STUDIES ON FLAVINS IN ORGANIC SOLVENTS-I*. SPECTRAL CHARACTERISTICS OF RIBOFLAVIN,RIBOFLAVIN TETRABUTYRATE AND LUMICHROME

Abstract— The absorption and fluorescence spectra of riboflavin, riboflavin tetrabutyrate and lumichrome have been studied in organic solvents of different polarity including ethanol, acetone, dioxane, pyridine, acetic acid and in cyclohexane-dioxane mixtures. The molar extinction coefficients of the compounds were measured and the character of solvent sensitive changes observed in the absorption spectra are discussed. The effect of hydrogen bonding and/or intramolecular charge transfer complex are proposed as possible explanation. Changes in the shape of fluorescence emission spectra are less evident; in the case of riboflavin tetrabutyrate, mirror symmetry to the shape of the longest wavelength maximum was observed in low-polar solvents. Quantum yields of fluorescence are higher the lower the polarity of the solvent used; the value for riboflavin tetrabutyrate is greater than for riboflavin. Changes were also observed in the fluorescence emission spectra of lumichrome in solvents of different polarity. Fairly good agreement was demonstrated for positions of the observed absorption maxima of riboflavin in 98% dioxane, riboflavin tetrabutyrate and lumichrome in 80% cyclohexane in mixture with dioxane and the values calculated from energies of molecular orbitals of flavins computed by B. Pullman and A. Pullman.     

PHOTCHEMICAL DEGRADATION OF FLAVINS—III. HYDROXYETHYL AND FORMYLMETHYL ANALOGS OF RIBOFLAVIN*

Abstract— The anaerobic photolysis of the flavins containing hydroxyethyl and formythmethyl side-chains in place of the ribityl group of riboflavin has been studied employing spectro-photmetry, thin-layer chromatography and other mehtods. Both flavins are shown to decompose, in part, directly to lumichrome. Acetaldehyde arises from the hudoxyethyl by the flavin nucleus as has been observed previously for riboflavin. The side-chain of the hydroxyethylflavin, is shown to be oxidized to the formylmethyl group. Formylmethylflavin is converted in part to lumichrome and unknown products from the side-chain and in part to a reduced flavin. Formaldehyde could not be detected among the products. Possible mechanisms are discussed briefly.     

Photochemical degradation of flavins. IV. Studies of the anaerobic photolysis of riboflavin

Abstract A variety of substances which quench the fluorescence of riboflavin decrease the rate of anaerobic photobleaching (photolysis) of the flavin at concentrations which have little effect on the fluorescence. A semi-quantitative estimate of the yield of various products of photolysis using thin layer chromatography and autoradiography with C¹⁴-labelled riboflavin shows that at least ten radioactive products are formed. Whereas the yield of most of these is decreased by low concentrations of quenchers, such as phenol, some are decreased only at quencher concentrations high enough to decrease the fluorescence significantly. The effects of quenchers on the phosphorescence and ESR signal induced by light were also observed. It is concluded that lumichrome arises, in part, from degradation of the excited singlet state, but that 9-formylmethylflavin and other products arise through the triplet state. A major product of anaerobic photolysis is a substance moving just ahead of riboflavin on chromatograms and which, like 9-formylmethylflavin, is destroyed by treatment with sodium hydroxide. Effects of varying the nature of the solvent, the flavin structure and the pH are reported, and the kinetics of processes involved in the primary photochemical acts and in quenching are discussed.     

PHOTOCHEMICAL REACTIONS IN RIGID MEDIA-I. THE EFFECT OF GELATIN ON THE AEROBIC PHOTOCHEMISTRY OF RIBOFLAVIN IN AQUEOUS SOLUTION

Abstract— The addition of potassium iodide to dilute aqueous solutions of riboflavin reduced both the rate of the aerobic photolysis and the fluorescence quantum yield of riboflavin in the same proportions. This indicated that under these conditions the photolysis proceeded from the singlet excited state. The addition of gelatin to aqueous solutions of riboflavin also reduced the rate of the aerobic photolysis but increased slightly the quantum yield of fluorescence. The rates and the fluorescence of solutions of riboflavin to which gelatin had been added were also reduced by the addition of potassium iodide but in this case the effect on the rate was proportionately greater than the effect on the fluorescence. The data suggests that in the presence of gelatin the mechanism of the reaction is changed and that the triplet state becomes more important.     

