Search

An operationally simple protocol is disclosed to facilitate entry to benzo-3,4-coumarins directly from biaryl carboxylic acids without the need for substrate prefunctionalization. Complementary to classic lactonization strategies, this disconnection relies on the oxidation competence of photoactivated (-)-riboflavin (vitamin B2) to generate the heterocyclic core via photoinduced single electron transfer. Collectively, the inexpensive nature of the catalyst, ease of execution, and absence of external metal additives are a convincing endorsement for the incorporation of simple vitamins in contemporary catalysis. [ABSTRACT FROM AUTHOR]

We study the effect of hydrotrope nicotinamide on the solubilization of sparingly soluble riboflavin drug molecule. Nicotinamide molecules self-associate through stacking of their pyridine rings, and they also form complexes with riboflavin molecules. Water molecules only prefer to stay at the periphery of the riboflavin molecules, and they are replaced by the hydrotrope molecules with increasing concentration of the solution. The analyses of orientation distributions and distance measurements reveal that the riboflavin and nicotinamide molecules form 1:2 sandwich complexes. It is demonstrated that the self-aggregation of nicotinamide and the complexation between riboflavin and nicotinamide does not have much influence on the number of water-water average hydrogen bonds but they influence the riboflavin-water, nicotinamide-water, riboflavin-riboflavin, riboflavin-nicotinamide, and nicotinamide-nicotinamide hydrogen bonds. Favorable van der Waals interaction energy between riboflavin and nicotinamide plays an important role in the 1:1 or 1:2 complex formation between drug and hydrotrope molecules. The electrostatic energy component of drug and hydrotrope interaction also contributes to the solubilization process. The negative Flory-Huggins interaction parameter value suggests the favorable interactions between the hydrotrope and drug molecules. [ABSTRACT FROM AUTHOR]

Redox active cofactors play a dynamic role inside protein binding active sites because the amino acids responsible for binding participate in electron transfer (ET) reactions. Here, we use femtosecond transient absorption (FsTA) spectroscopy to examine the ultrafast ET between quinacrine (Qc), an antimalarial drug with potential anticancer activity, and riboflavin binding protein (RfBP) with a known K d = 264 nM. Steady-state absorption reveals a ~ 10 nm red-shift in the ground state when QcH3 2+ is titrated with RfBP, and a Stern-Volmer analysis shows ~84% quenching and a blue-shift of the QcH3 2+ photoluminescence to form a 1:1 binding ratio of the QcH3 2+-RfBP complex. Upon selective photoexcitation of QcH3 2+ in the QcH3 2+-RfBP complex, we observe charge separation in 7 ps to form ¹[QcH3_red •+-RfBP•+], which persists for 138 ps. The FsTA spectra show the spectroscopic identification of QcH3_red • +, determined from spectroelectrochemical measurements in DMSO. We correlate our results to literature and report lifetimes that are 10-20× slower than the natural riboflavin, Rf-RfBP, complex and are oxygen independent. Driving force (ΔG) calculations, corrected for estimated dielectric constants for protein hydrophobic pockets, and Marcus theory depict a favorable one-electron ET process between QcH3 2+ and nearby redox active tyrosine (Tyr) or tryptophan (Trp) residues. [ABSTRACT FROM AUTHOR]

The effect of graphene (G) and graphene oxide (GO) dispersions in a triblock copolymer, Kolliphor P407 (poloxamer-407), on the fluorescence of vitamin B2 (riboflavin) has been comparatively investigated. The quality of the dispersions was assessed by measuring the thickness of their flakes and visualizing their distribution within the copolymer and degree of exfoliation via scanning and transmission electron microscopies (SEM and TEM). The influence of G or GO and copolymer concentration on the fluorescence intensity was studied, and fluorescence quenching phenomena were observed; these become more effective with increasing G or GO content for Kolliphor concentrations equal to or above the CMC, the diminution in intensity being stronger for G dispersions, which is related to the different hydrophobicity of the nanomaterials, because these modify the distribution equilibrium of the vitamin between the solution and the Kolliphor micellar aggregates. Further, for a given G or GO concentration, the intensity decreases with increasing copolymer concentration. The ratio between the fluorescence intensity in the absence and the presence of G fits to a second-order polynomial, suggesting a combined mechanism of static and dynamic quenching, while for GO dispersions it follows the Stern-Volmer linear equation in the low concentration range. The quenching observed herein could be useful in the development of optical sensors for riboflavin determination. G-based sensors are expected to have better performance in terms of sensitivity and repeatability than GO-based ones due to their stronger nanomaterial-vitamin interaction, superior quenching efficiency, and higher signal-to-noise ratio. [ABSTRACT FROM AUTHOR]

Roseoflavin is a naturally occurring riboflavin analogue with antibiotic properties. It is biosynthesized from riboflavin in a reaction involving replacement of the C8 methyl with a dimethylamino group. Herein we report the identification of a flavin-dependent enzyme that converts flavin mononucleotide (FMN) and glutamate to 8-amino-FMN via the intermediacy of 8-formyl-FMN. A mechanistic proposal for this remarkable transformation is proposed. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb) Rv2671 is annotated as a 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate (AROPP) reductase (RibD) in the riboflavin biosynthetic pathway. Recently, a strain of Mtb with a mutation in the 5' untranslated region of Rv2671, which resulted in its overexpression, was found to be resistant to dihydrofolate reductase (DHFR) inhibitors including the anti-Mtb drug para-aminosalicylic acid (PAS). In this study, a biochemical analysis of Rv2671 showed that it was able to catalyze the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), which explained why the overexpression of Rv2671 was sufficient to confer PAS resistance. We solved the structure of Rv2671 in complex with the NADP+ and tetrahydrofolate (THF), which revealed the structural basis for the DHFR activity. The structures of Rv2671 complexed with two DHFR inhibitors, trimethoprim and trimetrexate, provided additional details of the substrate binding pocket and elucidated the differences between their inhibitory activities. Finally, Rv2671 was unable to catalyze the reduction of AROPP, which indicated that Rv2671 and its closely related orthologues are not involved in riboflavin biosynthesis. [ABSTRACT FROM AUTHOR]

Generating molecular complexity using a single catalyst, where the requisite activation modes are sequentially exploited as the reaction proceeds, is an attractive guiding principle in synthesis. This requires that each substrate transposition exposes a catalyst activation mode (AM) to which all preceding or future intermediates are resistant. While this concept is exemplified by MacMillan's beautiful merger of enamine and iminium ion activation, examples in other fields of contemporary catalysis remain elusive. Herein, we extend this tactic to organic photochemistry. By harnessing the two discrete photochemical activation modes of (--) -riboflavin, it is possible to sequentially induce isomerization and cyclization by energy transfer (ET) and single-electron transfer (SET) activation pathways, respectively. This catalytic approach has been utilized to emulate the coumarin biosynthesis pathway, which features a key photochemical E → Z isomerization step. Since the ensuing SET-based cyclization eliminates the need for a prefunctionalized aryl ring, this constitutes a novel disconnection of a pharmaceutically important scaffold. [ABSTRACT FROM AUTHOR]

