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The riboflavin biosynthetic pathway is a promising target for the development of novel antimycobacterial drugs given the lack of riboflavin transporter in M. tuberculosis. Herein, a series of riboflavin derivatives was designed, synthesized and screened for their antimycobacterial and antibacterial activity. The compounds 1a, 1b, 2a, 3a and 5a displayed noticeable antitubercular activity against M. tuberculosis with minimum inhibitory concentration (MIC 99 ) in the range of 6.25 to 25 μM. The lead compound 5a had a selectivity index of 10.7 in the present study. The compounds 2a, 2b, 2c, 4c and 4d showed relatively low to moderate antibacterial activity (MIC = 100-200 μM) against gram-positive strains. Notably, the compounds do not show any inhibition against gram-negative strains even at 200 μM concentration. Further, molecular docking and binding experiments with representative flavin mononucleotide (FMN) riboswitch suggested that the riboflavin analogs exhibited antimycobacterial activity plausibly through FMN riboswitch-mediated repression of riboflavin biosynthesis. In addition to FMN riboswitch, flavoproteins involved in the flavin biosynthesis could also be target of riboflavin derivatives. In conclusion, the potency and low toxicity of riboflavin analogs particularly 5a (MIC 99  = 6.25) make it a lead compound for the synthesis of new analogs for antimycobacterial therapy., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

We report an effective, operationally simple, and environmentally friendly system for the synthesis of tertiary amides by the oxidative coupling of aromatic or aliphatic aldehydes with amines mediated by riboflavin tetraacetate ( RFTA ), an inexpensive organic photocatalyst, and visible light using oxygen as the sole oxidant. The method is based on the oxidative power of an excited flavin catalyst and the relatively low oxidation potential of the hemiaminal formed by amine to aldehyde addition.

In the panorama of modified G-quadruplexes (G4s) with interesting proprieties, here, it has been reported the synthesis of new modified d(TGGGAG) sequences forming G-quadruplexes, with the insertion of a riboflavin unit (Rf, vitamin B 2 ). Exploiting the flavin similarity with the hydrogen bond pattern of guanine and aiming at mimic a typical nucleoside scaffold, the synthesis of the riboflavin building block 3 it has been efficiently carried out. The effect of insertion of riboflavin mimic nucleoside on the G-quadruplex properties has been here, for the first time investigated. A biophysical characterization of Rf-modified sequences (A-D) has been carried out by circular dichroism (CD), fluorescence spectroscopy, differential scanning calorimetry (DSC) and native gel electrophoresis. CD and electrophoresis data have suggested that Rf-modified sequences are able to form parallel tetramolecular G4 structures similar to that of the unmodified sequence. Analysis of the DSC thermograms has revealed that all modified G-quadruplexes have a higher thermal stability compared with the natural sequence, particularly the stabilisation is higher when the Rf residue is introduced at the 3'-end. Further, DSC analysis has revealed that the Rf residues introduced at the 3'-end are able to form additional stabilising interactions, energetically almost comparable to the enthalpic contribution of a G-tetrad. Fluorescence measurement are consistent with this result showing that the Rf residues introduced at 3'-end are able to form stacking interactions with the adjacent bases within the G-quadruplex structure. The whole of data suggested that the introduction of Rf unit can stabilize G-quadruplex structures and can be a promising candidate for future theranostic applications., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Riboflavin (vitamin B 2 ) is an essential nutrient for humans and animals that must be obtained from the diet. To ensure an optimal supply, riboflavin is used on a large scale as additive in the food and feed industries. Here, we describe a historical overview of the industrial process of riboflavin production starting from its discovery and the need to produce the vitamin in bulk at prices that would allow for their use in human and animal nutrition. Riboflavin was produced industrially by chemical synthesis for many decades. At present, the development of economical and eco-efficient fermentation processes, which are mainly based on Bacillus subtilis and Ashbya gossypii strains, has replaced the synthetic process at industrial scale. A detailed account is given of the development of the riboflavin overproducer strains as well as future prospects for its improvement.

