Riboflavin 5 -phosphate, synthesis

The procedure to phosphorylate riboflavin derivatives on a preparative scale has recently been improved . These preparations, and also commercial FMN, contain a considerable amount of riboflavin phosphate isomers, which are difficult to separate by column chromatography. This problem is emphasized in the chemical synthesis of FAD where the yield is rather low (20-25 %). In this context, it is surprising that a modification of the synthesis of FAD from FMN published by Cramer and Neuhoeffer has not been noticed by workers in the flavin field. According to Cramer and Neuhoeffer, the yield of the chemical synthesis of FAD is drastically improved ( 70 % pure FAD). The procedure was successfully applied in the author s own laboratory (yield 60-70%). It is expected that the improved procedure of the FAD synthesis will stimulate the active-site directed studies on flavoproteins because the problem of separating FMN or FAD from their synthetic by-products has already been solved by use of FMN- or FAD-specific affinity column... 

Most tissues contain very little free riboflavin and, except in the kidneys, where 30% is as riboflavin phosphate, more than 80% is FAD, almost all bound to enzymes. Isolated hepatocytes (and presumably other tissues) show saturable concentrative uptake of riboflavin. The of the uptake process is the same as that of flavokinase, and uptake is inhibited by inhibitors of flavokinase, suggesting that tissue uptake is the result of carrier-mediated diffusion, fol-lowedbymetabolic trapping as riboflavin phosphate, then onward metabolism to FAD, catalyzed by FAD pyrophosphorylase. FAD is a potent inhibitor of the pyrophosphorylase and acts to limit its own synthesis. FAD, which is not protein bound is rapidly hydrolyzed to riboflavin phosphate by nucleotide pyrophosphatase unbound riboflavin phosphate is similarly rapidly hydrolyzed to riboflavin by nonspecific phosphatases (Aw et al., 1983 Yamada et al., 1990). 

Control over tissue concentrations of riboflavin coenzymes seems to be largely by control of the activity of flavokinase, and the synthesis and catabolism of flavin-dependent enzymes. Almost all the vitamin in tissues is enzyme bound, and free riboflavin phosphate and FAD are rapidly hydrolyzed to riboflavin. If this is not rephosphorylated, it rapidly diffuses out of tissues and is excreted. 

The activities of a variety of flavin-dependent enzymes are depressed in hypothyroidism. They are increased by the administration of thyroxine or triiodothyronine, as a result of increased synthesis of riboflavin phosphate and... 

Other Coenzymes and Cofactors.— The chemical synthesis of riboflavin phosphates and their acetyl derivatives has been reinvestigated. Riboflavin 4 -monophosphate (10) is an important contaminant of commercial flavin mononucleotide (FMN), and... 

Interestingly, the synthesis of riboflavin phosphate has taken a shghtly different course. As reported in 1950 (Scheme 12.116), when the reduced Al-ribosyl derivative of 2,3-dimethylaniline is phosphorylated with phosphorus oxychloride and then carefully hydrolyzed, the corresponding monophosphate is capable of isolation. Then, treatment of that ester with diazotized aniline yielded l-D-l -ribitylamino-6-phenylazo-3,4-dimethylbenzene. Reduction to the corresponding amine followed by condensation with alloxan then yielded riboflavin monophosphate. 

Scheme 12.116. A representation of the synthesis of riboflavin phosphate (after Flexser, L. A. US. Patent 2,610,176,1950).

These reactions lead to a synthesis of cozymase or flavine -adenine dinucleotide from adenosine triphosphate with nicotinamide-ribofuranoside-5-phosphoric acid or riboflavine phosphate respectively. 

Kornberg has hypothesized that UDPG might arise from a p3rro-phosphorylase mechanism analogous to the synthesis of FAD from ATP and riboflavin phosphate. Thus, uridine triphosphate plus glucose-l-phos-... 

Introduction of the cobalt atom into the corrin ring is preceeded by conversion of hydrogenobyrinic acid to the diamide (34). The resultant cobalt(II) complex (35) is reduced to the cobalt(I) complex (36) prior to adenosylation to adenosylcobyrinic acid i7,i -diamide (37). Four of the six remaining carboxyhc acids are converted to primary amides (adenosylcobyric acid) (38) and the other amidated with (R)-l-amino-2-propanol to provide adenosylcobinamide (39). Completion of the nucleotide loop involves conversion to the monophosphate followed by reaction with guanosyl triphosphate to give diphosphate (40). Reaction with a-ribazole 5 -phosphate, derived biosyntheticaHy in several steps from riboflavin, and dephosphorylation completes the synthesis. 