Riboflavin (Rf) is a class of importantvitamins (Vitamin B2) and a well-known antioxidant. Herewe have synthesized nanohybridsof Rf with a number of inorganic nanoparticles (NPs); namely zincoxide (ZnO), titanium oxide (TiO2), aluminum oxide (Al2O3) and gold NPs of similar sizes. While high resolutiontransmission electron microscopy (HRTEM) confirms integrity and sizesof the NPs, intactness of the molecular structure of the drug Rf isrevealed from absorption and steady-state emission spectra of thedrug in the nanohybrid. Raman spectroscopy on the nanohybrids showsthe nature of molecular complexation of the drug with the inorganicNPs. For the semiconductor and insulator NPs, the complexation isfound to be noncovalent, however, a covalent attachment of the drugwith the dangling bonds of metal atoms at the surface is observed.In order to investigate antioxidant activity of the nanohybrids, wehave performed 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay of thenanohybrids in dark as well as under blue light irradiation. Whereaschange of the antioxidant activity of the nanohybrids with respectto free riboflavin in the absence of light is observed to be insignificant,a drastic change in the activity in the case of TiO2andZnO in the presence of light is evident. No change in the case ofAl2O3and a significant decrease in the antioxidantactivity for gold nanohybrids are also remarkable. Picosecond-resolvedfluorescence studies on the nanohybrids reveal a molecular pictureof the differential antioxidant activities. An ultrafast photoinducedelectron transfer from Rf to ZnO and TiO2are clearly evidentfrom the corresponding fluorescence transients. We have compared thepicosecond-resolved transients with that of Rf in the presence ofa well-known electron acceptor benzoquinone (BQ) and found similartime scales. No temporal change in the fluorescence transient of riboflavinin Al2O3nanohybrids compared to that of freeRf is observed indicating uneventful excited state relaxation of thenanohybrids. Nanosurface energy transfer (NSET) over Försterresonance energy transfer (FRET) is found to be the prevailing de-excitationmechanism in the case of gold nanohybrids, because of the strong spectraloverlap between Rf emission and surface plasmon absorption of thegold NPs. Different excited state mechanisms as revealed from ourstudies are expected to be useful for the design of NP-sensitizeddrugs, which are reported sparsely in the literature. [ABSTRACT FROM AUTHOR]

Energy drinks, as familiar consumer products, have been widely used in laboratory courses to help promote student interest, as well as to connect lecture concepts with laboratory work. Energy drinks contain B vitamins: pyridoxine (vitamin B6) and riboflavin (vitamin B2) of which amounts are high enough to be of concern. In this work, a fast and inexpensive high-performance liquid chromatography (HPLC) coupled with fluorescence detection method for determining pyridoxine and riboflavin simultaneously in energy drinks is developed. It takes advantage of the native fluorescence of B vitamins and provides high selectivity and sensitivity with low background noise. The method is suitable for undergraduate students, but it could be used for graduate level too including extra tasks such optimization and validation stages. [ABSTRACT FROM AUTHOR]

Isotope-labeled flavins are crucial reporters for many biophysical studies of flavoproteins. A purine-deficient Escherichia coli strain engineered for expression of the ribAGH genes of Bacillus subtilis converts isotope-labeled purine supplements into the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, with yields up to 40%. The fermentation products can subsequently be converted into isotope-labeled riboflavin and the cognate flavocoenzymes, FMN and FAD, by in vitro biotransformation with better than 90% yield. Using this approach, more than 100 single or multiple 13C-, 15N-, 17O-, and 18O-labeled isotopologues of these cofactors and ligands become easily accessible, enabling advanced ligand-based spectroscopy of flavoproteins and lumazine receptor proteins at unprecedented resolution. [ABSTRACT FROM AUTHOR]

The renewed use of arsenicals as chemotherapeutics has rekindled interest in the biochemistry of As(III) species. In this work, simple bis- and tris-arsenical derivatives were synthesized with the aim of exploiting the chelate effect in the inhibition of thiol-disulfide oxidoreductases (here, Quiescin sulfhydryl oxidase, QSOX, and protein disulfide isomerase, PDI) that utilize two or more CxxC motifs in the catalysis of oxidative protein folding. Coupling 4-aminophenylarsenoxide (APAO) to acid chloride or anhydride derivatives yielded two bis-arsenical prototypes, BA-1 and BA-2, and a tris-arsenical, TA-1. Unlike the monoarsenical, APAO, these new reagents proved to be strong inhibitors of oxidative protein folding in the presence of a realistic intracellular concentration of competing monothiol (here, 5 mM reduced glutathione, GSH). However, this inhibition does not reflect direct inactivation of QSOX or PDI, but avid binding of MVAs to the reduced unfolded protein substrates themselves. Titrations of reduced riboflavin-binding protein with MVAs show that all 18 protein -SH groups can be captured by these arsenicals. With reduced RNase, addition of substoichiometric levels of MVAs is accompanied by the formation of Congo Red- and Thioflavin T-positive fibrillar aggregates. Even with Kd values of ~50 nM, MVAs are ineffective inhibitors of PDI in the presence of millimolar levels of competing GSH. These results underscore the difficulties of designing effective and specific arsenical inhibitors for folded enzymes and proteins. Some of the cellular effects of arsenicals likely reflect their propensity to associate very tightly and nonspecifically to conformationally mobile cysteine-rich regions of proteins, thereby interfering with folding and/or function. [ABSTRACT FROM AUTHOR]

This paper describes the detrimental effect of photosynthetically evolved oxygen on anodic current generation in the presence of riboflavin upon illumination of a mixed phototrophic culture enriched from a freshwater pond at +0.6 V vs standard hydrogen electrode. In the presence of riboflavin, the phototrophic biomass in the anodic compartment produced an electrical current in response to light/dark cycles (12 h /1 2 h) over 12 months of operation, generating a maximum current density of 17.5 mA.m-2 during the dark phase, whereas a much lower current of approximately 2 mA.m-2 was generated during illumination. We found that the low current generation under light exposure was caused by high rates of reoxidation of reduced riboflavin by oxygen produced during photosynthesis. Quantification of biomass by fluorescence in situ hybridization images suggested that green algae were predominant in both the anode-based biofilm (55.1%) and the anolyte suspension (87.9%) with the remaining biovolume accounted for by bacteria. Genus-level sequencing analysis revealed that bacteria were dominated by cyanobacterium Leptolyngbia (∼35%), while the prevailing algae were Dictyosphaerium, Coelastrum, and Auxenochlorella. This study offers a key comprehension of mediator sensitivity to reoxidation by dissolved oxygen for improvement of microbial solar cell performance. [ABSTRACT FROM AUTHOR]

