Enzymatic Synthesis of Riboflavin and FMN Specifically Labeled with 13C in the Xylene Ring

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Abstract
The condensation of 3-hydroximino-2-butanone (1) with 5-amino-6-ribitylamino-2,4(1H,3H)- pyrimidinedione (2) yields 6,7-dimethyl-8-ribityllumazine (3). At slightly alkaline pH, the car­bonyl group of 1 reacts preferentially with the 5-amino group of 2 (regioselectivity, 4:1). Under acidic conditions, the reaction occurs with higher yield and marginal regioselectivity of opposite direction (1:1.4). Appropriately 13C-labeled samples of 1 afford 3 labeled at C-6α, C-6, C-7 or C-7α. [6α,7α-13C2]-3 was prepared by condensation of 2 with [1,4-13C2]diacetyl. The lumazines 3 were converted to riboflavin by the enzyme, riboflavin synthase, with almost quantitative yield. By this procedure, any C-atom of the carbocyclic moiety of riboflavin can be selectively labeled with 13C at high abundance. Phosphorylation yields the respectively 13C-labeled FMN samples.