Consumers are increasingly becoming aware of their health and nutritional requirements, and in this context, vitamins produced in situ by microbes may suit their needs and expectations. B groups vitamins are essential components of cellular metabolism and among them riboflavin is one of the vital vitamins required by bacteria, plants, animals and humans. Here, we focus on the importance of microbial production of riboflavin over chemical synthesis. In addition, genetic abilities for riboflavin biosynthesis by lactic acid bacteria are discussed. Genetically modified strains by employing genetic engineering and chemical analogues have been developed to enhance riboflavin production. The present review attempts to collect the currently available information on riboflavin production by microbes in general, while placing greater emphasis on food grade lactic acid bacteria and human gut commensals. For designing riboflavin-enriched functional foods, proper selection and exploitation of riboflavin-producing lactic acid bacteria is essential. Moreover, eliminating the in situ vitamin fortification step will decrease the cost of food production., (© 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Photodynamic inactivation of bacteria (PIB) proves to be an additional method to kill pathogenic bacteria. PIB requires photosensitizer molecules that effectively generate reactive oxygen species like singlet oxygen when exposed to visible light. To allow a broad application in medicine, photosensitizers should be safe when applied in humans. Substances like vitamin B2, which are most likely safe, are known to produce singlet oxygen upon irradiation. In the present study, we added positive charges to flavin derivatives to enable attachment of these molecules to the negatively charged surface of bacteria. Two of the synthesized flavin derivatives showed a high quantum yield of singlet oxygen of approximately 75%. Multidrug resistant bacteria like MRSA (Methicillin resistant Staphylococcus aureus), EHEC (enterohemorrhagic Escherichia coli), Pseudomonas aeruginosa, and Acinetobacter baumannii were incubated with these flavin derivatives in vitro and were subsequently irradiated with visible light for seconds only. Singlet oxygen production in bacteria was proved by detecting its luminescence at 1270 nm. After irradiation, the number of viable bacteria decreased up to 6 log10 steps depending on the concentration of the flavin derivatives and the light dosimetry. The bactericidal effect of PIB was independent of the bacterial type and the corresponding antibiotic resistance pattern. In contrast, the photosensitizer concentration and light parameters used for bacteria killing did not affect cell viability of human keratinocytes (therapeutic window). Multiresistant bacteria can be safely and effectively killed by a combination of modified vitamin B2 molecules, oxygen and visible light, whereas normal skin cells survive. Further work will include these new photosensitizers for topical application to decolonize bacteria from skin and mucosa.

Robust chemical routes towards valuable bioactive entities such as riboflavines, quinoxalinones and benzodiazepines are described. These make use of modern flow hydrogenation protocols enabling the chemoselective reduction of nitro group containing building blocks in order to rapidly generate the desired amine intermediates in situ. In order to exploit the benefits of continuous processing the individual steps were transformed into a telescoped flow process delivering selected benzodiazepine products on scales of 50 mmol and 120 mmol respectively.

Two different qualities of riboflavin (RF) i.e., synthetic (RFs) and biosynthetic riboflavin (RFbs) have been investigated with respect to photoinduced color change in the solid state. Several methods (XRD, FT-IR, VIS-, NIR- and fluorescence spectroscopy) were employed to elucidate the properties of the crystalline structure of RFs and RFbs and the influence of irradiation on the color and structural changes of the samples in the solid state. It was shown that RFs an RFbs represent two different crystal modifications of riboflavin and that RFbs can easily be transformed into a dihydrate upon exposure to humidity. Based on the observed irreversible color change and reduction in fluorescence intensity upon irradiation, an irreversible photoreduction of the molecule was assumed in case of RFs. A more pronounced, reversible color change and reversible reduction in fluorescence intensity indicated a reversible photoreduction process in the case of RFbs. The mechanism of these processes was further investigated by means of NIR and FT-IR spectrophotometry. It is apparent from the current study that the crystal modification of RF can strongly influence the solid state photochemistry of this molecule.