We investigated the lumazine proteinfrom Photobacteriumleiognathiin complex with its biologically active cofactor,6,7-dimethyl-8-ribityllumazine, at different redox states and comparedthe results with samples containing a riboflavin cofactor. Using anaerobicphotoreduction, we were able to record optical absorption kineticsfrom both cofactors in similar protein environments. It could be demonstratedthat the protein is able to stabilize a neutral ribolumazine radicalwith ∼35% yield. The ribolumazine radical state was furtherinvestigated by W-band continuous-wave EPR and X-band pulsed ENDORspectroscopy. Here, both the principal values of the g-tensor and an almost complete mapping of the proton hyperfine couplings(hfcs) could be obtained. Remarkably, the g-tensor’sprincipal components are similar to those of the respective riboflavin-containingprotein; however, the proton hfcs show noticeable differences. Comparingtime-resolved optical absorption and fluorescence data from ribolumazine-containingsamples, solely fluorescence but no signs of any intermediate radicalor a triplet state could be identified. This is in contrast to lumazineprotein samples containing the riboflavin cofactor, for which a highyield of the photogenerated triplet state and some excited flavinradical could be detected using time-resolved spectroscopy. Theseresults clearly demonstrate that ribolumazine is a redox-active moleculeand could, in principle, be employed as a cofactor in other enzymaticreactions. [ABSTRACT FROM AUTHOR]

Riboflavin receptors are overexpressedin malignant cells fromcertain human breast and prostate cancers, and they constitute a groupof potential surface markers important for cancer targeted deliveryof therapeutic agents and imaging molecules. Here we report on thefabrication and atomic force microscopy (AFM) characterization ofa core–shell nanocomposite consisting of a gold nanoparticle(AuNP) coated with riboflavin receptor-targeting poly(amido amine)dendrimer. We designed this nanocomposite for potential applicationssuch as a cancer targeted imaging material based on its surface plasmonresonance properties conferred by AuNP. We employed AFM as a techniquefor probing the binding interaction between the nanocomposite andriboflavin binding protein (RfBP) in solution. AFM enabled precisemeasurement of the AuNP height distribution before (13.5 nm) and afterchemisorption of riboflavin-conjugated dendrimer (AuNP–dendrimer;20.5 nm). Binding of RfBP to the AuNP–dendrimer caused a heightincrease to 26.7 nm, which decreased to 22.8 nm when coincubated withriboflavin as a competitive ligand, supporting interaction of AuNP–dendrimerand its target protein. In summary, physical determination of sizedistribution by AFM imaging can serve as a quantitative approach tomonitor and characterize the nanoscale interaction between a dendrimer-coveredAuNP and target protein molecules in vitro. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) is an essential messenger in human physiology, mediating cellular processes ranging from proliferation to apoptosis. The effects of NO are concentration dependent, and control over the instantaneous amount of NO available to cells is essential for determining the therapeutic NO dosages for various applications. As such, the development of NO therapeutic materials relies on accurate quantitative NO measurements that provide both total NO release from the NO donor as well as instantaneous NO concentrations. On the basis of the complexity of the cell media environment, inaccurate NO reporting often occurs for in vitro studies. These inaccuracies result from using inert media such as phosphate buffer saline (PBS), failing to account for the reactivity of media components. In this work, we describe a method for directly quantifying the instantaneous and total amounts of NO from commonly used NO donors in commercially available cell media routinely used for endothelial and neural cell lines. A riboflavin-tryptophan complex found in the media was identified as the major scavenger of NO in the cell media and likely reacts with NO via a radical—radical reaction. This finding significantly impacts the amount of available NO. The scavenging effects are concentration dependent on the riboflavin—tryptophan complex and the NO release rate from the NO donor. The results of this study provide insights on the exogenous amounts of NO that are present in cell media and may provide an explanation for differences in NO dosages between buffer experiments and in vitro and in vivo studies. [ABSTRACT FROM AUTHOR]

Experiments were conducted to investigate the effect of riboflavin on the reactivity of nanoscale zerovalent iron (NZVI) during three reaction cycles of carbon tetrachloride (CT) degradation. The degradation kinetics of CT by NZVI without riboflavin (0.556 ± 0.044 h-1) was 1.5 times higher than that with riboflavin (0.370 ± 0.012 h-1) in the first cycle. Riboflavin was rapidly reduced (65.0 ± 7.0 h-1) by NZVI during CT degradation, resulting in the slow degradation kinetics of CT in the first cycle due to competition for electrons from NZVI between riboflavin and CT. These results indicate that riboflavin is not effective as an electron shuttle for reduction of CT by NZVI. On the other hand, the degradation kinetics of CT by NZVI without riboflavin decreased to 0.122 ± 0.033 h-1 in the third cycle, while that with riboflavin was significantly enhanced (0.663 ± 0.005 h-1). The results from X-ray analyses and transmission electron microscopy suggest that the decline in reactivity of NZVI without riboflavin in the third cycle resulted from continuous Fe(0) oxidation to iron oxides on the NZVI surface. In contrast, riboflavin enhanced the reactivity of NZVI by reductive dissolution of passive iron oxides on NZVI surface by reduced riboflavin. The experimental results suggest that riboflavin can play a pivotal role in the prolongation of NZVI reactivity in long-term in situ and ex situ applications of NZVI. [ABSTRACT FROM AUTHOR]

An assay for quantification of riboflavin (Rf) in milk-based products has been developed using the principle of surface plasmon resonance with on-chip measurement. The quantification was done indirectly by measuring excess of Rf binding protein (RBP) that remains free after complexation with Rf molecules originally present in the sample solution. The chip was modified with covalently immobilized Rf in order to bind the RBP in excess. A chemical modification was performed to introduce a reactive ester group at the N-3 position of the natural Rf to bind amino groups present on the chip surface. Calibration solutions were prepared by mixing a range of Rf standard solutions with an optimized concentration of RBP. The Rf content in the milk-based products was then measured by comparison of the response against the calibration. Results obtained were very close to those from an official HPLC-fluorescence procedure. The limit of quantification was determined to 234 [micro]g/L and the limit of detection to 70 [micro]g/L by an injection volume of 160 [micro]L.

Molecular recognition mediated by hydrogen-bonding interactions at a water/C[Cl.sub.4] interface was investigated directly by means of time-resolved total internal reflection (TIR) fluorescence spectroscopy. The TIR fluorescence decay profile of riboflavin (RF) in the absence of a guest in the C[Cl.sub.4] phase was fitted satisfactorily by a single-exponential function. In the presence of N,N-dioctadecyl[1,3,5]triazine-2,4,6-triamine (DTT) as a guest in the C[Cl.sub.4] phase, on the other hand, the fluorescence decay profiles were best fitted by double-exponential functions with the relevant amplitude ([A.sub.i]) being varied with the concentration of DTT. Furthermore, the rotational reorientation time of RF at the interface determined by fluorescence dynamic anisotropy was 210 ps in the absence of DTT, while fast (160-220 ps) and slow (670-750 ps) rotational reorientation times were observed in the presence of DTT. This slow rotational reorientation time was shown to ascribe to that of the RF-DDT complex formed at the water/C[Cl.sub.4] interface. These results indicate that molecular recognition mediated by complementary hydrogen bonding takes place effectively at the water/C[Cl.sub.4] interface, which was observed directly by both fluorescence dynamics and fluorescence dynamic anisotropy measurements under the TIR conditions.

