Flash photolysis of flavins. I. Photoreduction in non-aqueous solvents

Abstract— The photoreduction of riboflavin, FMN, and lumiflavin in a series of hydroxylic solvents has been examined using flash photolysis. A comparison of the relative quantum yields for the photoxidation of several alcohols and esters by lumiflavin demonstrated that the hydroxyl hydrogen of the alcohol is abstracted approximately twice as readily as is the alpha hydrogen. The use of glycerol as both solvent and reductant provided direct evidence that the initial reaction proceeds by a one-electron reduction to form the flavin semi-quinone. In the case of riboflavin (and FMN) the kinetic results are consistent with an initial intramolecular hydrogen atom transfer, analogous to photobleaching in aqueous solution, followed by a reaction of the semi-quinone with the reductant which prevents degradation of the ribityl side-chain. Quenching by iodide indicates that all the reactions proceed via the flavin triplet state, as is the case in aqueous systems.     

The solvent effect on polar compounds containing acidic hydrogens

The effect of aromatic and non-aromatic solvents on the proton chemical shifts of 23 polar compounds has been determined. The protons which are activated by electron-withdrawing groups show large highfield shifts in benzene (relative to the isotropic solvents). Based on evidence provided by infrared data, the highfield shifts of the acidic protons in benzene solution are interpreted in terms of a model involving C? Hπ hydrogen bonding. This model successfully interprets the data reported previously for steroidal ketones. The same model can be extended to benzene solutions of other polar compounds containing strongly electron-deficient sites to which alkyl groups are attached. It is observed that the use of CCl₄ as a reference solvent in studies of benzene induced shifts may have greater significance, since these two solvents have similar dielectric constants.     

Model systems for flavoenzyme activity: interplay of hydrogen bonding and aromatic stacking in cofactor redox modulation

[structure: see text] A model system has been developed to study the synergy between aromatic stacking and hydrogen bonding in the binding of a flavin derivative. The results show that the identity of both the hydrogen bonding and pi-stacking units strongly determine the overall receptor affinity for flavin in both the oxidized and radical anion forms.     

Effects of solvents on the electron configurations of the low-spin dicyano[meso-tetrakis(2,4,6-triethylphenyl)porphyrinato]iron(III) complex: importance of the C-H...N weak hydrogen bonding

There are two types of electron configurations, (d(xy))(2)(d(xz), d(yz))(3) and (d(xz), d(yz))(4)(d(xy))(1), in low-spin iron(III) porphyrin complexes. To reveal the solvent effects on the ground-state electron configurations, we have examined the (13)C- and (1)H-NMR spectra of low-spin dicyano[meso-tetrakis(2,4,6-triethylphenyl)porphyrinato]ferrate(III) in a variety of solvents, including protic, dipolar aprotic, and nonpolar solvents. On the basis of the NMR study, we have reached the following conclusions: (i) the complex adopts the ground state with the (d(xz), d(yz))(4)(d(xy))(1) electron configuration, the (d(xz), d(yz))(4)(d(xy)())(1) ground state, in methanol, because the d(pi) orbitals are stabilized due to the O-H...N hydrogen bonding between the coordinated cyanide and methanol; (ii) the complex also exhibits the (d(xz), d(yz))(4)(d(xy))(1) ground state in nonpolar solvents, such as chloroform and dichloromethane, which is ascribed to the stabilization of the d(pi) orbitals due to the C-H...N weak hydrogen bonding between the coordinated cyanide and the solvent molecules; (iii) the complex favors the (d(xz), d(yz))(4)(d(xy))(1) ground state in dipolar aprotic solvents, such as DMF, DMSO, and acetone, though the (d(xz), d(yz))(4)(d(xy))(1) character is less than that in chloroform and dichloromethane; (iv) the complex adopts the (d(xy))(2)(d(xz), d(yz))(3) ground state in nonpolar solvents, such as toluene, benzene, and tetrachloromethane, because of the lack of hydrogen bonding in these solvents; (v) acetonitrile behaves like nonpolar solvents, such as toluene, benzene, and tetrachloromethane, though it is classified as a dipolar aprotic solvent. Although the NMR results have been interpreted in terms of the solvent effects on the ordering of the d(xy) and d(pi) orbitals, they could also be interpreted in terms of the solvent effects on the population ratios of two isomers with different electron configurations. In fact, we have observed the unprecedented EPR spectra at 4.2 K which contain both the axial- and large g(max)-type signals in some solvents such as benzene, toluene, and acetonitrile. The observation of the two types of signals has been ascribed to the slow interconversion on the EPR time scale at 4.2 K between the ruffled complex with the (d(xz), d(yz))(4)(d(xy))(1) ground state and, possibly, the planar (or nearly planar) complex with the (d(xy))(2)(d(xz), d(yz))(3) ground state.     

