Riboflavin Derivatives Research Articles

Antimutagenic effects and possible mechanisms of action of vitamins and related compounds against genotoxic heterocyclic amines from cooked food

Possible antimutagenic activity of 26 vitamins and related compounds — ascorbic acid, β-carotene, cyanocobalamin, folic acid, nicotinic acid, nicotinamide, pantothenic acid, pyridoxale, pyridoxamine, pyridoxine, retinal, retinol, retinoic acid, retinyl acetate, retinyl palmitate, riboflavin, riboflavin 5′-phosphate, flavin adenine dinucleotide (FAD), α-tocopherol, α-tocopherol acetate, vitamins K 1, K 3, K 4, 1,4-naphthoquinone, and coenzyme Q 10 — was tested against six heterocyclic amine (HCA) mutagens, i.e., 2-amino-3-methyl-imidazo[4,5- f]quinoline (IQ), 2-amino-3,4-dimethyl-imidazo[4,5- f]quinoline (MeIQ), 2-amino-3,8-dimethyl-imidazo[4,5- f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP), 2-amino-6-methyl-dipyrido[1,2- a:3′,2′- d]imidazole (Glu-P-1) and 3-amino-1-methyl-5 H-pyrido[4,3- b]indole (Trp-P-2) in the Salmonella/reversion assay using tester strains Salmonella typhimurium TA 98 and TA 100. Retinol, retinal, riboflavin, riboflavin 5′-phosphate, FAD, vitamins K 1, K 3, K 4, 1,4-naphthoquinone, and coenzyme Q 10 caused a concentration-dependent decrease in the mutagenicity of all six mutagens in both tester strains. Quantification of antimutagenic potencies by calculating ID 50 values (inhibitory dose for 50% reduction of mutagenic activity) from dose–response curves resulted in the following data (nmol/plate): retinol: 480–1400; retinal: 268–441; riboflavin: 25–>100; riboflavin 5′-phosphate: 800–4723; FAD: 970–>1000; vitamin K 1: 401–740; vitamin K 3 (menadione): 85–590; vitamin K 4: 45–313; 1,4-naphthoquinone: 170–290; coenzyme Q 10: 490–860. In general, there were no major differences between HCAs tested except in part with Trp-P-2 nor between the two tester strains. In enzyme kinetic experiments with Salmonella, retinol, vitamins K 3, and K 4 behaved as competitive inhibitors of IQ induced mutagenesis. However, at the highest concentration of menadione (200 nmol/plate) and of riboflavin 5′-phosphate (2000 nmol/plate), non-competitive inhibition was observed. At other concentrations of riboflavin 5′-phosphate and at all concentrations of FAD, meaningful interpretation of enzyme kinetics were not possible. Reduction of the activity of 7-ethoxy- and 7-methoxyresorufin- O-dealkylases with IC 50 values of 2.03–30.8 μM indicated strong inhibition of 1A1 and 1A2 dependent monooxygenases by menadione and retinol. Riboflavin 5′-phosphate and FAD were less effective (IC 50: 110–803.7 μM). Nicotinamide-adenine-dinucleotidephosphate (NADPH) cytochrome P-450 reductase was not affected by retinoids but stimulated by naphthoquinones and both riboflavin derivatives up to about 50 and 80%, respectively. Again, the mutagenic activity of N-hydroxy-2-amino-3-methyl-imidazo[4,5- f]quinoline ( N-OH-IQ) in Salmonella was not suppressed by K-vitamins but marginally reduced by retinol, retinal, and FAD but distinctly by riboflavin 5′-phosphate. In various experiments designed for modulation of the mutagenic response, inhibition of metabolic activation of IQ to N-OH-IQ was found to be the only relevant mechanism of antimutagenesis of menadione while a weak contribution of an other way seemed possible for retinol and FAD.

And One for Migraine Prophylaxis.

Since no treatment for established migraine headaches is likely to approach 100 percent success, the search for effective and safe preventive interventions continues. This study examines an unlikely candidate, riboflavin (vitamin B2). Riboflavin derivatives …

Dynamically modified, biospecific optical fiber sensor for riboflavin binding protein based on hydrophobically associated 3-octylriboflavin.