Effect of light intensity and wavelengths on photodegradation reactions of riboflavin in aqueous solution

A study of the effect of light intensity and wavelengths on photodegradation reactions of riboflavin (RF) solutions in the presence of phosphate buffer using three UV and visible radiation sources has been made. The rates and magnitude of the two major photodegradation reactions of riboflavin in phosphate buffer (i.e., photoaddition and photoreduction) depend on light intensity as well as the wavelengths of irradiation. Photoaddition is facilitated by UV radiation and yields cyclodehydroriboflavin (CDRF) whereas photoreduction results from normal photolysis yielding lumichrome (LC) and lumiflavin (LF). The ratios of the photoproducts of the two reactions at 2.0 M phosphate concentration, CDRF/RF (0.09-0.22) and CDRF/LC (0.54-1.75), vary with the radiation source and are higher with UV radiation than those of the visible radiation. On the contrary, the ratios of LF/LC (0.15-0.25) increase on changing the radiation source from UV to visible. The rate is much faster with UV radiation causing 25% degradation of a 10(-5) M riboflavin solution in 7.5 min compared to that of visible radiations in 150-330 min.     

PHOTOLYSIS OF PHENACYL ESTERS IN A TWO-PHASE SYSTEM

ABSTRACT

Phenacyl esters are useful photoremovable protecting groups for carboxylic acids in organic synthesis and biochemistry. In this work, simple one-pot arrangements of the phenacyl and 2,5-dimethylphenacyl ester photolysis are proposed. The reactions were performed in both the benzene/water two-phase system and in water. Cetyltrimethylammonium bromide was found to increase substantially the efficiency of the deprotection as well as the purity of the products by lowering the interfacial tension between the phases. Utilizing water as a medium significantly reduced the necessity to use environmentally malign organic solvents. The overall yields varied from 72 to 98% depending on the reaction conditions.     

OXYGEN QUENCHING IN MICELLAR SOLUTION AND ITS EFFECT ON THE PHOTOCYCLIZATION OF N-METHYLDIPHENYLAMINE

Abstract— Oxygen effects on the photocyclization of N-methyldiphenylamine to N-methylcarbazole were investigated in n-hexane, water, and aqueous surfactant solutions by steady state irradiations and flash photolysis measurements. The reaction sequence in micelles was found to involve the same intermediate steps as in homogeneous solutions. In aerated micellar solutions, the quantum yield of N-methylcarbazole is significantly higher than in n-hexane, while the rate constants of the unimolecular reaction steps show no solvent dependence. The bimolecular dehydrogenation of the intermediate 4a, 4b-dihydro-N-methylcarbazole by oxygen is enhanced in aqueous and micellar solutions, whereas the quenching rate of triplet intermediates by oxygen was not affected. The lesuhs are interpreted using a dispersed phase model of micellar solutions. Special 'micellar effects' need not be invoked since the dependence of the quantum yield on the solvent is shown to be due to the difference in the overall oxygen concentration.     

Determination of picomolar levels of flavins in natural waters by solid-phase ion-pair extraction and liquid chromatography

A method is described for the rapid determination of flavins in sea water, based on solid-phase extraction followed by ion-pair high-performance liquid chromatography (HPLC) with fluorescence detection. Riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) and their photochemical breakdown products, lumiflavin, formylmethylflavin, and lumichrome can be determined with subpicomolar detection limits. The method was used at sea in the analysis of coastal and open ocean waters. In both environments, riboflavin, lumiflavin and lumichrome were routinely observed at concentrations in the picomolar range; lumiflavin and lumichrome were generally confined to the photic zone while riboflavin was present throughout the water column. Formylmethylflavin, FMN, and FAD were only occasionally observed; when present, these flavins were observed at consistently higher concentrations than riboflavin, lumiflavin and lumichrome.     

PHOTOCHEMICAL-LIKE BEHAVIOUR OF RIBOFLAVIN IN THE DARK PROMOTED BY ENZYME-GENERATED TRIPLET ACETONE

Abstract— Triplet acetone obtained enzymically by the isobutyraldehyde/peroxidase/O₂ system transfers its energy to the riboflavin, which changes into a decomposition product of lumichrome type and produces aggregates. When riboflavin and tryptophan are added to this system, further products—formylkynurenine and an adduct between the riboflavin and the tryptophan—are formed. The adduct can be separated by gel filtration and ion exchange chromatography and is similar to that prepared by irradiating riboflavin in the presence of tryptophan.     