Research has been conducted on riboflavin isotopomers. The preparation of these compounds labeled with (super)13 C in any position of xylene moiety has been carried out via highly versatile one-pot methods and the details are described.

Excited-stateprotonation of riboflavin in the oxidized form isstudied in water. In the −1 < pH < 2 range, neutral andN(1)-protonated riboflavin coexist in the electronic ground state.Transient absorption shows that the protonated form converts to theground state in <40 fs after optical excitation. Broadband fluorescenceupconversion is therefore used to monitor solvation and protonationof the neutral species in the excited singlet state exclusively. Aweak fluorescence band around 660 nm is assigned to the product ofprotonation at N(5). Its radiative rate and quantum yield relativeto neutral riboflavin are estimated. Protonation rates agree withproton diffusion times for H+concentrations below 5 Mbut increase at higher acidities, where the average proton distanceis below the diameter of the riboflavin molecule. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We describe a solid state electrode structure based on a biologically derived proton-active redox center, riboflavin (RFN). The redox reaction of RFN is a pH-dependent process that requires no water. The electrode was fabricated using our previously described 'stuffing' method to entrap RFN into vapor phase polymerized poly(3,4-ethylenedioxythiophene). The electrode is shown to be capable of measuring the proton activity in the form of an effective pH over a range of different water contents including nonaqueous systems and ionic liquids (ILs). This demonstrates that the entrapment of the redox center facilitates direct electron communication with the polymer. This work provides a miniaturizable system to determine pH (effective) in nonaqueous systems as well as in ionic liquids. The ability to measure pH (effective) is an important step toward the ability to customize ILs with suitable pH (effective) for catalytic reactions and biotechnology applications such as protein preservation. [ABSTRACT FROM AUTHOR]

The article describes the use of multifunctional chemical analysis (MCA) systems, Vernier and MeasureNet, in undergraduate chemistry laboratory curriculum. It illustrates experiments showing the accuracy of Vernier and MeasureNet in detecting salicylate in aspirin tablets by flow injection analysis and liquid chromatographic separation of a mixture of riboflavin and fluorescein. Information on the features of the MCA systems and their comparison is provided.

Hunanamycin A, the first natural product with a pyrido[1,2,3-de]quinoxaline-2,3-dione core, was isolated from a marine-derived Bacillus hunanensis. Hunanamycin A is related to a degradation product of riboflavin but has undergone an N-prenylation and subsequent cyclization. The structure, including stereochemistry, was determined by NMR and MS methods. Hunanamycin A exhibits a minimum inhibitory concentration (MIC) of 12.4 μM against the bacterial pathogen Salmonella enterica. [ABSTRACT FROM AUTHOR]

Guanine is a major target for oxidation in DNA, with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) as a major product. 8-oxodG is itself significantly more susceptible to oxidation than guanine, with the resulting damage consisting of more than 10 different products. This complexity has hampered efforts to understand the determinants of biologically relevant DNA oxidation chemistry. To address this problem, we have developed a high mass accuracy mass spectrometric method to quantify oxidation products arising site specifically in DNA. We applied this method to quantify the role of sequence context in defining the spectrum of damage products arising from oxidation of 8-oxodG by two oxidants: nitrosoperoxycarbonate (ONOOCO2-), a macrophage-derived chemical mediator of inflammation, and the classical one-electron oxidant, riboflavin-mediated photooxidation. The results reveal the predominance of dehydroguanidinohydantoin (DGh) in 8-oxodG oxidation by both oxidants. While the relative quantities of 8-oxodG oxidation products arising from ONOOCO2- did not vary as a function of sequence context, products of riboflavin-mediated photooxidation of 8-oxodG were highly sequence dependent. Several of the 8-oxodG oxidation products underwent hydrolytic conversion to new products with half-lives of 2-7 h. The results have implications for understanding the chemistry of DNA oxidation and the biological response to the damage, with DNA damage recognition and repair systems faced with a complex and dynamic set of damage targets. [ABSTRACT FROM AUTHOR]

A novel optically active polymer consisting of riboflavin units as the main chain (poly-1) was prepared from naturally occurring riboflavin (vitamin B2) 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. [ABSTRACT FROM AUTHOR]

Lumazine synthase catalyzes the penultimate step in the biosynthesis of riboflavin, while riboflavin synthase catalyzes the last step. O-Nucleoside, S-nucleoside, and N-nucleoside analogues of hypothetical lumazine biosynthetic intermediates have been synthesized in order to obtain structure and mechanism probes of these two enzymes, as well as inhibitors of potential value as antibiotics. Methods were devised for the selective cleavage of benzyl protecting groups in the presence of other easily reduced functionality by controlled hydrogenolysis over Lindlar catalyst. The deprotection reaction was performed in the presence of other reactive functionality including nitro groups, alkenes, and halogens. The target compounds were tested as inhibitors of lumazine synthase and riboflavin synthase obtained from a variety of microorganisms. In general, the S-nucleosides and N-nucleosides were more potent than the corresponding O-nucleosides as lumazine synthase and riboflavin synthase inhibitors, while the C-nucleosides were the least potent. A series of molecular dynamics simulations followed by free energy calculations using the Poisson-Boltzmann/surface area (MM-PBSA) method were carried out in order to rationalize the results of ligand binding to lumazine synthase, and the results provide insight into the dynamics of ligand binding as well as the molecular forces stabilizing the intermediates in the enzyme-catalyzed reaction. [ABSTRACT FROM AUTHOR]