A new approach to the production of optical fiber sensors is described which is based on a dynamic modification procedure. In this approach, the optical fiber surface is rendered hydrophobic through the covalent attachment of a C18 moiety. Specific sensing ligands are then associated with this surface through either their inherent or designed hydrophobicity. To investigate the feasibility of the approach, an optical fiber sensor has been constructed for riboflavin binding protein in which 3-octylriboflavin is associated with the fiber surface. Fluorescence quenching occurs upon binding of the protein to the immobilized riboflavin derivative. The sensor possesses a minimum measurable quantity of 7.3 pmol of binding protein in a probe volume which is less than 10 microL. With this approach, the sensing surface was repetitively regenerated 15 times over a 1-h period with less than a 5% variation in sensor-to-sensor performance. The approach is general, and with minor variations it can be used in a variety of sensing situations.

8-thiocyanatoflavins as active-site probes for flavoproteins.

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.

Lampteromyces bioluminescence — 5 chemical synthesis of lampteroflavin as mushroom light emitter

Lampteroflavin was synthesized through coupling between 5-trityl-2,3-(p-methoxy)benzylidene-1-β-D-ribofuranosyl trichloacetimidate and 2′,4′-(p-methoxy)benzylidene derivative of riboflavin to produce the crucial alpha riboside. Chloroethyl group was removed under neutral condition.

Carbon-13, nitrogen-15, and phosphorus-31 NMR studies on 6-hydroxy-L-nicotine oxidase from Arthrobacter oxidans

The interaction between the apoprotein of 6-hydroxy-L-nicotine oxidase from Arthrobacter oxidans and the prosthetic group FAD has been investigated by 13C, 15N, and 31P NMR techniques. The FAD prosthetic group was selectively enriched in 13C and 15N isotopes by adding isotopically labeled riboflavin derivatives to the growth medium of riboflavin-requiring mutant cells. In the oxidized state the chemical shift of the C(7) and C(8) atoms indicates that the xylene moiety of the isoalloxazine ring is embedded in a hydrophobic environment. The polarization of the isoalloxazine ring as a whole is, however, much more comparable to that of free flavin in a polar and protic environment than to free flavin in an apolar environment. The polarization of the ring system can be ascribed to strong hydrogen bonds between the apoprotein and the two carbonyl groups. The binding of the competitive inhibitor, 6-hydroxy-D-nicotine, influences the resonances of the C(4a) and the N(5) atoms strongly. It is suggested that these shifts are due to a strong hydrogen-bonding interaction between the N(5) atom and the inhibitor. On reduction all resonances, except those of the C(10a) and the N(1) atoms, shift upfield, indicating the increased electron density in the ring system. In the dithionite-reduced enzyme, the ring system is bent at the N(5) position. Due to the bending of the N(5) atom and the sp2 hybridized N(10) atom, electron density from the N(10) atom is reallocated at the C(4) carbonyl group. In contrast, in the substrate-reduced enzyme the N(5) atom is almost completely sp2 hybridized, yielding a rather planar isoalloxazine ring.(ABSTRACT TRUNCATED AT 250 WORDS)

Flavin-sensitized photo-oxidation of unsaturated fatty acids

Photo-oxidation of unsaturated fatty acids or esters (LH = oleic acid, linoleic acid, methyl linoleate, and linolenic acid) by oxygen proceeds efficiently in the presence of a riboflavin derivative, riboflavin-2′,3′,4′,5′-tetra-acetate (Fl), in acetonitrile by a type II (singlet oxygen) mechanism with a limiting quantum yield (φ= 0.65) which corresponds to the quantum yield of the intersystem crossing of the flavin. The addition of HClO4—by which Fl is protonated to FlH+—showed a remarkable inhibitory effect on the Fl-sensitized photo-oxidation of LH, since FlH+ is photo-reduced by LH to yield the dihydroflavin radical cation FlH2+˙ which has no ability to sensitize the energy transfer to oxygen in its ground state.