Flavocoenzymes with selective or universal stable isotope labeling are important tools for the investigation of flavoproteins using a variety of spectroscopic methods. Numerous selectively labeled flavin isotopologs can be generated by the combined application of chemical synthesis and in vitro biotransformation using commercially available enzymes and/or recombinant riboflavin biosynthesis enzymes. Notably, the complex reaction sequences can be rapidly carried out using enzyme-assisted one-pot reaction strategies.

The article presents extended computer investigations of various sulfate derivatives of riboflavin. A number of physicochemical parameters such as total energy, binding energy and formation heat were calculated via semi-empirical methods AM1 and PM3 for the different derivatives of riboflavin. Their analysis made it possible to determine the sequence of formation of sulfate derivatives--esterification is the easiest at hydroxyl groups at the farthest positions from the ring. This methodology may be used to study biologically active compounds.

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.

The discovery of vitamins as essential factors in the diet was a scientific breakthrough that changed the world. Diseases such as scurvy, rickets, beriberi, and pellagra were recognized to be curable with an adequate diet. These diseases had been prevalent for thousands of years and had a dramatic impact on societies as well as on economic development. This Review highlights the key achievements in the development of industrial processes for the manufacture of eight of the 13 vitamins., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A novel optically active polymer consisting of riboflavin units as the main chain (poly-1) was prepared from naturally occurring riboflavin (vitamin B(2)) in three steps. The riboflavin residues of poly-1 were converted to 5-ethylriboflavinium cations (giving poly-2), which could be reversibly transformed into the corresponding 4a-hydroxyriboflavins (giving poly-2OH) through hydroxylation/dehydroxylation reactions. This reversible structural change was accompanied by a visible color change along with significant changes in the absorption and circular dichroism (CD) spectra. The nuclear Overhauser effect spectroscopy (NOESY) and CD spectra of poly-2 revealed a supramolecularly twisted helical structure with excess one-handedness through face-to-face stacking of the intermolecular riboflavinium units, as evidenced by the apparent NOE correlations between the interstrand riboflavin units and intense Cotton effects induced in the flavinium chromophore regions. The hydroxylation of poly-2 at the 4a-position proceeded in a diastereoselective fashion via chirality transfer from the induced supramolecular helical chirality assisted by the ribityl pendants, resulting in a 83:17 diastereomeric mixture of poly-2OH. The diastereoselectivity of poly-2 was remarkably higher than that of the corresponding monomeric model (64.5:35.5), indicating amplification of the chirality resulting from the supramolecular chirality induced in the stacked poly-2 backbones. The optically active poly-2 efficiently catalyzed the asymmetric organocatalytic oxidation of sulfides with hydrogen peroxide, yielding optically active sulfoxides with up to 60% enantiomeric excess (ee), whose enantioselectivity was higher than that catalyzed by the monomeric counterpart (30% ee). In addition, upon exposure to primary and secondary amines, poly-2 exhibited unique high-speed vapochromic behavior arising from the formation of 4a-amine adducts in the film.

Riboflavin synthase catalyzes the transfer of a four-carbon fragment between two molecules of the substrate, 6,7-dimethyl-8-ribityllumazine, resulting in the formation of riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Earlier, a pentacyclic adduct formed from two substrate molecules was shown to be a catalytically competent intermediate, but the mechanism of its formation is still poorly understood. The present study shows that the recombinant N-terminal domain of riboflavin synthase from Escherichia coli interacts specifically with the exomethylene-type anion of 6,7-dimethyl-8-ribityllumazine but not with any of the tricyclic adduct-type anions that dominate the complex anion equilibrium in aqueous solution. Whereas these findings can be implemented into previously published mechanistic hypotheses, we also present a novel, hypothetical reaction sequence that starts with the transfer of a hydride ion from the 6,7-dimethyl-8-ribityllumazine exomethylene anion to an electroneutral 6,7-dimethyl-8-ribityllumazine molecule. The pair of dehydrolumazine and dihydrolumazine molecules resulting from this hydride transfer is proposed to undergo a 4 + 2 cycloaddition, affording the experimentally documented pentacyclic intermediate. In contrast to earlier mechanistic concepts requiring the participation of a nucleophilic agent, which is not supported by structural and mutagenesis data, the novel concept has no such requirement. Moreover, it requires fewer reaction steps and is consistent with all experimental data.

