresistable

“…[39] Density functional theory calculations were used to evaluate the following mechanistic pathways: nucleophilic catalysis, hydride transfer followed by subsequent Diels-Alder, hydrogen atom transfer, and a nucleophilic addition mechanism. [39] While the Diels-Alder transformation cannot yet be ruled out, the alternative pathways are more likely to proceed with nucleophilic addition being of the lowest energy. Although, it should be noted that due to high-energy intermediates such as 22 , and the likelihood of a stepwise mechanism, the synthesis of Riboflavin probably does not proceed via a Diels-Alderase although it formally constitutes a “[4+2]” construction.…”

“…Although, it should be noted that due to high-energy intermediates such as 22 , and the likelihood of a stepwise mechanism, the synthesis of Riboflavin probably does not proceed via a Diels-Alderase although it formally constitutes a “[4+2]” construction. [39] …”

Biocatalytic Strategies towards [4+2] Cycloadditions

“…This reaction leads to the biosynthesis of the essential cofactor riboflavin via pentacyclic intermediate 29 (Figure A) . Several mechanisms have been proposed for the riboflavin synthase‐catalysed dimerisation of 6,7‐dimethyllumazine 30 but DFT calculations in water have ruled out most of them . Among the energetically disfavoured routes were a radical tautomerization followed by stepwise cyclization and hydride transfer followed by Diels–Alder cyclization .…”

“…Several mechanisms have been proposed for the riboflavin synthase‐catalysed dimerisation of 6,7‐dimethyllumazine 30 but DFT calculations in water have ruled out most of them . Among the energetically disfavoured routes were a radical tautomerization followed by stepwise cyclization and hydride transfer followed by Diels–Alder cyclization . The mechanism is favoured on computational grounds, compatible with kinetic isotope effects and involves a tautomerization of one reaction partner to diene‐diamine 31 and sequential nucleophilic additions of the 6‐ and 7‐methylene groups to the 7‐ and 6‐carbon atoms of the other reaction partner.…”