Liquid-assisted grinding protocol has been successfullyemployedto identify (1) the use of riboflavin as a sensing molecule for melamineand acetoguanamine and (2) the chelation of riboflavin on a silveratom. Riboflavin is then chelated onto a visible light-emitting, silver-containingmetal–organic framework, AgL {i.e., [AgL]n·nH2O (L = 4-cyanobenzoate)}to produce (AgL)-(riboflavin) chelation crystals. These chelationcrystals can be used for the detection of melamine and acetoguanamineby forming binding crystals of (AgL)-(riboflavin)-(melamine) and (AgL)-(riboflavin)-(acetoguanamine).The Commission Internationale de l'Eclairage (CIE) 1931 chromaticitycoordinates of AgL crystals, (AgL)-(riboflavin) chelation crystals,(AgL)-(riboflavin)-(melamine) binding crystals, and (AgL)-(riboflavin)-(acetoguanamine)binding crystals based on their solid-state photoluminescence (PL)emission spectra are calculated to be approximately (0.16, 0.16),(0.32, 0.44), (0.25, 0.37), and (0.23, 0.15), respectively. The originalPL emission of AgL may be attributed to ligand-centered luminescence.However, the chelation with riboflavin may cause (1) a bathochromaticspectral shift (i.e., red shift), (2) an emission broadening, and(3) a quenching effect through the mode of Förster resonanceenergy transfer. The binding with the amine analytes (i.e., good electrondonors), such as melamine and acetoguanamine, may alter the redoxpotential of riboflavin inhibiting quenching and enhancing luminescenceof binding crystals of (AgL)-(riboflavin)-(melamine) and (AgL)-(riboflavin)-(acetoguanamine).π → π* energy in the riboflavin-melamine or riboflavin-acetoguanaminebinding complex is then enhanced. Consequently, hypsochromic spectralshifts (i.e., blue shift) are observed in their PL emission responses. [ABSTRACT FROM AUTHOR]

The electrochemical behavior of the naturally occurring vitamin B2, riboflavin (Flox), was examined in detail in dimethyl sulfoxide solutions using variable scan rate cyclic voltammetry (ν = 0.1 - 20 V s-1) and has been found to undergo a series of proton-coupled electron transfer reactions. At a scan rate of 0.1 V s-1, riboflavin is initially reduced by one electron to form the radical anion (Flrad•-) at E0f = -1.22 V versus Fc/Fc+ (E0f = formal reduction potential and Fc = ferrocene). Flrad•- undergoes a homogeneous proton transfer reaction with the starting material (Flox) to produce FlradH• and Flox-, which are both able to undergo further reduction at the electrode surface to form FlredH- (E0f = -1.05 V vs Fc/Fc+) and Flrad•2- (E0f = -1.62 V vs Fc/Fc+), respectively. At faster voltammetric scan rates, the homogeneous reaction between Flrad•- and Flox begins to be outrun, which leads to the detection of a voltammetric peak at more negative potentials associated with the one-electron reduction of Flrad•- to form Flred2- (E0f = -1.98 V vs Fc/Fc+). The variable scan rate voltammetric data were modeled quantitatively using digital simulation techniques based on an interconnecting "scheme of squares" mechanism, which enabled the four formal potentials as well as the equilibrium and rate constants associated with four homogeneous reactions to be determined. Extended time-scale controlled potential electrolysis (t > hours) and spectroscopic (EPR and in situ UV-vis) experiments confirmed that the chemical reactions were completely chemically reversible. [ABSTRACT FROM AUTHOR]

The present study describes the biophysical characterizationofgeneration-five poly(amidoamine) (PAMAM) dendrimers conjugated withriboflavin (RF) as a cancer-targeting platform. Two new series ofdendrimers were designed, each presenting the riboflavin ligand attachedat a different site (isoalloxazine at N-3 and d-ribose atN-10) and at varying ligand valency. Isothermal titration calorimetry(ITC) and differential scanning calorimetry (DSC) were used to determinethe binding activity for riboflavin binding protein (RfBP) in a cell-freesolution. The ITC data shows dendrimer conjugates have KDvalues of ≥465 nM on a riboflavin basis, an affinity∼93-fold lower than that of free riboflavin. The N-3 seriesshowed greater binding affinity in comparison with the N-10 series.Notably, the affinity is inversely correlated with ligand valency.These findings are also corroborated by DSC, where greater protein–conjugatestability is achieved with the N-3 series and at lower ligand valency. [ABSTRACT FROM AUTHOR]

Light-induced degradation of the riboflavin derivatives is reported, including 5-deaza-riboflavin, iso-6,7-riboflavin, 3-methyl-tetra-acetyl-riboflavin (3MeTARF), and 3-benzyl-lumiflavin. The studied compounds undergo photolytic processes with considerable quantum yields in methanolic solutions (φ ≈ 10-3-10-4 mol eins-1). Photolysis in anaerobic conditions is more efficient than that in the presence of oxygen. Experiments show that triplet excited state of flavin molecules is involved in the reactions. The main photo- products are the respective alloxazinic and isoalloxazinic derivatives of the substrates. 3MeTARF reveals higher photostability than riboflavin. The values of photodegradation quantum yields were estimated for the four studied derivatives. [ABSTRACT FROM AUTHOR]

Flavodoxins are bacterial electron transport proteins whose redox competence is due to the presence of a tightly but noncovalently bound FMN molecule. While the thermodynamics of the complex are understood, the mechanism of association between the apoflavodoxin and the redox cofactor is not so clear. We investigate here the mechanism of FMN binding to the apoflavodoxin from Helicobacter pylori, an essential protein that is being used as a target to develop antimicrobials. This flavodoxin is structurally peculiar as it lacks the typical bulky residue interacting with the FMN re face but bears instead a small alanine. FMN binding is biphasic, regardless of the presence of phosphate molecules in solution, while riboflavin binding takes place in a single step, the rate constant of which coincides with the fast phase of FMN binding. A mutational study at the isoalloxazine and phosphate subsites for FMN binding clearly indicates that FMN association is always limited by interaction with the isoalloxazine subsite because mutating residues that interact with the phosphate moiety of FMN in the native complex hardly changes the observed rate constants and amplitudes. In contrast, replacing tyr92, which interacts with the isoalloxazine, greatly lowers the rate constants. Our analysis indicates that the two FMN binding phases observed are related neither with alternative or sequential interaction with the two binding subsites nor with the presence of bound phosphate. It is possible that they reflect the intrinsic conformational heterogeneity of the apoflavodoxin ensemble. [ABSTRACT FROM AUTHOR]

As more protein biopharmaceuticals are produced using mammalian cell culture techniques, it becomes increasingly important for the biopharmaceutical industry to have tools to characterize the cell culture media and evaluate its impact on the cell culture performance. Exposure of the cell culture media to light, temperature stress, or adventitious introduction of low-level organisms during preparation can lead to the generation of chemical degradants or metabolites of the media components, which are potentially detrimental to the cell culture process. In this work, we applied a liquid chromatography—mass spectrometry based metabolomics methodology for the investigation of a media lot used for a mammalian cell culture process that had resulted in low growth rate and failure to meet required viable cell density (VCD). The study led to the observation of increased levels of tryptophan oxidation products and a riboflavin degradant, lumichrome, in the malfunctioning media lot, relative to working media lots. A compound found 7-fold higher in the working media lots appeared to be tetrahydropentoxyline, a condensation product of glucose and tryptophan. A second compound found at an over 50-fold higher level in the malfunctioning media lot with a proposed molecular formula of C21H17N3O3 from high-resolution mass spectrometry (HRMS) analysis remains unknown, although it is confirmed to be a degradant of tryptophan in the media. A study of the cell culture media performed under stress conditions using fluorescent light and heat showed that the media powder was highly resistant to light-induced degradation, while solution media could be easily degraded after brief light exposure. It is therefore suspected that inadvertent exposure of the media to light during preparation and storage has resulted in the poor performance of the media causing the low growth and VCD in the cell culture process. [ABSTRACT FROM AUTHOR]