A series of novel dexibuprofen derivatives covalently linked via alkylene spacers of variable length to tetraacetylated riboflavin have been developed. The target compounds became accessible by reaction of the chloromethyl ester of dexibuprofen with tetraacetylriboflavin (compound 7) or by synthesis of the appropriate N3-(omega-iodoalkyl)-2',3',4',5'-Tetraacetylriboflavin followed by treatment with dexibuprofen (derivatives 8-11), respectively. Biological screening revealed that the target compounds exhibit antiproliferative effects on MCF-7 breast cancer and HT-29 colon carcinoma cells with IC50 values in the range of 8-15 microM. Enzymatic studies on human platelets indicated significant COX-1 inhibitory activities of the target compounds.

An accurate and simple analysis method by high-performance liquid chromatography-mass spectrometry for the riboflavin crude product of semisynthesis is established in this study. In this method, a C(18) column is used, and the mobile phase is methanol-water. Under the optimal analysis conditions established in this study, all components in the riboflavin crude product are well separated and identified, respectively. The external standard method is used for the quantitative analysis, and the results are proven to be accurate.

D-Ribose is a functional five-carbon sugar, which has been used for the commercial production of riboflavin. Mechanisms of d-ribose biosynthesis from xylose were investigated in the genetically engineered Bacillus subtilis JY200 with a deficiency in transketolase. A transketolase gene (tkt) disruption cassette in plasmid pUNKC was introduced into the chromosomal tkt gene in the wild type B. subtilis 168. Analysis of culture broth by thin layer chromatography confirmed that the disruption of tkt allowed B. subtilis JY200 to produce d-ribose. In a batch culture of B. subtilis JY200, a loss of cell viability was observed after glucose depletion. Fed-batch cultivation by feeding 400 gl(-1) glucose solution as a co-substrate was carried out to supply energy to xylose metabolism and to maintain cell viability throughout cultivation. Fed-batch cultivation of B. subtilis JY200 in a complex medium containing 11 gl(-1) xylose and 5 gl(-1) glucose initially gave the best result of 10.1 gl(-1)D-ribose concentration, 0.24 gg(-1)D-ribose yield and 0.29 gl(-1)h(-1) productivity, corresponding to 40-, 5- and 12-fold increases compared with those in the batch culture. A kinetic study of D-ribose production in fed-batch cultivations of B. subtilis JY200 suggested that xylose uptake might be critical to maximize D-ribose biosynthesis from xylose.

The cofactor flavin adenine dinucleotide (FAD) is required for the catalytic activity of a large class of enzymes known as flavoenzymes. Because flavin cofactors participate in catalysis via a number of different mechanisms, isoalloxazine analogues are valuable for mechanistic studies. We report improved chemical syntheses for the preparation of the two key analogues, 5-deazariboflavin and 1-deazariboflavin.

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%.

Chemical riboflavin production, successfully used for decades, is in the course of being replaced by microbial processes. These promise to save half the costs, reduce waste and energy requirements, and use renewable resources like sugar or plant oil. Three microorganisms are currently in use for industrial riboflavin production. The hemiascomycetes Ashbya gossypii, a filamentous fungus, and Candida famata, a yeast, are naturally occurring overproducers of this vitamin. To obtain riboflavin production with the gram-positive bacterium Bacillus subtilis requires at least the deregulation of purine synthesis and a mutation in a flavokinase/FAD-synthetase. It is common to all three organisms that riboflavin production is recognizable by the yellow color of the colonies. This is an important tool for the screening of improved mutants. Antimetabolites like itaconate, which inhibits the isocitrate lyase in A. gossypii, tubercidin, which inhibits purine biosynthesis in C. famata, or roseoflavin, a structural analog of riboflavin used for B. subtilis, have been applied successfully for mutant selections. The production of riboflavin by the two fungi seems to be limited by precursor supply, as was concluded from feeding and gene-overexpression experiments. Although flux studies in B. subtilis revealed an increase both in maintenance metabolism and in the oxidative part of the pentose phosphate pathway, the major limitation there seems to be the riboflavin pathway. Multiple copies of the rib genes and promoter replacements are necessary to achieve competitive productivity.