FEMTOSECOND STUDIES OF HEMATOPORPHYRIN DERIVATIVE IN SOLUTION: EFFECT OF pH AND SOLVENT ON THE EXCITED STATE DYNAMICS

Abstract We report direct femtosecond measurements of the excited state dynamics of hematoporphyrin derivative (HpD) in solution. The dynamics are found to be very sensitive to the solvent and pH of aqueous solutions. The decay of the excited singlet states is much faster in acidic and pH 7 buffer aqueous solutions (<230 ps) than in basic aqueous solutions or organic solvents (> 10 ns). The dynamical results show strong correlation with static fluorescence measurements: weaker fluorescence in acidic and pH 7 buffer solutions corresponding to shorter-lived excited states. A new fast decay component with a time constant around 5 ps is identified both in acidic aqueous solutions and in organic solvents such as acetone and attributed to internal conversion from the second to the first excited singlet state of aggregates or certain oligomers in HpD, in accord with the observation that the fast decay component is larger at a higher concentration. Oxygen is found to have no effect on the dynamics on the time scale investigated, 1 ns, indicating that oxygen quenching of the singlet excited states is insignificant on this time scale. The sensitive solvent and pH dependence of the excited state dynamics has important clinical implications in the use of HpD as a photosensitizing agent.     

Effect of phosphate buffer on photodegradation reactions of riboflavin in aqueous solution

The effect of phosphate buffer on aerobic photodegradation reactions of riboflavin (RF) at pH 7.0 has been studied. The photoproducts of the two major reactions, viz., intramolecular photoreduction and intramolecular photoaddition, have been determined by a specific multicomponent spectrophotometric method. The overall photodegradation of riboflavin in the presence of phosphate buffer involves the participation of both H2PO4-and HPO4(2-) species. The second-order rate constants for the H2PO4(-)-catalysed photodegradation of riboflavin (normal photolysis) to lumichrome (LC) and HPO4(2-)-catalysed photodegradation of riboflavin (photoaddition) to cyclodehydroriboflavin (CDRF) are 0.93 x 10(-4) and 4.0 x 10(-4) M(-1) s(-1), respectively. The addition of 0.25-2.00 M phosphate to RF solutions at pH 7.0 gives rise to RF-HPO4(2-) complex and hence the quenching of 4-36% fluorescence, respectively. This results in the suppression of normal photolysis leading to the formation of LC in favour of photoaddition to yield CDRF. The present study shows the involvement of H2PO4- anions in the base-catalysed degradation of riboflavin by normal photolysis vis-a-vis the involvement of HPO42- anions in photoaddition reactions of riboflavin suggested earlier [M. Schuman Jorns, G. Schollnhammer, P. Hemmerich, Intramolecular addition of the riboflavin side chain. Anion-catalysed neutral photochemistry, Eur. J. Biochem. 57 (1975) 35-48].     

ULTRAVIOLET PHOTOLYSIS OF RIBOFLAVIN IN PYRIDINE: A PRELIMINARY STUDY

Abstract— Ultraviolet photolysis of riboflavin in pyridine leads to the formation of a red photoproduct. This photoreaction is apparently attributed to the photochemical interaction of pyridine with riboflavin. It is found that the rate of the photoreaction follows 1/2 order kinetics with respect to pyridine. ESR spectrum of the irradiated reaction mixture appears to be identical with that of riboflavin semiquinone obtained from visible light irradiation. Whether or not such a flavin radical is the responsible intermediate in the photoreaction is not established. Possible modes of the reaction are discussed. Absorption, excitation, and emission spectra of the red photoproduct strongly suggest that the structure of the photoproduct is considerably different from that of riboflavin or pyridine, indicating an extensive photolytic process involving covalent bond alterations.     

REVERSIBLE EPR PHOTOSIGNALS IN CHARGE-TRANSFER-COMPLEXES

Abstract— Various organic solutions were irradiated in a search for rapidly reversible EPR photosignals. Using an averaging technique, the rise and decay of the photosignals of CT-complexes formed between chloranil and organic solvents were shown to be of the order of tens of msec. However, the mechanism involved could not be fully elucidated without additional data such as those obtained from flash photolysis and other techniques.     