The addition of photosensitizers to water can accelerate disinfection in sunlight-based systems by enhancing oxidation of target compounds through direct reaction with the excited sensitizer or through production of another oxidant, such as singlet oxygen (¹O2). The kinetics of the oxidation of selected amino acids in the presence of the sensitizer riboflavin (Vitamin B2), its primary photoproduct lumichrome, and its derivative riboflavin tetraacetate (2',3',4',5'-tetraacetylriboflavin; RTA) were quantified and the mechanisms of reaction were determined during exposure to 365 ± 9 nm light. ¹O2-mediated reactions contributed to the rapid photodegradation of the four amino acids, but its contribution was sensitizer-dependent and varied from 5.4-10.2% for tyrosine, 7.1-12.4% for tryptophan, 18.7-69.0% for methionine, and 64.7-100.2% for histidine. Riboflavin was subject to rapid photodegradation (t1/2 <8 min), while the half-lives of lumichrome and RTA were 100 and 30 times longer, respectively. Lumichrome and RTA also were more efficient ¹O2 sensitizers (quantum yield (Φ) = 0.63 and 0.66) compared to riboflavin (Φ = 0.48). Of the three flavin-based compounds, RTA shows the most promise as a sensitizer in sunlight-based disinfection systems because it absorbs both visible and UV light, is an efficient ¹O2 sensitizer, is a strong oxidant in its triplet state, and exhibits greater photostability. [ABSTRACT FROM AUTHOR]

Isotope-labeled riboflavin in DMSO was employed in conjunction with femtosecond time-resolved infrared vibrational spectroscopy and quantum chemical calculations to analyze and assign the electronically excited state vibrational modes of the isoalloxazine unit as a prototype for the cofactors in flavin binding blue-light receptors. Using the riboflavin 13C-analogues RF-2-13C and RF-4,10a-13C, the carbonyl vibrations, in particular, were studied. Various quantum chemical models were applied that take into account a polarizable environment or the impact of hydrogen bonds. The CIS quantum-chemistry method was successfully applied to describe the lowest singlet excited electronic state in riboflavin. The experimentally observed frequencies and isotope-shifts as well as their variability in the diverse model calculations are discussed. On these grounds, a consistent assignment of the electronic ground and excited state vibrations is presented. [ABSTRACT FROM AUTHOR]

The article presents a chemistry experiment on a polymeric monolithic column. It notes that the high-performance liquid chromatography (HPLC) was used to test the polymer-based monolithic column. It states that the ion-pairing reagent was used to sort thiamine from riboflavin. It also mentions the technique of retaining sodium dodecyl sulfate.

In blue-light photoreceptors using flavin (BLUF), the signaling state is formed already within several 100 ps after illumination, with only small changes of the absorption spectrum. The accompanying structural evolution can, in principle, be monitored by femtosecond stimulated Raman spectroscopy (FSRS). The method is used here to characterize the excited-state properties of riboflavin and flavin adenine dinucleotide in polar solvents. Raman modes are observed in the range 90−1800 cm−1for the electronic ground state S0and upon excitation to the S1state, and modes >1000 cm−1of both states are assigned with the help of quantum-chemical calculations. Line shapes are shown to depend sensitively on resonance conditions. They are affected by wavepacket motion in any of the participating electronic states, resulting in complex amplitude modulation of the stimulated Raman spectra. Wavepackets in S1can be marked, and thus isolated, by stimulated-emission pumping with the picosecond Raman pulses. Excited-state absorption spectra are obtained from a quantitative comparison of broadband transient fluorescence and absorption. In this way, the resonance conditions for FSRS are determined. Early differences of the emission spectrum depend on excess vibrational energy, and solvation is seen as dynamic Stokes shift of the emission band. The nπ* state is evidenced only through changes of emission oscillator strength during solvation. S1quenching by adenine is seen with all methods in terms of dynamics, not by spectral intermediates. [ABSTRACT FROM AUTHOR]

The blue light using flavin (BLUF) domain photosensors, such as the transcriptional antirepressor AppA, utilize a noncovalently bound flavin as the chromophore for photoreception. Since the isoalloxazine ring of the chromophore is unable to undergo large-scale structural change upon light absorption, there is intense interest in understanding how the BLUF protein matrix senses and responds to flavin photoexcitation. Light absorption is proposed to result in alterations in the hydrogen-bonding network that surrounds the flavin chromophore on an ultrafast time scale, and the structural changes caused by photoexcitation are being probed by vibrational spectroscopy. Here we report ultrafast time-resolved infrared spectra of the AppA BLUF domain (APPABLUF) reconstituted with isotopically labeled riboflavin (RI) and flavin adenine dinucleotide (FAD), which permit the first unambiguous assignment of ground and excited state modes arising directly from the flavin carbonyl groups. Studies of model compounds and DFT calculations of the ground state vibrational spectra reveal the sensitivity of these modes to their environment, indicating that they can be used as probes of structural dynamics. [ABSTRACT FROM AUTHOR]

We report the selective detection of single nitric oxide (NO) molecules using a specific DNA sequence of d(AT)15 oligonucleotides, adsorbed to an array of near-infrared fluorescent semiconducting single-walled carbon nanotubes (AT15-SWNT). While SWNT suspended with eight other variant DNA sequences show fluorescence quenching or enhancement from analytes such as dopamine, NADH, L-ascorbic acid, and riboflavin, d(AT)15 imparts SWNT with a distinct selectivity toward NO. In contrast, the electrostatically neutral polyvinyl alcohol enables no response to nitric oxide, but exhibits fluorescent enhancement to other molecules in the tested library. For AT15-SWNT, a stepwise fluorescence decrease is observed when the nanotubes are exposed to NO, reporting the dynamics of single-molecule NO adsorption via SWNT exciton quenching. We describe these quenching traces using a birth-and-death Markov model, and the maximum likelihood estimator of adsorption and desorption rates of NO is derived. Applying the method to simulated traces indicates that the resulting error in the estimated rate constants is less than 5% under our experimental conditions, allowing for calibration using a series of NO concentrations. As expected, the adsorption rate is found to be linearly proportional to NO concentration, and the intrinsic single-site NO adsorption rate constant is 0.001 s-1 μM NO-1. The ability to detect nitric oxide quantitatively at the single-molecule level may find applications in new cellular assays for the study of nitric oxide carcinogenesis and chemical signaling, as well as medical diagnostics for inflammation. [ABSTRACT FROM AUTHOR]