Riboflavin, or vitamin B2, is a constituent and precursor of both riboflavin 5 -phosphate, also known as flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). The name riboflavin is a synthesis of the names for the molecule s component parts, ribitol and flavin. The structures of riboflavin. 

Group-transfer reactions often involve vitamins3, which humans need to have in then-diet, since we are incapable of realizing their synthesis. These include nicotinamide (derived from the vitamin nicotinic acid) and riboflavin (vitamin B2) derivatives, required for electron transfer reactions, biotin for the transfer of C02, pantothenate for acyl group transfer, thiamine (vitamin as thiamine pyrophosphate) for transfer of aldehyde groups and folic acid (as tetrahydrofolate) for exchange of one-carbon fragments. Lipoic acid (not a vitamin) is both an acyl and an electron carrier. In addition, vitamins such as pyridoxine (vitamin B6, as pyridoxal phosphate), vitamin B12 and vitamin C (ascorbic acid) participate as cofactors in an important number of metabolic reactions. 

The precursors for riboflavin biosynthesis in plants and microorganisms are guanosine triphosphate and ribulose 5-phosphate. As shown in Figure 7.3, the first step is hydrolytic opening of the imidazole ring of GTP, with release of carbon-8 as formate, and concomitant release of pyrophosphate. This is the same as the first reaction in the synthesis ofpterins (Section 10.2.4), but utilizes a different isoenzyme of GTP cyclohydrolase (Bacher et al., 2000, 2001). 

An alternative which is attractive for large scale work is the electrochemical reduction of aldonolac-tones.42 43 Particular attention has been paid to the electroreductive synthesis of ribose from ribonolac-tone because of the importance of the former in the synthesis of riboflavin (vitamin 62). Processes generally involve a mercury cathode and maintenance of an acidic pH, often with the assistance of a phosphate or borate buffer. It has been reported that alkali metal ions are also necessary, suggesting that the reduction occurs via metal amalgam formation. However, other accounts make no mention of metal... 

A similar reaction sequence led to the synthesis of riboflavin 5-phosphate and, earlier, to the 6-phosphate of methyl a-D-glucopyranoside. ... 

Guanosine triphosphate and ribulose-5-phosphate are recruited in a 1 2 stoichiometric ratio by GTP cyclohydrolase II and DHBP synthase, respectively, for riboflavin biosynthesis. Since at substrate saturation the activity of B. subtilis DHBP is twice the activity of B. suhtilis cyclohydrolase II (DSM, unpublished observations) and since both enzymatic activities are associated with the same bifunctional protein encoded by rihA, the balanced formation of the pyrimidinedione and the dihydroxybutanone intermediates is ensured. However, the ifg.s constant of DHBP synthase ( 1 mmol is about 100-fold higher than the ifg.s constant of GTP cyclohydrolase II imposing the risk of excessive synthesis of the pyrimidinone and pyrimidinedione intermediates in case of reduced intracellular concentrations of pentose phosphate pathway intermediates. This can be expected, for instance, in glucose-limited fed-batch fermentations, which are frequentiy used in industrial applications. The pyrimidinone and pyrimidinedione intermediates are highly reactive, oxidative compounds, which can do serious damage on the bacteria. 

The mechanism of synthesis of the coenzyme forms of riboflavin has been studied by a number of investigators. Purified enzymes capable of catalyzing the formation from riboflavin of riboflavin 5 -phosphate and flavin adenine dinucleotide have been described. The formation of a number of glycoades of riboflavin by animal tissue preparations and microorganisms has been reported. 

Until recently the synthesis of flavin adenine dinucleotide had not been studied with purified enzymes from animal tissues. However, the ability of animal tissues to synthesize this coenzyme has been known for a number of years. Klein and Kohn 138) observed formation of flavin adenine dinucleotide in red blood cells in vivo and in vitro, and Trufanov 139) obtained synthesis of flavin adenine dinucleotide in rat tissue slices. Makino et al. 130) obtained formation of flavin adenine dinucleotide from vitamin Ba and ATP in the presence of pig kidney acetone powders, and Yagi 129) reported the synthesis of the coenzyme by the action of acetone powders of rabbit liver or kidney from riboflavin 5 -phosphate, but not riboflavin, and ATP. 

---