FT-IR studies of solvent and temperature effects on poly(diglycidal ether of bisphenol A) (epoxy)

The infrared spectra of poly(diglycidal ether of bisphenol A) (epoxy) were studied in solutions of chloroform, benzene, and carbon tetrachloride separately to investigate the solvent effect through the behavior of the hydroxyl stretching band. The effect of temperature on the epoxy resin was also studied in this region. The results indicate that there is a varying degree of the weakening of the hydrogen bonding of the hydroxyl group in the epoxy, depending on the solvent. There is evidence also for the formation of hydrogen bonding between chloroform and the hydroxyl group of the epoxy and to a lesser extent between epoxy and benzene.     

亚油酸与核黄素或FAD激发态之间电荷转移的直接证据

根据荧光显微镜方法,我们首次发现核黄素(维生素B2)主要分布在细胞核的膜上和核的内部,故核黄素光敏化与辐射化的靶位置主要集中在细胞核内;当核黄素的浓度较大时,细胞膜上也有药物的分布,即在高浓度时,细胞膜也是光敏化与辐射敏化的作用位点一。应用308nn激光光解时间分辨吸收方法,以亚油酸作为脂质的模型化合物,研究了亚油酸与核黄素和黄素腺嘌呤二核苷酸(FAD)的激发三重态之间的电荷转移过程,首次给出了电荷转移的直接证据。     

有机溶剂与氢化可的松的溶剂—溶质作用

通过测定氢化可的松在多种溶剂／水体系中的分配系数研究了氢化可的松与溶剂的相互作用机制．其中醇、酮与氢化可的松有较强的氢键作用，酯、醚、氯代烷烃等溶剂与氢化可的松的氢键作用较弱，四氯化碳、苯、环己烷不能与氢化可的松形成氢键．     

1,2,4,5‐Tetrakis(2‐vinyl­pyridyl)­benzene–di­chloro­methane (1/2)

The title compound, C₃₄H₂₆N₄·2CH₂Cl₂, lies about an inversion center. The solvent mol­ecules interact with the benzene mol­ecule both through C—H⃛N hydrogen bonding to span pyridine N atoms of adjacent vinyl groups, possibly stabilizing the rotational conformation observed, and through a π interaction between a dichloromethane Cl atom and a pyridyl ring C—C bond of a c‐glide‐related mol­ecule. The benzene mol­ecules form stacks along the a axis such that two of the four olefin groups are properly oriented for photoreactivity (2+2 cyclo­dimerization).     

ELECTROSTATIC EFFECTS IN THE PHOTOREDUCTION OF FLAVINS BY EDTA

The anaerobic photoreduction of riboflavin, flavin mononucleotide, N(3)-carboxymethylriboflavin, N(3)-methyl-lumiflavin, and lumiflavin by EDTA was studied in aqueous solution over the pH range 2.5–10. The electrostatic effects of the electron donor-acceptor pair produce a secondary effect on the reactivity, and this effect can be predicted from the product of the charges (Z_D x Z_A). The trianonic and tetraanonic species of EDTA have nitrogens which are free from intramolecular hydrogen bonding, and these species are potentially the most reactive. However, in some pH regions the electrostatic effect can become the dominant factor when both the electron donor and acceptor become negatively charged. The excited states of flavins are susceptible to charge effects whether the charge is localized on the side chain or involves the isoalloxazine ring system.     