Computing accurate absorption spectra of riboflavin (RBF) has proven a difficult task for computational chemistry. Time-dependent density functional theory have herein been employed using a wide range of recent range-separated and hybrid meta functionals to investigate ultraviolet and visible spectra of RBF to determine if any progress has been made through recent developments. It is concluded that B3LYP and PBE0 perform the best throughout the entire test set. However, since all methods deviate from experimental results by at least 40 nm when computing the spectra in vacuum, two approaches to describe aqueous solution are implemented together with the MPWB1K, B3LYP, and PBE0 functionals: implicitly using integral equation formulation of the polarized continuum model (minor improvement) and explicitly through molecular dynamics (MD) simulations of the molecule embedded in a water cluster whereafter snapshots of RBF−water clusters are extracted and time-dependent density functional theory calculations performed. The resulting averaged spectra from the MD-simulated clusters show a constant blue-shift for all peaks by ∼20 nm compared to experimental data at the TD-B3LYP/6-31(d,p) level. [ABSTRACT FROM AUTHOR]

The interaction of the macrocyclic host, cucurbit[7]uril (CB7), with riboflavin (RF) and its derivative, flavin adenine dinucleotide (FAD), has been investigated using absorption and steady-state and time-resolved fluorescence measurements. Interestingly, in the presence of CB7, the fluorescence intensity of RF is quenched, whereas the fluorescence intensity of FAD is enhanced. It is proposed that the fluorescence quenching of RF results from the tautomerization of its isoalloxazine moiety from the lactam to the lactim forms, upon binding to CB7. Such a tautomerization can be brought about since the two lactim forms have higher dipole moments than the lactam form of RF, and thus experience much stronger dipole−dipole interactions (and hence greater binding affinities) with CB7 in the former cases than in the latter. This tautomerization in the presence of CB7 leads to a significant reduction in the observed radiative decay rate and hence a reduction in the fluorescence intensity of RF. Binding of CB7 with RF is confirmed by an increase in the rotational correlation time of RF in the presence of CB7. Geometry optimization studies indicate the formation of an exclusion complex between CB7 and RF, possibly stabilized by H-bonding interactions, as also suggested by the characteristic red shift in the absorption spectra of the CB7−RF system. In the case of FAD, both the isoalloxazine ring and the adenine moiety can interact with the CB7 host. In aqueous solutions, a good fraction of FAD molecules exists in a “closed” conformation with the adenine and isoalloxazine rings stacked together, thus leading to very efficient fluorescence quenching due to the ultrafast intramolecular electron transfer from adenine to the isoalloxazine moiety. Binding of the adenine and/or the isoalloxazine moiety of FAD with CB7 inhibits the stacking interaction and changes the “closed” conformation to the “open” conformation, wherein the adenine and isoalloxazine moieties are widely separated, thus prohibiting the electron transfer process. This reduces the inherent fluorescence quenching of FAD molecule and results in the observed fluorescence enhancement. As observed for RF, the interaction of CB7 with the isoalloxazine ring of FAD should cause fluorescence quenching due to the lactam to lactim tautomerization process. However, in the interplay between the above two opposing effects, the fluorescence enhancement due to the modulation in the conformational dynamics of FAD by the CB7 host predominates. The conformational change is in fact supported by the observation of a long lifetime component in the fluorescence decay of FAD in the presence of CB7. Moreover, at acidic pH, when FAD is already present mainly in the “open” form, the conformational dynamics no longer plays any major role and the fluorescence of FAD is quenched by CB7, as expected, due to the tautomerization at the isoalloxazine moiety. [ABSTRACT FROM AUTHOR]

The photophysics and photochemistry of flavin molecules are of great interest due to their role for the biological function of flavoproteins. An important analysis tool toward the understanding of the initial photoexcitation step of flavins is electronic and vibrational spectroscopy, both in frequency and time domains. Here we present quantum chemical [(time-dependent) density functional theory ((TD-)DFT)] calculations for vibrational spectra of riboflavin, the parent molecule of biological blue-light receptor chromophores, in its electronic ground (S0) and lowest singlet excited states (S1). Further, vibronic absorption spectra for the S0→ S1transition and vibronic emission spectra for the reverse process are calculated, both including mode mixing. Solvent effects are partially accounted for by using a polarizable continuum model (PCM) or a conductor-like screening model (COSMO). Calculated vibrational and electronic spectra are in good agreement with measured ones and help to assign the experimental signals arising from photoexcitation of flavins. In particular, upon photoexcitation a loss of double bond character in the polar region of the ring system is observed which leads to vibronic fine structure in the electronic spectra. Besides vibronic effects, solvent effects are important for understanding the photophysics of flavins in solution quantitatively. [ABSTRACT FROM AUTHOR]

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-6ribitylamino-2,4(1 H,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 co/i 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. [ABSTRACT FROM AUTHOR]

Various olefins can be hydrogenated quantitatively with neutral flavin 2catalysts in the presence of 1−2 equiv of hydrazine under 1 atm of O2. Vitamin B2derivative 2gacts as a highly efficient and robust catalyst for the present environmentally benign process producing water and nitrogen gas as the only waste products. [ABSTRACT FROM AUTHOR]

Four different riboflavin (RF) derivatives, two electronically modified compounds (1- and 5-deazariboflavin, 1DRF and 5DRF) and two sterically modified compounds (7,8-didemethyl- and 8-isopropylriboflavin, DMRF and iprRF), were subjected to a combination of time-resolved measurements (absorption and fluorescence) and high-level quantum chemical investigations. Both alkyl-modified flavins showed similar fluorescence properties as the parent compound, yet 5DRF had a larger quantum yield of fluorescence (ΦF= 0.52) than RF (ΦF= 0.27). Interestingly, 1DRF did not show fluorescence at all under these steady state conditions. The triplet quantum yield was different for the modified flavins such that no triplet formation was found for 1DRF, whereas the other compounds all formed triplet states (ΦTRfor 5DRF of 0.64 and 0.50 and 0.23 for iprRF and DMRF, respectively). The triplet states of the two alkyl-modified flavins decayed with similar time constants as the parent compound, whereas a shorter lifetime was measured for 5DRF (τTR= 15 μs, compared to τTR= 29 μs for RF). In the calculations, the flavin derivatives were modeled as lumiflavins, that is, without the ribityl chain. We conclude that for aqueous solutions of DMRF, iprRF, and 5DRF intersystem crossing (ISC) takes place from the S11(ππ*) to the T23(ππ*) state by a vibronic spin−orbit coupling mechanism, a process common to most flavins, whereas ISC is slow in excited 1DRF due to the absence of a close-by triplet state. [ABSTRACT FROM AUTHOR]