PHOTOCHEMISTRY OF FLAVINS. II. PHOTOPHYSICAL PROPERTIES OF ALLOXAZINES AND ISOALLOXAZINES

Abstract. Pulsed laser photolysis at 347nm has been used to study the transient spectroscopy of alloxazine, lumichrome, lumiflavin, and riboflavin in acidic (pH 2.2) aqueous solution and in ethanol. Intersystem crossing quantum yields (φ_ISC) were determined by a modification of the comparative laser excitation method which utilizes the variation of the triplet yield with intensity in conjunction with a kinetic model for the various photophysical and photochemical processes occurring during the pulse. Fluorescence quantum yields and lifetimes are also reported. Correction for quenching of the excited singlet state by H⁺ ions shows that, in neutral aqueous solution, intersystem crossing for flavins is an efficient process (φ_ISC˜ 0.7) which, in conjunction with fluorescence, accounts for the fate of all absorbed photons. For alloxazine (φ_ISC˜ 0.45) and lumichrome (φ_ISC˜ 0.7) the results are more difficult to interpret owing to interconversion between alloxazine and isoalloxazine structures in the singlet excited state. For all four compounds, the quantum yield of products derived from the singlet excited state is estimated as ˜0.04. There is evidence of biphotonic product formation at high laser energies. In ethanol, where φ_ISC for lumichrome is about twice that of lumiflavin, internal conversion between the excited singlet and ground states appears to be a significant process. Complete triplet-triplet absorption spectra in the region 260–750nm are reported. For lumichrome at pH 2.2 there is spectral evidence for isomeric triplet states which appear to be in equilibrium.     

On the High Viscosity of Aqueous Solution of Lysozyme Induced by Some Organic Solvents

A remarkably high viscosity has been induced in protein aqueous solutions by the addition of certain structurally related organic solvents. The effect has been observed for lysozyme aqueous solutions containing tetramethylurea (TMU), dimethylsulfoxide (DMSO), dimethylformamide, and hexamethylphosphortriamide. The effect has also been induced in ferrocytochrome c aqueous solutions by TMU. Critical concentrations for both the protein and organic solvent were verified for the onset of the viscosity increase. A common feature of the solvents which were able to induce the effect is a dipolar moiety (C=O, S → O and P → O) and a nonpolar region represented by the methyl groups. The resulting fluids show an extremely restricted flow and a typical non-Newtonian, pseudoplastic behavior. Use was made of¹H nuclear magnetic resonance (NMR) and Raman spectroscopy to characterize protein structural modifications and of¹³C NMR to investigate changes in relaxation times and chemical shifts in the solvent/water solutions. A systematic rheological characterization of the systems was undertaken for some of the solvents, and unusual patterns of viscous effects were identified for the solvent/water systems both with and without protein. The process was found to be at least partially reversible, as concluded from the recovered original solution rheological characteristics and the original protein¹H NMR spectrum, after eliminating the organic solvent by ultrafiltration. The whole process was characterized as consisting of two mutually independent stages. The first involves an extensive conformational transition of the polypeptide backbone, from a predominantly α-helical to increased random coiled and β-sheet structures, with the occurrence of nonorthodox protein secondary structures at regions above the solvent critical point. The second stage consists of short-lived interchain contacts leading to an entanglement of the macromolecular system as a whole. A microphase reversion in the organic solvent/water mixture, supported by¹³C NMR and rheological results, is proposed as the driving force causing the observed behavior.     

THE PHOTOCHEMISTRY OF RIBOFLAVIN—V. THE PHOTODEGRADATION OF ISOALLOXAZINES IN ALCOHOLIC SOLVENTS

Abstract— Riboflavin and other N -10 substituted isoalloxazines have been irradiated anaerobically in alcohols and alcohol-water mixtures. Deuterium substitution was used in several different ways to determine the hydrogen kinetic isotope effect. Riboflavin and 10-(3'-hydroxypropyl)-isoalloxazine were photobleached in 2-propanol, 2-propanol-0-d, 2-propanol-2-d, t -butyl alcohol, and t -butyl alcohol-0-d. No isotope effect significantly different from 1.0 was found for any of the nondeuterated to deuterated solvent comparisons. When the anaerobic irradiation of 10-(3'-hydroxypropyl-3',3'-d₂)-isoalloxazine and 10-(3'-hydroxypropyl)-isoalloxazine in either 2-propanol or t-butyl alcohol were compared, kinetic isotope effect ratios for H to D of 2.8 were obtained. Thin layer chromatography in conjunction with color return at 445 nm during oxidation showed that lumichrome and formylmethylflavin, which are irreversible photodegradation products, were formed from the photoreaction in alcohols. The photochemistry of isoalloxazines in alcohols, while somewhat different from the aqueous photochemistry, does not involve a primary photoreduction with the solvent, and photo degradation still dominates. This was an unexpected result and it illustrates the unusual photochemical behavior of isoalloxazines. The anaerobic irradiation of riboflavin in 2-propanol-water mixtures showed that the efficiency of photodegradation is linked to the physical properties of the solvent.     