Attachment of riboflavin derivatives to electrode surfaces

A procedure is described for the site-specific immobilization of riboflavin cofactors on platinum, gold and carbon electrode surfaces. The cofactors were bound to each electrode surface through the 8-methyl group of the flavin moiety. Stable monolayer coverages of flavin coenzymes were formed by initially coating each electrode surface with 3-mercaptopropylsilane followed by reaction with 8-α-bromo- O-tetra-acetylriboflavin. The mild derivatization conditions developed in this study were necessary to avoid competing side reactions, including the decomposition of flavin derivatives. Different methods of synthesizing these flavin-silane-modified electrodes were examined. In addition, the potential versus pH behavior of these flavin-modified electrodes is reported.

Use of a polymer‐bound flavin derivative for the rapid regeneration of NAD(P)+ from NAD(P)H in dehydrogenase systems

An aldehyde derivative of riboflavin was covalently attached by reductive alkylation to soluble polycationic supports. The flavopolymers so obtained were stable under operational conditions. The catalytic efficiency towards oxidation of NADH by these flavopolymers was demonstrated, and the kinetic parameters (Km and kcat) revealed an overall catalytic efficiency (kcat/Km) 185-fold greater compared to riboflavin. Various factors affecting the chemical regeneration of NAD+ from NADH such as pH, ionic strength, nature of the buffer etc. were studied. The most interesting result was the highly favourable influence of borate ions which increased the reaction rate by a factor 2-4 compared to the other buffers. The flavopolymers are very effective for in situ recycling of NAD(P)+. With up to 300-fold NADH----NAD+ conversions for the system using yeast alcohol dehydrogenase and up to 1500-fold NADPH----NADP+ regenerations for the system using glucose-6-phosphate dehydrogenase. These flavopolymers are superior to previous chemical recycling systems.

Clarification and Quantitation of Primary (Tissue) and Secondary (Microbial) Catabolites of Riboflavin That Are Excreted in Mammalian (Rat) Urine

Riboflavin derivatives were quantitated and identified in urine of rats fed 0, 2 and 6 µg riboflavin/g diet per day both with and without added succinyl sulfathiazole for 6 wk. Two rats from each dietary group were placed in metabolic cages and urine was collected in the dark for 24 h. On the fourth week, a third animal from each group received an i.p. injection of [2-14C]riboflavin before being placed in a metabolic cage and urine collected in the dark for 48 h. Urine samples were extracted with phenol for flavin components and with chloroform for lumichrome and derivatives. Riboflavin was the predominant flavin excreted by rats in all dietary groups, followed by hydroxymethylriboflavins and smaller amounts of flavin mononucleotide (FMN), lumiflavin and 10-hydroxyethylflavin. Carboxylumichromes accounted for 5–10% of the total flavin-derived fluorescence in urine of rats fed 2 and 6 µg riboflavin/g diet and were reduced to ∼3% when sulfathiazole was added to the base diets. Carboxylumichromes were absent from urine of riboflavin-deficient rats. Riboflavin accounted for 85–90% of the recovered radioactivity of all radioactive urine extracts; no radioactively labeled carboxylumichromes were detected. These results indicate that hydroxymethylriboflavins are primary catabolites of riboflavin derived from tissue microsomal oxidations, whereas carboxylumichromes reflect the continued oxidation of ring hydroxymethyl functions plus gut microbial cleavage of the side chain of flavin.

PHOTOCHEMICAL PROPERTIES OF FLAVIN DERIVATIVES

Abstract— The triplet yields of 15 derivatives of riboflavin modified either in position 8 or in the ribityl side chain have been determined. The triplet yields measured range from 0.002 to 0.9. In addition, estimates for the rate constants of the photoreaction with EDTA and for the complex formation constants with EDTA at pH 7.0 are given. Evidence for an interaction in solution of at least the 2′ and 5′ hydroxyl groups of RF with the isoalloxazine ring is presented.

6-Thiocyanatoflavins and 6-mercaptoflavins as active-site probes of flavoproteins.