Chromatographic determination of riboflavin and its derivatives in food

Three elution methods on two different reversed-phase C18 columns were developed to determine flavin derivatives in raw egg white, raw egg yolk, egg powder, pasteurised milk, fermented milk products and liver (chicken, calf and pig). Additionally, 11 thin-layer chromatography solvent systems were used to confirm presence of flavins detected in assessed products. It was found that an Alphabond C18 column was not as effective as a Symmetry C18 column. Method A (mobile phase gradient of methanol-0.05 M ammonium acetate, pH 6.0 applied on an Alphabond C18 column) can be used for determination of flavin adenine dinucleotide, flavin mononucleotide, riboflavin 4',5'-cyclic phosphate, riboflavin, 10-formylmethylflavin and 10-hydroxyethylflavin in products that do not contain 7alpha-hydroxyriboflavin. Method B (mobile phase gradient of methanol-demineralized water, on an Alphabond C18 column) can be useful to separate flavin coenzymes from other flavin compounds or to confirm the presence of 7alpha-hydroxyriboflavin and 10-hydroxyethylflavin in analysed samples. Method C (mobile phase gradient of methanol-0.05 M ammonium acetate, pH 6.0, on a Symmetry C18 column) allows separation of all flavins detected in tested products: flavin adenine dinucleotide, flavin mononucleotide, riboflavin 4',5'-cyclic phosphate, riboflavin, 10-formylmethylflavin, 10-hydroxyethylflavin, 7alpha-hydroxyriboflavin, riboflavin-beta-D-galactoside and riboflavin-alpha-D-glucoside.     

Effect of molecular association on solubility,diffusion, and permeability in polymeric membranes

The filling‐type membrane is composed of grafted polymer and solvent‐resistant substrate; the calculation of solubility, diffusivity and swelling‐suppression effect by the substrate permits the prediction of solvent permeability. As noted in our previous article, the use of this approach, called membrane design, resulted in accurate prediction of the permeability of aromatic compounds. In this study, the influence of hydrogen bonding on solubility and diffusivity is investigated both theoretically and experimentally. The solubility of chloroform and dichloromethane in poly(acrylate)s increases, and their diffusivity decreases, compared with that estimated without considering the hydrogen‐bonding effect. Solubilities predicted by the lattice‐fluid hydrogen‐bonding (LFHB) model show good agreement with the results of vapor sorption. Comparison of diffusion coefficients measured by vapor permeation with those predicted from free volume theory reveals that the decrease of solvent diffusion coefficient is approximately proportional to the fraction of associated molecules. Fluxes of chloroform and dichloromethane were measured by vapor permeation experiments through filling‐type acrylate membranes, and predictions agree well with experiments. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 38: 171–181, 2000     

A flavin analogue with improved solubility in organic solvents

We report the synthesis and initial electrochemical characterization of a benzene-soluble flavin analogue: N(10)-2,2-dibenzylethyl-7,8-dimethylisoalloxazine (DBF, 1). This analogue, which has an unmodified flavin headgroup, is intended for use in the spectroscopic examination of the electronic effects of flavin hydrogen bonding in simple model systems in aprotic, non-hydrogen bonding solvents. With future spectroscopic studies in mind, we have developed a synthetic route, which allows the incorporation of isotopic labels using inexpensive starting materials.     

Does Urea Preferentially Interact with Amide Moieties or Nonpolar Sidechains? A Question Answered Through a Judicious Selection of Model Systems

The transfer model suggests that urea unfolds proteins mainly by increasing the solubility of the amide backbone, probably through urea-induced increase in hydrogen bonding. Other studies suggest that urea addition increases the magnitude of solvent-solute van der Waals interactions, which increases the solubility of nonpolar sidechains. More recent analyses hypothesize that urea has a similar effect in increasing the solubility of backbone and sidechain groups. In this work, we compare the effects of urea addition on the solvation of amides and alkyl groups. At first, we study the effects of urea addition upon solvent hydrogen bonding acidity and basicity through the perturbation in the fluorescence spectrum of probes 1-AN and 1-DMAN. Our results demonstrate that the solvent's hydrogen bonding properties are minimally affected by urea addition. Subsequently, we show that urea addition does not perturb the intra-molecular hydrogen bonding in salicylic acid significantly. Finally, we investigate how urea preferentially interacts with amide and alkyl groups moieties in water by comparing the effects of urea addition upon the solubility of acetaminophen and 4-tertbutylphenol. We show that urea affects amide and t-butyl solubility (lowers the transfer free energy of both amide (backbone) and alkyl (sidechain) groups) in a similar fashion. In other words, preferential interaction of urea with both moieties contributes to protein denaturation.     

