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.     

Probing Protonation Sites of Isolated Flavins Using IR Spectroscopy: From Lumichrome to the Cofactor Flavin Mononucleotide

Infrared spectra of the isolated protonated flavin molecules lumichrome, lumiflavin, riboflavin (vitamin B₂), and the biologically important cofactor flavin mononucleotide are measured in the fingerprint region (600–1850 cm^?1) by means of IR multiple‐photon dissociation (IRMPD) spectroscopy. Using density functional theory calculations, the geometries, relative energies, and linear IR absorption spectra of several low‐energy isomers are calculated. Comparison of the calculated IR spectra with the measured IRMPD spectra reveals that the N10 substituent on the isoalloxazine ring influences the protonation site of the flavin. Lumichrome, with a hydrogen substituent, is only stable as the N1‐protonated tautomer and protonates at N5 of the pyrazine ring. The presence of the ribityl unit in riboflavin leads to protonation at N1 of the pyrimidinedione moiety, and methyl substitution in lumiflavin stabilizes the tautomer that is protonated at O2. In contrast, flavin mononucleotide exists as both the O2‐ and N1‐protonated tautomers. The frequencies and relative intensities of the two C?O stretch vibrations in protonated flavins serve as reliable indicators for their protonation site.     

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.     

PROTONATION AND DEPROTONATION EQUILIBRIA OF LUMICHROME

Abstract— In order to investigate the possibility of the tautomerization of alloxazine to isolloxazine in its ground state, the parameters affecting the redistribution of charges in the lumichrome molecule were studied. The absorption and emission spectra of lumichrome as a function of pH in the range H₀ = - 6 to pH = 12 were recorded. At extreme pH conditions the spectra of lumichrome are similar to those of the isoalloxazine system. At high acid concentration ( H ₀ < - 3.0) the absorption spectrum of lumichrome protonated at N₁₀, is practically identical to that of lumiflavin. The fluorescence quantum yield of the two cations is negligible at room temperature.
At pH = 10.5 lumichrome is deprotonated at positions N₁ or N₃, The two monoanions have different excitation spectra. Except for slight differences in the extinction coefficients, the absorption of the anion deprotonated at N₃ is very similar to the lumichrome spectrum. The absorption spectra of the N₁ monoanion and of the di-anion are similar to the spectrum of lumiflavin except for a blue shift of about 20 nm. Furthermore, the emission spectrum of the N₁ -monoanion is identical to that of the isoalloxazine system. These results indicate that the charge distribution in the lumichrome molecule depends on the protonation and deprotonation of the nitrogen atoms at positions 10 and l. Both processes cause a redistribution of charges so that an isoalloxazine ring system is formed.     

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.     

Electron ionization mass spectrometric study of substituted alloxazine‐5‐oxides and iso‐alloxazine‐5‐oxide

The fragmentation pathways in electron ionization (EI) mass spectra of a series of new N(5)‐oxides of alloxazines and iso‐alloxazine are presented, and compared with those of substituted alloxazines and iso‐alloxazine. The EI mass spectra of these compounds showed characteristic fragmentation pathways A, B and C, started by the ejection of atomic oxygen, a HNCO molecule and an OH ^. radical, respectively. On the basis of B/E and B²/E spectra, the mechanism of elimination of the OH ^. radical is discussed. The influence of the methyl substituent in the benzene ring of alloxazine on the mass fragmentation pathways is described. Copyright © 2009 John Wiley & Sons, Ltd.     

Photophysical properties of lumichromes in water

The photophysics of lumichrome, 1-methyllumichrome, and lumiflavin in water solutions have been investigated. Fluorescence lifetimes of 2.7 and 2.2 ns were observed for lumichrome and 1-methyllumichrome, respectively, the corresponding triplet state lifetimes of 17 and 18 μs have been obtained from the transient absorption spectra. Evidence for long lived species with absorption maxima near 450 nm and lifetimes of ca. 400 μs has been found in the transient absorption spectra of both lumichromes. Quantum yields for the sensitised production of singlet oxygen, φ_Δ, are 0.36 and 0.41 for lumichrome and 1-methyllumichrome, respectively, in D₂O.     