6-Thiocyanatoflavins have been found to be susceptible to nucleophilic displacement reactions with sulfite and thiols, yielding respectively the 6-S-SO3--flavin and 6-mercaptoflavin, with rate constants at pH 7.0, 20 degrees C, of 55 M-1 min-1 for sulfite and 1000 M-1 min-1 for dithiothreitol. The 6-SCN-flavin binds tightly to riboflavin-binding protein as the riboflavin derivative, to apoflavodoxin, apo-lactate oxidase, and apo-Old Yellow Enzyme as the FMN derivative, and to apo-D-amino acid oxidase as the FAD derivative. The riboflavin-binding protein derivative is inaccessible to dithiothreitol attack, and the lactate oxidase and D-amino acid oxidase derivatives show only limited accessibility. However, the flavodoxin and Old Yellow Enzyme derivatives react readily with dithiothreitol, indicating that the flavin 6-position is exposed to solvent in these proteins. The lactate oxidase and D-amino acid oxidase derivatives convert slowly but spontaneously to the 6-mercaptoflavin enzyme forms in the absence of any added thiol, indicating the presence of a thiol residue in the flavin binding site of these proteins. The reaction rates have been investigated of 6-mercaptoflavins with iodoacetamide, N-ethylmaleimide, methyl methanethiosulfonate, H2O2, and m-chloroperbenzoate, in both the free and protein-bound state. The results confirm the conclusions drawn from the studies with 6-SCN-flavins described above and from 6-N3-flavins [Massey, V., Ghisla, S., & Yagi, K. (1986) Biochemistry (preceding paper in this issue)]. The spectral properties of the protein-bound 6-mercaptoflavin vary widely among the five proteins studied and show stabilization of the neutral flavin with flavodoxin and riboflavin-binding protein and of the anionic species by Old Yellow Enzyme, lactate oxidase, and D-amino acid oxidase. In the case of the latter two enzymes, the stabilization appears to be due to interaction of the negatively charged flavin with a positively charged protein residue located near the flavin pyrimidine ring. This positively charged residue appears to be responsible also for the strong stabilization of the two-electron oxidation state of the mercaptoflavin as the 6-S-oxide. With the other flavoproteins studied this oxidation level is stabilized as the 6-sulfenic acid or 6-sulfenate.

Lnfrared spectra and molecular association of lumiflavin and riboflavin derivatives

Infrared spectra of lumiflavin (Lf), [3-ND]Lf, [3-NMe]Lf, [2- 13C]Lf, [4a- 13C]Lf, [1,3- 15N]Lf, [1,3,5- 15N]Lf, riboflavin (Rf), [3-NMe]Rf, riboflavin-2′,3′,4′,5′-tetraacetate (RfT) and [3-NMe]RfT were measured in their solid and solution states. The vibrational assignments of lumiflavin have been made on the basis of the observed isotope frequency shifts in the i.r. spectra. The differences between the skeletal vibrations of Lf and those of Rf are discussed. The modes of molecular association of Lf and Rf are also proposed based on the i.r. spectra of the carbonyl stretching region.

Molecular interaction between a reduced riboflavin derivative and salicylic acid derivatives

The interaction of reduced riboflavin-2′,3′,4′,5′-tetrabutyrate with salicylic acid, aspirin, and salicylamide has been spectroscopically investigated to determine the binding mechanism. Hydrogen-1 and carbon-13 nuclear magnetic resonance, infrared, and absorption spectra were measured in chloroform-d and chloroform. The association of the reduced riboflavin with salicylic acid derivatives is different from that oxidized one. Salicylic acid and the reduced riboflavin form a cyclic hydrogen bonded complex through the imino (3-N, 5-N) protons and the carbonyl (2-C, 4-C) oxygens of the isoalloxazine ring of the latter, and the carboxylic hydroxyl proton and carbonyl oxygen of the former. Aspirin and the reduced riboflavin form a complex by the same mode as salicylic acid. Salicylamide forms a cyclic hydrogen bonded complex with the reduced riboflavin through the imino (3-N, 5-N) protons and the carbonyl (2-C, 4-C) oxygens of the isoalloxazine ring, and the amino proton and the carbonyl oxygen of salicaylmide. It appears that both the oxidized and reduced form of riboflavin are associated with salicylic acid derivatives.