Design and synthesis of a library of BODIPY-based environmental polarity sensors utilizing photoinduced electron-transfer-controlled fluorescence ON/OFF switching

We systematically examined the mechanism of the solvent polarity dependence of the fluorescence ON/OFF threshold of the BODIPY (boron dipyrromethene) fluorophore and the role of photoinduced electron transfer (PeT). In a series of BODIPY derivatives with variously substituted benzene moieties at the 8-position, the oxidation potential of the benzene moiety became more positive and the reduction potential of the BODIPY fluorophore became more negative as the solvent polarity was decreased; consequently, the free energy change of PeT from the benzene moiety becomes larger in a more nonpolar environment. Utilizing this finding, we designed and synthesized a library of probes in which the threshold of fluorescence ON/OFF switching corresponds to different levels of solvent polarity. These environment-sensitive probes were used to examine bovine serum albumin (BSA) and living cells. The polarity at the surface of albumin was concluded to be similar to that of acetone, while the polarity of the internal membranes of HeLa cells was similar to that of dichloromethane.     

ASSESSMENT OF DIELECTRIC ENRICHMENT AROUND TWO FLUOROPHORES IN BINARY SOLVENTS

Abstract We investigated dielectric enrichment, or the preferential interaction between a probe and a polar solvent, using two different fluorophores in binary solvents that will not form strong hydrogen bonds with the probe. The first probe, 6-propionyl( N,N -dimethylamino)naphthalene (PRODAN), undergoes a large change in dipole moment in the excited state. We found that its emission energy varies linearly with the dielectric parameter f(D) [f(D) = 2(D - 1)/(2D + 1)] in acetone/benzene and acetonekyclohexane mixtures. The emission of the second probe, N-acetyl tryptophanamide (NATA), also varies linearly with f (D) in ethylether/acetonitrile solvents. This lack of preferential solvation was further investigated by studies of NATA in acetonitrile/methanol mixtures where hydrogen bonding is possible. We found that NATA-methanol interactions are not energetic enough to give rise to significant preferential solvation. Since preferential solvation does not occur even for weakly hydrogen bonding solvents it is thus highly unlikely that dielectric enrichment, which takes place in the absence of specific interactions, occurs with any frequency. We postulate that previous reports of dielectric enrichment were due to a lack of consideration of changes in f (D) of the solvent.     

THE EFFECT OF 8α-SUBSTITUTION ON FLAVIN TRIPLET STATE AND SEMIQUINONE PROPERTIES AS INVESTIGATED BY FLASH PHOTOLYSIS*

Abstract— Flash photolysis techniques have been used to study the effect of 8α-substitution on flavin triplet state formation and decay and on the properties of neutral and anionic serniquinones. Compared with riboflavin, the N(1) and N(3) isomers of 8α-histidylriboflavin show a lower triplet yield (?10%) and a faster rate of decay (? 4-Cfold). Acetylation of the histidyl a-amino groups and of the flavin ribityl side chain results in a 2-fold increase in triplet yield and a 2-fold slower rate of decay. The yield of neutral 8α-substituted flavin semiquinones upon flash photolysis in the presence of EDTA was approximately 50% that given by riboflavin. These substituted flavin neutral semiquinones dismutated at a rate 2–3 times slower than the corresponding unsubstituted form, although the anionic semiquinones dismutated at approximately the same rate. In the presence of oxygen, the kinetics of semiquinone decay changed from second order to pseudo-first order upon raising the pH, thus showing anionic semiquinone oxidation as seen previously with unsnbstituted flavins. The pK values for the ionization of the neutral 8α-substituted Aavin semiquinones are 1–1.5 units lower than the unsubstituted form. The anionic 8α-substituted flavin semiquinones react with oxygen at a rate 2–10 times more slowly than does the riboflavin form. Such alterations in properties probably reflect the electron-withdrawing effect of the 8α-substituents on the flavin ring system.     