Nekoflavin, which was found in cat's choroids [Matsui, K. (1965) J. Biochem. 57, 201-206], was identified as 7 alpha-hydroxyriboflavin by comparing the physicochemical properties of nekoflavin acetate with those of chemically synthesized 7 alpha-hydroxyriboflavin pentaacetate. 7 alpha-Hydroxyriboflavin was synthesized from 4-chloro-2-methylbenzonitrile through 7-cyano-7-demethylriboflavin and 7 alpha-aminoriboflavin, which are also new flavins.

With the goal of generating a novel fluorine-containing flavin analogue with a reduction potential the same as normal flavin, 2'-fluoro-2'-deoxy-D-arabinoflavin has been synthesized. In its riboflavin and FAD forms, UV-visible spectral properties are similar to those of normal flavins, and tight binding to riboflavin binding protein and mercuric ion reductase occurs with very similar spectral changes. The reduction potential of the 2'-FaFAD analogue is determined to be -207 mV compared with -206 mV for FAD, indicating that the intervening 1'-methylene group insulates the redox-active isoalloxazine from the 2'-fluorine. With the intent of using the analogue as a fluorine NMR probe of the active site environments of two-electron-reduced mercuric ion reductase, apoenzyme was reconstituted and its behavior under reducing conditions examined. Whereas with normal enzyme, addition of two electrons gives rapid formation of a charge-transfer species where FAD remains oxidized and a disulfide is reduced to a thiol/thiolate pair, with the 2'-FaFAD enzyme, addition of two electrons gives rapid reduction of the flavin followed by slow transfer of electrons to the disulfide with very little development of the typical charge-transfer absorption. Analysis of crystal structure data suggests that having the fluorine in the alternate arabino stereochemistry places it much nearer the flavin-proximal cysteine/cystine sulfur, where it may inhibit both electron transfer from reduced flavin and the charge-transfer interaction between reduced thiolate and FAD.

The reduction potential of flavin bearing a methylsulfonyl moiety (MeSO2) in place of a methyl group at position 8 is increased by more than 150 mV as compared with normal flavin. This substitution is accompanied by a substantial increase in reactivity with various reductants, including NADH, and greatly (10(3)-fold) enhanced susceptibility toward nucleophilic attack by sulfite at N(5). 1,5-Dihydro-8-(methylsulfonyl)riboflavin exhibits two intense, well-resolved absorption bands (lambda max = 310, 362 nm) in a region where most other reduced flavins exhibit weak, characterless absorption. This unusual spectrum is attributable to a shift of pi-electron density from the N(5) atom into the benzene ring. It is observed only with reduced flavins bearing a strongly electronegative substituent (MeSO2, CN) at the 8-position. The effect is abolished by replacing the hydrogen at N(5) with a bulky group, like sulfite, which interferes with sp2 hybridization at N(5). Reaction of 8-MeSO2-substituted flavins with thiols results in nucleophilic displacement of MeSO2- in a reaction that is about 10(3)-fold faster than an analogous nucleophilic displacement reaction observed with 8-halo-substituted flavins. The flavin ring acts as a redox switch in controlling electrophilicity at the 8-position, as judged by the fact that the displacement reactions are observed only with the oxidized flavins. Initial studies to evaluate 8-MeSO2-substituted flavins as active site probes were conducted with flavodoxin from Clostridium beijerinckii MP. 8-MeSO2FMN is rapidly bound to apoflavodoxin, accompanied by absorbance and fluorescence changes similar to those observed for FMN binding. 1,5-Dihydro-8-MeSO2FMN flavodoxin exhibits spectral properties (lambda max = 323, 382 nm) similar to those of the corresponding free flavin, except for a bathochromic shift due to a change in the polarity of the flavin environment. As judged by peak resolution and intensity, the spectral properties of 1,5-dihydro-FMN flavodoxin (lambda max = 311, 362 nm) appear to lie about midway between those observed for the free 1,5-dihydro forms of FMN versus 8-MeSO2FMN. This suggests that the protein environment may favor enhanced resonance delocalization of pi-electron density into the benzene ring of bound 1,5-dihydro-FMN, as compared with the free flavin. This hypothesis is consistent with previous NMR studies and with a proposal that electron transfer from reduced flavodoxin to other redox proteins occurs through this region of the ring. 8-MeSO2FMN bound to flavodoxin reacts readily with exogenous thiols but does not react with sulfite.(ABSTRACT TRUNCATED AT 400 WORDS)