Interaction between flavins and alcohols

The effect of alcohols on the spectral properties of riboflavin derivatives in non-polar solvent was studied by various spectroscopic methods in order to support the view point that alcohol may directly interact with the isoalloxazine moiety of FAD and enhance the catalytic activity of D-amino acid oxidase (DAAO). The most likely association complex between alcohol and riboflavin is 1 : 1 stoichiometric complex through the 3-N imino and the 2-C carbonyl groups of the isoalloxazine ring and the hydroxyl group of alcohols. It appears that methanol has a larger association constant than any other alcohols, and the association constant decreases with the increase in carbon number and with the steric requirement of the alkyl group of alcohols.

Thermodynamics of association of 8-substituted riboflavins with egg white riboflavin binding protein.

Association constants of 14 species of riboflavin derivatives with hen egg-white riboflavin binding protein were measured by a fluorometric titration method using flavin and protein fluorescence in a temperature range of 10--42 degrees C. The derivatives were riboflavins whose 8-CH3 group was substituted with alkylamino, alkyloxy, halogen or hydrogen. The flavin derivatives were classified into four groups, i.e., RR'N, HRN, RO, and halogen, on the basis of the linear relation between enthalpy and entropy changes (linear free energy relationship), and from the chemical structures of the 8-substituents. The relation between the entropy change and the bulkiness of 8-substituents suggested burying of the substituents in a cavity of the protein, and provided an indication of the size of the substituent-accepting cavity. The relation also suggested an electric repulsion between halogen substituents and the binding site. A very good correlation was found between the constant term of the linear free energy relation and the wavelength of the visible absorption peak of flavins, and this correlation suggested the electronic nature of the flavin-protein interaction.

Interaction of flavins and some alcohols on the molecular level

The effect of some alcohols on the riboflavin derivatives in non-polar solvent was studied by various spectroscopic method in order to support the viewpoint that alcohol may directly interact with the isoalloxazine moiety of FAD, the coenzyme of D-amino-acid oxidase. The most possible association complex between alcohol and riboflavin is the 1∶1 complex through the 2-C carbonyl function of the isoalloxazine ring and the hydroxyl proton of alcohol. It is appeared that methanol has a larger association constant than any other alcohols, and the association constant decreases with the carbon number increases and being bulkier in the alkyl group of alcohols.

Changes in fluorescence spectra during early development of chick embryo

1. 1. As a first step toward identification of the photoreceptor(s) involved in the photoacceleration of chick embryo development, a flourospectrophotometric study was performedon the post-nuclear supernatant from homogenate of embryos 3–6 days old. 2. 2. The cranial lenght of the embryo was used as a staging method, since strict correlation was found between cranial lenght, wet body weight and Hamburger-Hamilton stage within the embryonic growth studied. 3. 3. The stage 16 embryo homogenate exhibited three predominant fluorescence:348nm (exited at 286 nm, proteins), 428 nm (excited at 347 nm, unknown compound) and 520 nm (excited at 380 nm, riboflavin derivative). 4. 4. 348 and 520 nm emissions increased in intensity with age, accompanied by a slight red-shift due to a concentration effect. The 430 nm fluorescence showed a dramatic shift toward 473 nm (excited at 347 nm) by stage 30. 5. 5. The majority of these fluorophores were found in the soluble protein fraction. 6. 6. The photoacceleration of embryonic growth was examined in terms of the changes in proportion of the three fluorophores. No imbalance in accumulation of the fluorophoreswas detectable by fluorometry.

Modification of Riboflavin for coupling to glassy carbon

Riboflavin was acetylated and then modified at the position 8 methyl group to give the 8-bromo and then the 8-triphenylphosphonium bromide derivatives. The phosphonium salt, after the addition of a strong base, was incubated with pieces of glassy carbon that had been oxidized, acylated, and reduced to form surface aldehyde groups. The objective here was to couple the riboflavin derivative to the activated glassy carbon through a double bond bridge by a Wittig type reaction. Cyclic voltammetry measurements on the glassy carbon following the Wittig incubation, showed the appearance of new redox groups with E′ o values of — 0.16 V and — 0.44 V, indicating that coupling may have been accomplished. NMR spectra of the riboflavin derivatives agreed with literature results where comparable data were available. Additional studies are planned for verification of the riboflavin to glassy carbon coupling and for subsequent testing of the coupled riboflavin for cofactor activity with an appropriate apoenzyme.

Chemical synthesis and some properties of 6-substituted flavins.

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.