The m/z 91 rearrangement ion in the mass spectra of some 1,4-benzodiazepines

With the aid of isotope labeling and by substituent shifts, the relatively strong m/z 91 ion in the mass spectra of 7-chloro-2-methoxy-5-phenyl-3H-1,4-benzodiazepine and related compounds was shown to contain the 5-phenyl ring and the 3-CH₂ group. Mechanisms involving the opening of the 7-membered ring and the migration of the phenyl ring from C-5 to C-3 are postulated for the formation of this ion. This rearrangement ion was also observed in the mass spectra of some 1-alkyl-7-chloro-1,3-dhydro-5-phenyl-2H-1,4-benzodiazepin-2-ones.     

Femtosecond pump-probe experiments on trapped flavin: optical control of dissociation

Femtosecond pump-probe experiments are performed on flavin biomolecules isolated in an ion trap. Mass spectra of the photoinduced fragments show that the fragmentation pathways can be modified using two-color two-photon excitation. In particular, when an infrared probe pulse (810 nm) is added subsequent to the first excitation step (excitation of the S(1) state of flavin mononucleotide at 405 nm), branching ratios between lumichrome and lumiflavin production are inverted relative to the single excitation case.     

Analogs of sparteine. IV. Mass spectral features of a series of N,N′-disubstituted bispidines

The mass spectra of a series of N, N′-disubstituted bispidinc derivatives have been investigated, and salient features analogous to those seen in the spectra of related azabieyelie compounds observed. The most important feature observed in the spectra is the common base peak (m/e 58), which results from the generation of N, N-dimethylformimmonium ion 24. The pathway by which this ion originates is discussed.     

Stereochemical effects in mass spectra—Studies on mass spectra of epimers of podophyllotoxin and its derivatives

Mass spectra of thirteen epimers of podophyllotoxin and its derivatives have been examined. By means of high resolution mass spectrometry, metastable ion scanning and isotopelabeling techniques, the effects of configuration of C₁-OH and condensed lactone ring on the fragmentation pattern and the relative abundances of characteristic ions in the mass spectra of these compounds have been studied and rationalized.     

Synthesis of 6-hydroxycarvone derivatives and their oxidative decyclization with lead tetraacetate

6-Hydroxy derivatives of R-(−)-carvone and 7,8-epoxycarvone were synthesized and subjected to oxidative ring cleavage by the action of lead tetraacetate. These reactions followed different patterns, depending on the substrate structure.     

The reaction of triplet flavin with indole. A study of the cascade of reactive intermediates using density functional theory and time resolved infrared spectroscopy

As a model for riboflavin, lumiflavin was investigated using density functional theory methods (B3LYP/6-31G* and B3LYP/6-31+G**) with regard to the proposed cascade of intermediates formed after excitation to the triplet state, followed by electron-transfer, proton-transfer, and radical[bond]radical coupling reactions. The excited triplet state of the flavin is predicted to be 42 kcal/mol higher in energy than the singlet ground state, and the pi radical anion lies 45.1 kcal/mol lower in energy than the ground-state flavin and a free electron in the gas phase. The former value compares to a solution-phase triplet energy of 49.8 kcal/mol of riboflavin. For the radical anion, the thermodynamically favored position to accept a proton on the flavin ring system is at N(5). A natural population analysis also provided spin density information for the radicals and insight into the origin of the relative stabilities of the six different calculated hydroflavin radicals. The resulting 5H-LF* radical can then undergo radical[bond]radical coupling reactions, with the most thermodynamically stable adduct being formed at C(4'). Vibrational spectra were also calculated for the transient species. Experimental time-resolved infrared spectroscopic data obtained using riboflavin tetraacetate are in excellent agreement with the calculated spectra for the triplet flavin, the radical anion, and the most stable hydroflavin radical.     

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.     