The 5,6-dimethylbenzimidazole unit of vitamin B12 is formed from riboflavin via FMN in aerobic and some aerotolerant bacteria. Thereby C-1' of the ribityl side chain gets C-2 of 5,6-dimethylbenzimidazole. Experiments with homogenates of Propionibacterium shermanii on the fate of C-atoms 2' to 5' of the ribityl side chain of riboflavin in this transformation are reported. It was found that [5'-3H]riboflavin leads to radioactive sugar phosphates. These were isolated, dephosphorylated, and separated. Thus 3H-D-fructose and 3H-D-glucose were detected. The degradation of 3H-D-glucose revealed that 14 per cent of the total radioactivity was located in C-1, and 86 per cent in C-6. This indicates that during 5,6-dimethylbenzimidazole biosynthesis a three carbon unit is formed from the ribityl side chain of riboflavin.

8-Thiocyanatoflavins at the riboflavin, FMN, and FAD level were prepared via the diazonium salt of the corresponding 8-aminoflavin and some of the physical and chemical properties studied. 8-Thiocyanatoriboflavin has a UV-visible spectrum similar to that of the native flavin with absorbance maxima at 446 nm (epsilon = 14,900 M-1 cm-1) and 360 nm. Reaction with thiols such as dithiothreitol and mercaptoethanol gives rise to an 8-mercapto- and an 8-SR-flavin, whereas reaction with sulfide yields only the 8-mercaptoflavin. The 8-SCN-flavin binds to riboflavin-binding protein as the riboflavin derivative, to apoflavodoxin, apo-Old Yellow Enzyme, and apo-lactate oxidase as the FMN derivative, and to apo-D-amino acid oxidase, apo-p-hydroxybenzoate hydroxylase, apo-glucose oxidase, apo-anthranilate hydroxylase, and apo-general acyl-CoA dehydrogenase as the FAD derivative. In two cases, namely, with anthranilate hydroxylase and D-amino acid oxidase, the 8-SCN-FAD was spontaneously and completely converted to the 8-mercapto-FAD derivative, suggesting the presence of a nucleophile (most likely the thiol of a cysteine residue) in the vicinity of the 8-position. It was also found that flavodoxin stabilizes the neutral radical and Old Yellow Enzyme the anionic radical of 8-SCN-FMN. Further studies with Old Yellow Enzyme, established that fully (two electron) reduced 8-SCN-FMN undergoes photoelimination of cyanide.

The chemical synthesis of riboflavin 5'-phosphorothioate (5'-FMNS) is described. 5'-FMNS is obtained from the alkaline hydrolysis of riboflavin 4',5'-cyclic phosphorothioate, which is produced upon reaction of riboflavin (RB) with thiophosphoryl chloride in trimethyl phosphate. 5'-FMNS has been tested for enzymatic reconstitution of NADPH-cytochrome P-450 reductase (EC 1.6.2.4) depleted of its FMN prosthetic group, but containing its full complement (1 equiv) of FAD. The synthesis, purification, and characterization of 5'-FMNS are reported, and documentation of its efficacy in reconstituting the reductase by fluorometric and absorbance spectrophotometric measurements, as well as enzymatic activity, is presented. Data indicate that 5'-FMNS is totally competent in reconstituting NADPH-cytochrome c reductase activity, which requires the presence of both FAD and a flavin mononucleotide, and its fluorescence is completely quenched upon addition to FMN-free NADPH-cytochrome P-450 reductase.

