Riboflavin and oxidation

Later, a completely different and more convenient synthesis of riboflavin and analogues was developed (34). It consists of the nitrosative cyclization of 6-(A/-D-ribityl-3,4-xyhdino)uracil (18), obtained from the condensation of A/-D-ribityl-3,4-xyhdine (11) and 6-chlorouracil (19), with excess sodium nitrite in acetic acid, or the cyclization of (18) with potassium nitrate in acetic in the presence of sulfuric acid, to give riboflavin-5-oxide (20) in high yield. Reduction with sodium dithionite gives (1). In another synthesis, 5-nitro-6-(A/-D-ribityl-3,4-xyhdino) uracil (21), prepared in situ from the condensation of 6-chloro-5-nitrouracil (22) with A/-D-ribityl-3,4-xyhdine (11), was hydrogenated over palladium on charcoal in acetic acid. The filtrate included 5-amino-6-(A/-D-ribityl-3,4-xyhdino)uracil (23) and was maintained at room temperature to precipitate (1) by autoxidation (35). These two pathways are suitable for the preparation of riboflavin analogues possessing several substituents (Fig. 4). 

One-electron oxidation of the adenine moiety of DNA and 2 -deoxyadenos-ine (dAdo) (45) gives rise to related purine radical cations 46 that may undergo either hydration to generate 8-hydroxy-7,8-dihydroadenyl radicals (47) or deprotonation to give rise to the 6-aminyl radicals 50. The formation of 8-oxo-7,8-dihydro-2 -deoxyadenosine (8-oxodAdo) (48) and 4,6-diamino-5-formamidopyrimidine (FapyAde) (49) is likely explained in terms of oxidation and reduction of 8-hydroxy-7,8-dihydroadenyl precursor radicals 47, respectively [90]. Another modified nucleoside that was found to be generated upon type I mediated one-electron oxidation of 45 by photoexcited riboflavin and menadione is 2 -deoxyinosine (51) [29]. The latter nucleoside is likely to arise from deamination of 6-aminyl radicals (50). Overall, the yield of formation of 8-oxodAdo 48 and FapyAde 49 upon one-electron oxidation of DNA is about 10-fold-lower than that of 8-oxodGuo 44 and FapyGua 43, similar to OH radical mediated reactions [91]. 

Other mediators which have been used in combination with diaphorase for the regeneration of NAD+ are riboflavin and Vitamin K3, which is 2,3-dimethyl-1,4-naphthoquinone. However, especially riboflavin is not stable enough for synthetic applications [40]. Better stability is exhibited by phenanthrolindiones as mediators. In combination with diaphorase, Ohshiro [41] showed the indirect electrochemical oxidation of cyclohexanol to cyclohexanone using the NAD+ dependent HLADH with a turnover frequency of two per hour. For an effective enzymatic synthesis, this turnover frequency, however, would be too small. In our own studies, we were able to accelerate the NAD(P)+ regeneration considerably by lowering the electron density within the... 

Note that this overall reaction requires three coenzymes that we encountered as metabolites of vitamins in chapter 15 NAD+, derived from lucotiiuc acid or nicotinamide FAD, derived from riboflavin and coenzyme A(CoASH), derived from pantothenic acid. In the overall process, acetyl-SCoA is oxidized to two molecules of carbon dioxide with the release of CoASH. Both NAD+ and FAD are reduced to, respectively, NADH and FADH2. Note that one molecule of guanosine triphosphate, GTP, functionally equivalent to ATP, is generated in the process. 

Riboflavin (vitamin B2) is a component of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), coenzymes that play a major role in oxidation-reduction reactions (see Section 15.1.1). Many key enzymes involved in metabolic pathways are actually covalently bound to riboflavin, and are thus termed flavoproteins. 

Babior, who has studied this enzyme at several stages of its purification, found in lysates of PMNs which were activated with zymosan that of eight potential biological reductants only reduced pyridine nucleotides supported the formation of O ". The K , for NADPH was less than the K , for NADH and the activity was decreased in preparations from three patients with chronic granulomatous disease. In accord with predictions based on reaction 7, 0.55 molecule of O7 was measured per molecule of NADPH oxidized under conditions of saturating concentrations of cytochrome c The enzyme which was extracted with Triton X-100 from a granule-rich fraction from activated PMNs, required an external source of FAD for the formation of O from NADPH . Riboflavin and FMN would not substitute. Flavin adenine dinucleotide was proposed as a necessary cofactor, which was probably lost when the enzyme was treated with the detergent. 

NAD+ and NADP+. This difference reflects the chemical difference between the vitamins riboflavin and nicotinamide which form the oxidation-reduction centers of the coenzymes. Another difference is that NAD+ and NADP+ tend to be present in free forms within cells, diffusing from a site on one enzyme to a site on another. These coenzymes are sometimes tightly bound but flavin coenzymes are usually firmly bound to proteins, fixed, and unable to move. Thus, they... 

Flavin coenzymes are usually bound tightly to proteins and cycle between reduced and oxidized states while attached to the same protein molecule. In a free unbound coenzyme the redox potential is determined by the structures of the oxidized and reduced forms of the couple. Both riboflavin and the pyridine nucleotides contain aromatic ring systems that are stabilized by resonance. Part of this resonance stabilization is lost upon reduction. The value of E° depends in part upon the varying amounts of resonance in the oxidized and reduced forms. The structures of the coenzymes have apparently evolved to provide values of E° appropriate for their biological functions. 

Current-reversal chronopotentiometry is also useful for detecting adsorption of the product generated during the forward electrolysis time. Complete adsorption of the product on the electrode surface causes tr to equal tf since no product is lost by diffusion from the electrode. This approach has been used to determine the amount of adsorbed material formed during the reduction of riboflavin and the oxidation of iodide [10]. 

The determination of vitamins in pharmaceutical preparations continues to receive considerable attention. The voltammetric oxidation of vitamin A at a carbon paste electrode in the presence of vitamin E, a potential source of error in the assay, has been described [142,143]. Other assays involve the polaro-graphic determination of niacinamide [144-146], menadione (vitamin K3) [147], riboflavin (vitamin B2) [148], thiamine, riboflavin, and nicotinamide in multivitamin preparations [149], and multivitamins [150]. 

Hu, P.C., Chen, B.H. 2002. Effects of riboflavin and fatty acid methyl esters on cholesterol oxidation during illumination. J. Agric. Food Chem. 50, 3572-3578. 

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