丙烯酰胺在聚乙二醇水溶液中的聚合动力学

用改进溴法对丙烯酰胺 (AM)在聚乙二醇 (PEG)水溶液中聚合动力学进行研究 .在单一和氧化还原引发体系中分别考察了引发剂、单体和PEG用量、不同HLB值乳化剂以及聚合温度等因素对动力学的影响 .得到AM聚合速率与过硫酸铵 (APS)浓度的 0 91次方成正比 ;单一APS和APS 三乙醇胺 (TEA)氧化还原引发体系中的AM聚合表观活化能分别为 96 1和 4 2 3kJ mol.     

Study of types and mixture ratio of organic solvent used to dissolve polymers for preparation of drug-containing PLGA microspheres

The effects of the types and the ratios of various organic solvents used as a mixtures to dissolve poly (lactide-co-glycolide) (PLGA) by using a solvent evaporation method, a technique used to prepare polymer particles, were carefully studied in order to investigate their advantages in developing drug delivery system (DDS) formulations for the prepared microspheres. The particle size and drug loading efficiency of drug-containing PLGA microspheres were found to be dependent on the types of solvent used due to the interfacial tension between the organic solvent and water phase. The drug loading efficiency of monodisperse microspheres prepared by using a membrane emulsification technique employing organic solvents and high interfacial tension for dissolving the PLGA was increased in a controlled manner. The organic solvents with high interfacial tension in the water phase used for the preparation of polymer particles by means of the solvent evaporation method were found to be suitable in terms of improvement in the properties of DDS formulations.     

Chemical and photolytical transformation of biomedically significant compounds in the presence of deuterated solvents

The effect of the nature of solvent on the properties of biomedically important compounds is of particular importance. The conversion of certain biomedical compounds with deuterated solvents is an area of research that has not been accorded adequate recognition in the literature. We explored this area in the interest of shedding some light on the possible effects of solvent on the nature of the solute. The transformation of specific medically important compounds such as bilirubin, thymine, uracil, dehydrocholesterol (7-DHC) and vitamin D(3) was observed in the presence of deuterated solvents such as heavy water and deuterated chloroform. The products of the relevant reactions were confirmed spectrophotometrically. An additional feature to our investigation involved the photolysis of the aforementioned compounds by solar irradiation. The pure samples were dissolved in solutions of the deuterated solvents, corresponding to concentrations of typically 10(-2) mM, and exposed to sunlight for about 15-30 min. The deuterated solvents caused chemical transformation in all chemical compounds tested, and produced intense characteristic absorbance maxima between 200 and 700 nm. Sunlight exposure was also effective in either augmenting the effects of deuterated solvent as in bilirubin and 7-DHC or reducing it as with thymine or having no effect as with uracil or completely changing it as in vitamin D(3). It has been shown that the use of deuterated solvents produces unique chemical and photochemical conversions of bilirubin, 7-DHC, thymine, uracil and vitamin D(3). This was attributed to the fact that deuterated compounds display a somewhat different chemistry to their ordinary counterparts and that possibly thermodynamic considerations could be responsible for the novel transformations.     

Formation and characterization of polymersomes made by a solvent injection method

In this article a solvent injection method is described for vesicle formation using poly(butadiene)‐ b‐poly(acrylic acid) diblock copolymers as the amphiphilic molecules. Vesicles composed of polymer bilayers are commonly referred to as polymersomes. Solvent injection is shown to be a rapid method for polymersome formation suitable to make large volumes of polymersome solution. The method can be manipulated to obtain a wide range of vesicle sizes depending on the polymer concentration and preparation conditions. Polymersome solutions in this study are characterized using dynamic light scattering (DLS), fluorescent microscopy, and electron microscopy. Polymersome sizes range from tens of nanometers to several microns. The membrane thickness of smaller polymersomes is found to lie between 14–20 nm. Larger polymersomes are found to have somewhat thicker membranes. The procedure involves the addition of polymers dissolved in an organic solvent to a stirred aqueous solution. The formation of polymersomes by this method is proposed to be governed by the limited mutual solubility of the two solvents and the simultaneous diffusion of solvent and water out of and in to initially formed organic solvent droplets. Copyright © 2007 John Wiley & Sons, Ltd.