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-6 alpha, C-6, C-7 or C-7 alpha. [6 alpha, 7 alpha-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.

Various 8 alpha-sulfur-linked peptides related to the flavinyl peptides isolated from mitochondrial monoamine oxidase were synthesized in high yield and purity. The peptides, protected by an acetyl-blocking group on the amino terminus, were synthesized by conventional liquid-phase techniques and coupled to a tetraacetylriboflavin derivative activated in the 8alpha position. In some cases, the ribityl side chains of the flavinyl peptides were selectively deacetylated. In other cases, the thioether functions were oxidized to form sulfones. These flavinyl peptides were studied by uv-visible absorption and circular dichroic spectroscopies. A close correspondence in spectroscopic and other chemical properties indicated the identity of the synthetic and naturally obtained flavinyl peptides. Differences between the tetraacetylriboflavinyl and riboflavinyl peptides indicate an interaction between the ribityl side chain and thioether function in aqueous media. Evidence was obtained for an intramolecular complex between the tyrosyl and isoalloxazine moieties in aqueous media. Substitution in the 8alpha position was accompanied by an impairment of the protonation of the N1 position of the isoalloxazine ring and a lowering of the redox potential relative to the parent 8-methyflavins.

The synthesis of the 2,4-diamino analogue of riboflavin is described. Inhibition analysis in a microbial assay system indicated that this compound has a weak antifolate activity that could be overcome with a minimal amount of folinic acid, but at higher concentrations both folinic acid and riboflavin were required for the reversal of its inhibitory effect.

Several new halogen-containing riboflavin analogs that are antagonists for the stock strain of Lactobacillus casei have been found to be utilized by a mutant strain of the microorganism for growth and acid production. Riboflavin analogs consisting of all combinations of a single aliphatic group (methyl or ethyl) and a single halogen group (chloro or bromo) in positions 7 and 8 of the isoalloxazine nucleus possess this property. The analog devoid of substituents in positions 7 and 8 and the analog possessing a methyl group in position 7 and a cysteinyl group in position 8, were inert for both strains of the microorganism.

A number of derivatives of riboflavin and of 3-methyllumiflavin substituted in the 6 position have been synthesized starting with 6-nitro flavins, reduction to the 6-amino flavin, and diazotization, followed by reaction with the appropriate nucleophile. The absorption spectra, oxidation-reduction potentials, and the electron spin resonance spectra of the radical cationic forms of several of these synthetic compounds have been determined, including 6-S-cysteinyl-3-methyllumiflavin and 6-S-cysteinylriboflavin. The latter has been shown to be identical with the dephosphorylated form of the aminoacyl flavin isolated from trimethylamine dehydrogenase [Steenkamp, D. J., Kenney, W. C. & Singer, T. P. (1978) J. Biol. Chem. 253, 2812-2817; Steenkamp, D. J., McIntire, W., & Kenney, W. C. (1978) J. Biol. Chem. 253, 2818-2824] in regard to absorption specturm, photochemical properties, and mobility in high-voltage electrophoresis and in thin-layer chromatography. An unusually pronounced interaction between the amino group and the isoalloxazine ring system was deduced from the absorption spectra of 6-amino-3-methyllumiflavin and 6-aminoriboflavin.

A yellow chromophore has been isolated from Avena coleoptiles grown in the dark. It had previously been shown by Zenk [Zenk, M. H. (1967) Z. Pflanzenphysiol. 56, 122 - 140] to be a flavin of still unidentified structure, and had been suggested to be the 'blue light' photoreceptor in this organism. The structure of this flavin (F1-X) has been identified as 5'-malonylriboflavin by a combination of physicochemical techniques, and by its identity with a sample obtained synthetically. The 5'-malonylester linkage is relatively labile towards hydrolysis and photolysis, it isomerizes to an equilibrium mixture containing probably the 4' isomer. The electronic spectra of 5'-malonylriboflavin (absorption and fluorescence) are practically identical to those of normal riboflavin (vitamin B2).