Structures of aza-macrocyclic ligands with polyphosphonated dangling groups

Two phosphonated hexaaza-macrocycles with 24- and 26-membered rings have been synthesized via a Mannich reaction and characterized. The novel crystal structures of the macrocyclic ligands show the extended 3-D hydrogen bonded structure in their solid states. These ligands have unusual ring conformations with two or four pendent arms wrapping around the macrocycle with alternating up and down orders. Strong hydrogen bonding between the protonated phosphonate groups and the deprotonated phosphonated groups was observed in those structures together with the complicated solvent hydrogen bonding networks.     

A study of photochemistry of flavins in pyridine and with a donor

Abstract— Both anaerobic and aerobic photolysis of riboflavin in pyridine yielded several photoproducts, analogous to the photochemical reaction in aqueous solution. Lumiflavin was also photoreduced in pyridine (an electron donor) without decomposition of the isoall-oxazine ring Differences in the reactivity of excited singlet and triplet states with respect to formation of photoproducts have been confirmed in pyridine. In the photoreduction of riboflavin by N, N'-dimethyl-N-benzylethylene diamine, an initial rate with a higher relative quantum yield than that of the reaction at a later stage was observed both in water and pyridine. Photolysis in D₂O with the donor showed no significant solvent isotope effect. These results strongly suggest that the photolysis of riboflavin in water does not involve the water molecule in the primary photochemical act. A detailed mechanism of flavin photoreaction in water to account for solvent oxygen incorporation into a photoproduct (benzaldehyde) from N, N'-dimethyl-N-benzylethylene diamine has been proposed without involving photochemical splitting of water based on our results and molecular orbital computations. Preliminary results on the kinetics of free radical decay were obtained using an ESR spectrometer and the significance of the results are discussed.     

Photoinduced intramolecular charge transfer (ICT) reaction in trans-methyl p-(dimethylamino) cinnamate: A combined fluorescence measurement and quantum chemical calculations

The photophysical behaviour of trans-methyl p-(dimethylamino) cinnamate (t-MDMAC) donor–acceptor system has been investigated by steady-state absorption and emission spectroscopy and quantum chemical calculations. The molecule t-MDMAC shows an emission from the locally excited state in non-polar solvents. In addition to weak local emission, a strong solvent dependent red shifted fluorescence in polar aprotic solvents is attributed to highly polar intramolecular charge transfer state. However, the formation of hydrogen-bonded clusters with polar protic solvents has been suggested from a linear correlation between the observed red shifted fluorescence band maxima with hydrogen bonding parameters (). Calculations by ab initio and density functional theory show that the lone pair electron at nitrogen center is out of plane of the benzene ring in the global minimum ground state structure. In the gas phase, a potential energy surface along the twist coordinate at the donor (–NMe₂) and acceptor (–CH = CHCOOMe) sites shows stabilization of S₁ state and destabilization S₂ and S₀ states. A similar potential energy calculation along the twist coordinate in acetonitrile solvent using non-equilibrium polarized continuum model also shows more stabilization of S₁ state relative to other states and supports solvent dependent red shifted emission properties. In all types of calculations it is found that the nitrogen lone pair is delocalized over the benzene ring in the global minimum ground state and is localized on the nitrogen centre at the 90° twisted configuration. The S₁ energy state stabilization along the twist coordinate at the donor site and localized nitrogen lone pair at the perpendicular configuration support well the observed dual fluorescence in terms of proposed twisted intramolecular charge transfer (TICT) model.     

A stacking interaction between a bridging hydrogen atom and aromatic pi density in the n-B18H22-benzene system

The structures of n-B18H22 and of n-B18H22 x C6H6 were determined by single-crystal X-ray analysis at -60 degrees C. The geometry of the boron cluster itself does not seem to be appreciably affected by solvation. There does, however, appear to be an unusual interaction of a polyborane bridging hydrogen atom with the benzene pi system, giving rise to an extended stacked structure. The 1H{11B} spectrum of n-B18H22 in [D6]benzene differs from that in [D12]cyclohexane most noticeably in the bridging proton region. Upon moving from the aliphatic to the aromatic solvent, the greatest increase in shielding was for the signal corresponding to the bridge hydrogen atom that interacts with the pi system of benzene; the signal was shifted upfield by 0.49 ppm. Density functional theory calculations were performed on 1:1 and 2:1 complexes of the n-B18H22 unit with benzene.