A high-throughput screening (HTS) hit compound displayed moderate inhibition of Mycobacterium tuberculosis and Escherichia coli riboflavin synthases. The structure of the hit compound provided by the commercial vendor was reassigned as [3-(4-chlorophenyl)-5-hydroxy-5-(trifluoromethyl)-4,5-dihydrolH-pyrazol-l-yll(o-tolyl)methanone (18). The hit compound had a kis of 8.7 μM vs. M. tuberculosis riboflavin synthase and moderate antibiotic activity against both M. tuberculosis replicating phenotype and non-replicating persistent phenotype. Molecular modeling studies suggest that two inhibitor molecules bind in the active Site of the enzyme, and that the binding is stabilized by stacking between the benzene rings of two adjacent ligands. The most potent antibiotic in the series proved to be [5(4-chlorophenyl)-5-hydroxy-3-(trifluoromethyl)-4,5-dihydroI H-pyrazolI -yl](m-tolyl)methanone (16), which displayed a minimum inhibitory concentration (MIC) of 36.6 μM vs. M. tuberculosis replicating phenotype and 48.9 μM vs. M. tuberculosis nonreplicating phenotype. The HTS hit compound and its analogues provide the first examples of riboflavin synthase inhibitors with antibiotic activity. [ABSTRACT FROM AUTHOR]

The early steps in the biosynthesis of 7,8-didemethyl-8-hydroxy-5-deazariboflavin (Fo) and riboflavin in the archaea differ from the established eukaryotic and bacterial pathways. The archaeal pathway has been proposed to begin with an archaeal-specific GTP cyclohydrolase III that hydrolyzes the imidazole ring of GTP but does not remove the resulting formyl group from the formamide [Graham, D. E., Xu, H., and White, R. H. (2002) Biochemistry 41, 15074-15084]. This enzyme is different than the bacterial GTP cyclohydrolase II which catalyzes both reactions. Here we describe the identification and characterization of the formamide hydrolase that catalyzes the second step in the archaeal Fo and riboflavin biosynthetic pathway. The Methanocaldococcus jannaschii MJ0116 gene was cloned and heterologously expressed, and the resulting enzyme was shown to catalyze the formation of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (APy) and formate from 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-monophosphate (FAPy). The MJ0116-derived protein has been named ArfB to indicate that it catalyzes the second step in archaeal riboflavin and Fo biosynthesis. ArfB was found to require ferrous iron for activity although metal analysis by ICP indicated the presence of zinc as well as iron in the purified protein. The identification of this enzyme confirms the involvement of GTP cyclohydrolase III (ArfA) in archaeal riboflavin and Fo biosynthesis. [ABSTRACT FROM AUTHOR]

The success of arsenic trioxide in the treatment of acute promyelocytic leukemia has renewed interest in the cellular targets of As(III) species. The effects of arsenicals are usually attributed to their ability to bind vicinal thiols or thiol selenols in prefolded proteins thereby compromising cellular function. The present studies suggest an additional, more pleiotropic, contribution to the biological effects of arsenicals. As(III) species, by avid coordination to the cysteine residues of unfolded reduced proteins, can compromise protein folding pathways. Three representative As(III) compounds (arsenite, monomethylarsenous acid (MMA), and an aryl arsenical (PSAO)) have been tested with three reduced secreted proteins (lysozyme, ribonuclease A, and riboflavin binding protein (RfBP)). Using absorbance, fluorescence, and pre-steady-state methods, we show that arsenicals bind tightly to low micromolar concentrations of these unfolded proteins with stoichiometries of 1 As(III) per 2 thiols for MMA and PSAO and I As(III) for every 3 thiols with arsenite. Arsenicals, at 10 μM, strongly disrupt the oxidative folding of RfBP even in the presence of 5 mM reduced glutathione, a competing ligand for As(llI) species. MMA catalyzes the formation of amyloid-like monodisperse fibrils using reduced RNase. These in vitro data show that As(H1) species can slow, or even derail, protein folding pathways. In vivo, the propensity of As(III) species to bind to unfolded cysteine-containing proteins may contribute to oxidative and protein folding stresses that are prominent features of the cellular response to arsenic exposure. [ABSTRACT FROM AUTHOR]

Lumazine synthase catalyzes the reaction of 5-amino-6-d-ribitylamino-2,4(1H,3H)-pyrimidinedione (1) with (S)-3,4-dihydroxybutanone 4-phosphate (2) to afford 6,7-dimethyl-8-d-ribityllumazine (3), the immediate biosynthetic precursor of riboflavin. The overall reaction implies a series of intermediates that are incompletely understood. The 15N{31P} REDOR NMR spectra of three metabolically stable phosphonate reaction intermediate analogues complexed to Saccharomyces cerevisiae lumazine synthase have been obtained at 7 and 12 T. Distances from the phosphorus atoms of the ligands to the side chain nitrogens of Lys92, His97, Arg136, and His148 have been determined. These distances were used in combination with the X-ray crystal coordinates of one of the intermediate analogues complexed with the enzyme in a series of distance-restrained molecular dynamics simulations. The resulting models indicate mobility of the Lys92 side chain, which could facilitate the exchange of inorganic phosphate eliminated from the substrate in one reaction, with the organic phosphate-containing substrate necessary for the next reaction. [ABSTRACT FROM AUTHOR]

Femtosecond dynamics of riboflavin, the parent chromophore of biological blue-light receptors, was measured by broadband transient absorption and stationary optical spectroscopy in polar solution. Rich photochemistry is behind the small spectral changes observed: (i) loss of oscillator strength around time zero, (ii) sub-picosecond (ps) spectral relaxation of stimulated emission (SE), and (iii) coherent vibrational motion along a′ (in-) and a″ (out-of-plane) modes. Loss of oscillator strength is deduced from the differences in the time-zero spectra obtained in water and DMSO, with stationary spectroscopy and fluorescence decay measurements providing additional support. The spectral difference develops faster than the time resolution (20 fs) and is explained by formation of a superposition state between the optically active (1ππ*) S1and closely lying dark (1nπ*) states via vibronic coupling. Subsequent spectral relaxation involves decay of weak SE in the blue, 490 nm, together with rise and red shift of SE at 550 nm. The process is controlled by solvation (characteristic times 0.6 and 0.8 ps in water and DMSO, respectively). Coherent oscillations for a′ and a″ modes show up in different regions of the SE band. a″ modes emerge in the blue edge of the SE and dephase faster than solvation. In turn, a′ oscillations are found in the SE maximum and dephase on the solvation timescale. The spectral distribution of coherent oscillations according to mode symmetry is used to assign the blue edge of the SE band to a 1nπ*-like state (A″), whereas the optically active 1ππ* (A′) state emits around the SE maximum. The following model comes out: optical excitation occurs to the Franck−Condon ππ* state, a ππ*−nπ* superposition state is formed on an ultrafast timescale, vibrational coherence is transferred from a′ to a″modes by ππ*−nπ* vibronic coupling, and subsequent solvation dynamics alters the ππ*/nπ* population ratio. [ABSTRACT FROM AUTHOR]