Rapid One-Pot Synthesis of Riboflavin Isotopomers

Abstract

Flavocoenzymes labeled with stable isotopes are important reagents for the study of flavoproteins using isotope-sensitive methods such as NMR, ENDOR, infrared, and Raman spectroscopy. We describe highly versatile one-pot methods for the preparation of riboflavin isotopomers labeled with (13)C in every desired position of the xylene moiety. The starting materials are commercially available (13)C-labeled glucose samples, which are converted into riboflavin using enzymes of the oxidative pentose phosphate pathway in combination with recombinant enzymes of the riboflavin biosynthetic pathway. The overall reaction comprises six enzyme-catalyzed reaction steps for the synthesis of the vitamin and two auxiliary enzymes for in situ recycling of cofactors. The overall yields of riboflavin based on isotope-labeled glucose are 35-50%.

Thermodynamic and Probabilistic Metabolic Control Analysis of Riboflavin (Vitamin B2) Biosynthesis in Bacteria

“…Furthermore, the finding that the reaction of GTP cyclohydrolase II is irreversible is consistent with a study on E. coli GTP cyclohydrolase II [63]. The reactions of DHBP synthase (reaction 5) and riboflavin synthase (reaction 7) are described as inherently thermodynamically irreversible in the literature [64][65][66]. Our results for these two reactions suggest the same.…”

“…Concerning lumazine synthase (reaction 6), this result is in contrast to the general view in the literature. Römisch et al described the reaction of lumazine synthase as inherently irreversible [64,67]. Efimov et al studied the riboflavin kinase reaction catalyzed by the bifunctional FAD synthetase of C. ammoniagenes [68].…”

Evolution of Vitamin B2 Biosynthesis. A Novel Class of Riboflavin Synthase in Archaea

“…The doublets at 139.2 ppm and 132.0 ppm are assigned as C-7 and C-9a, respectively, on the basis of a detailed analysis of the 13 C NMR spectrum of riboflavin which has been reported elsewhere. 27 The fine splitting of the signals can be explained by long-range carboncarbon coupling in the doubly 13 C-labeled product. The other 15 carbon atoms of the enzyme product have intensities below the level of detection due to the absence of 13 C labeling.…”

13C‐, 15N‐ and 31P‐NMR studies of oxidized and reduced low molecular mass thioredoxin reductase and some mutant proteins

“…Preparation of [U-13 C 17 , U- 15 N 4 ]riboflavin [U-13 C 17 , U- 15 N 4 ]riboflavin was prepared according to published procedures [24].…”