Mass spectra of chloro-, aminochloro- and ethylaminochloro-s-triazines

The mass spectra of cyanuric chloride and all of the possible amino-and ethylaminochloro-s-triazines have been investigated. The simpler compounds provide evidence for the structures of low mass fragment ions found in the spectra of the more complex derivatives. Fragmentation patterns are presented which account for most of the major ions. These pathways involve either the initial loss of a chlorine atom (important for the simple aminochlorotriazines) or ring cleavage with expulsion of cyanogen chloride (important for cyanuric chloride). The ethyl derivatives undergo cleavage of the side chain prior to either ring cleavage or chlorine expulsion.     

Electron ionization mass spectra and their reproducibility for trialkylsilylated derivatives of organic acids, sugars and alcohols

Mass spectra of trialkylsilyl derivatives of fatty acids, dicarboxylic acids, hydroxyacids, oxoacids, sugars, amino acids and alcohols were obtained. Amino acids were analyzed as tert-butyldimethylsilyl derivatives; all other model compounds were analyzed as trimethylsilyl derivatives. Reproducibility of the electron ionization (EI) mass spectra for the derivatives obtained was discussed. It was shown that, for many investigated derivatives, composition of the respective mass spectra depended greatly on ion source contamination. The trimethylsilylated alpha-tocopherol mass spectrum composition was most significantly influenced by ion source contamination. This compound can be used to test ion source contamination.     

Mass spectra of 2-thioacylaminothiazoles, 2-thioacylaminobenzothiazoles and their ‘fixed structure’ amino and imino tautomeric forms

The fragmentation patterns of the thioacyl derivatives of 2-aminothiazole and 2-aminobenzothiazole and of their ‘fixed structure’ imino and amino tautomeric forms give evidence of the predominance of the imino tautomer in the molecular ions of the trifluorothioacetyl compounds. On the other hand, the M⁺˙ ions of the thioacetyl and thiobenzoyl derivatives are mainly the amino tautomers. A rearrangement with elimination of RCN and formation of the 2-thiazolothione (2-benzothiazolothione) ion is characteristic for ail the compounds investigated. Additional evidence for the interpretation of the main fragmentation paths is provided by the mass spectra of the N-trideuteromethyl and N-deuterated derivatives, high-resolution mass spectrometry being used to determine the elemental composition of some selected peaks.     

Electron impact induced fragmentation of dibenzo-18-crown-6 polyether and its nitro derivatives

The mass spectra of dibenzo-18-crown-6 polyether and three of its nitro derivatives have been determined. The fragmentation pathways of all the compounds suggested that ring contraction was taking place. The molecular ion of the polyether dissociated by three competing processes, mainly through loss of C₂H₄O units. The molecular ions of the three derivatives dissociated with ring contraction, as well as through losses of O, No and NO₂.     

Synthesis, structure and photophysical properties of a flavin-based platinum(II) complex

We synthesized a thiosemicarbazone-functionalized flavin (Fl-(H)TSC: 2-[2-(3,4-dihydro-7,8-dimethyl-2,4-dioxobenzo[g]pteridin-10(2H)-yl)ethylidene]-hydrazinecarbothioamide) and its Pt(II) complex [Pt(Fl-TSC)(2)], and characterized it using X-ray diffraction, UV-visible absorption and luminescence spectroscopy. X-ray structural analysis for [Pt(Fl-TSC)(2)] revealed that the structure of the isoalloxazine part was almost the same as that in lumiflavin (7,8,10-trimethylisoalloxazine), and the thiosemicarbazone moiety acted as a bidentate ligand to form a PtS(2)N(2) planar conformation. UV-visible absorption and luminescence spectra of these compounds were very similar to those of riboflavin, but the emission intensity and the lifetime decreased considerably. Theoretical calculations suggested that the charge-separated state (Fl˙(-)-TSC˙(+)) contributed to the faster quenching from the (1)π-π* emission state.     

Mass spectra of branched long-chain aliphatic alcohols

The mass-spectral behaviour of long-chain aliphatic primary alcohols of the normal, iso-and anteiso-series have been discussed. The mass-spectral fragmentation of individual alcoholic types have been found to be structurally specific. The mass spectra of some deuterio derivatives have shown, however, the complicated nature of the fragmentation processes.