Tetramethylalloxazines as efficient singlet oxygen photosensitizers and potential redox-sensitive agents.

Tetramethylalloxazines (TMeAll) have been found to have a high quantum yield of singlet oxygen generation when used as photosensitizers. Their electronic structure and transition energies (S0 → Si, S0 → Ti, T1 → Ti) were calculated using DFT and TD-DFT methods and compared to experimental absorption spectra. Generally, TMeAll display an energy diagram similar to other derivatives belonging to the alloxazine class of compounds, namely π,π* transitions are accompanied by closely located n,π* transitions. Photophysical data such as quantum yields of fluorescence, fluorescence lifetimes, and nonradiative rate constants were also studied in methanol (MeOH), acetonitrile (ACN), and 1,2-dichloroethane (DCE). The transient absorption spectra were also analyzed. To assess cytotoxicity of new compounds, a hemolytic assay was performed using human red blood cells (RBC) in vitro. Subsequently, fluorescence lifetime imaging experiments (FLIM) were performed on RBC under physiological and oxidative stress conditions alone or in the presence of TMeAll allowing for pinpointing changes caused by those compounds on the intracellular environment of these cells.

Azodicarboxylate-free esterification with triphenylphosphine mediated by flavin and visible light: method development and stereoselectivity control.

Triphenylphosphine (Ph3P) activated by various electrophiles (e.g., alkyl diazocarboxylates) represents an effective mediator of esterification and other nucleophilic substitution reactions. We report herein an aza-reagent-free procedure using flavin catalyst (3-methyl riboflavin tetraacetate), triphenylphosphine, and visible light (448 nm), which allows effective esterification of aromatic and aliphatic carboxylic acids with alcohols. Mechanistic study confirmed that photoinduced electron transfer from triphenylphosphine to excited flavin with the formation of Ph3P˙+ is a crucial step in the catalytic cycle. This allows reactive alkoxyphosphonium species to be generated by reaction of an alcohol with Ph3P˙+ followed by single-electron oxidation. Unexpected stereoselectivity control by the solvent was observed, allowing switching from inversion to retention of configuration during esterification of (S)- or (R)-1-phenylethanol; for example with phenylacetic acid, the ratio shifting from 10 : 90 (retention : inversion) in trifluoromethylbenzene to 99.9 : 0.1 in acetonitrile. Our method uses nitrobenzene to regenerate the flavin photocatalyst. This new approach to flavin re-oxidation has also been successfully proved in benzyl alcohol oxidation, which is a "standard" process among flavin-mediated photooxidations.

Chemical quenching of singlet oxygen by betanin.

Betanin is the best known natural dye belonging to the betacyanin family. In this work, efficient singlet oxygen quenching by betanin in deuterated water with the rate constant 1.20 ± 0.15 × 10(8) M(-1) s(-1) is reported, deduced from the (1)O2 phosphorescence decays measured as a function of betanin concentration. The quenching occurs by a chemical mechanism, as confirmed by the analysis of the transient absorption kinetics at the probe λ ∼ 535 nm, by comparison of the initial triplet signal amplitude of perinaphthenone acting as the (1)O2 photosensitizer with the final bleaching signal of betanin. The main betanin oxidation product is 2-decarboxy-2,3-dehydrobetanin, with its formation observed as the transient absorption signal at λ ∼ 445 nm. LC-MS/MS analysis of the photolyzed solutions supports the product identification as 2-decarboxy-2,3-dehydrobetanin, based on the molecular ion [M](+) observed at m/z 505. Isobetanin also undergoes a similar photooxidation reaction.

Analytics of Quinine and its Derivatives.

The objective of this study was to perform a synthesis and analysis of the most important information on quinine and its derivatives, which are still very important in the treatment of malaria. The analysis of stereoisomers of quinine and its derivatives was conducted using two techniques, high-performance liquid chromatography and capillary electrophoresis. Particularly noteworthy is the technique used for the determination of isotachophoresis, referred to as one of the so-called green chemistry techniques. Particular attention was paid to properties and the use of quinine and its derivatives in the treatment of malaria. The analytical part will supplement knowledge about quinidine, quinine, and cinchonidine, and will contribute to the growth of research on the so-much-needed drugs against malaria.

Study of photophysical properties of 5-deazaalloxazine and 1,3-dimethyl-5-deazaalloxazine in dependence of pH using different spectral techniques.

The photophysical properties of 5-deazaalloxazine and 1,3-dimethyl-5-deazalloxazine at different pH values were characterized using absorption spectra, fluorescence emission spectra, fluorescence excitation spectra, synchronous fluorescence spectra and total fluorescence spectra. Their ionised and/or neutral forms were discussed in comparison with those obtained for other derivatives of 5-deazaalloxazine and/or 5-deazaisoalloxazine. Steady-state and time-resolved techniques were used to study the protonation/deprotonation equilibria between cationic and neutral forms of both compounds and between neutral and monoanionic forms of 5-deazalloxazine, as well as between monoanionic forms of this compound and its dianion. We estimated pKa values for these equilibria both in the ground and excited states. Our steady-state and time-resolved measurements indicate that the cation of 5-deazaalloxazine in its isoalloxazinic form exhibits fluorescence that is quenched by protons in a dynamic process. Contrary to that, the cation of 1,3-dimethyl-5-deazaalloxazine has almost no fluorescence. Additionally, we found that the neutral forms of 5-deazalloxazine and 1,3-methyl-5-deazalloxazine are also quenched in acidic conditions by protons. In basic conditions, 5-deazaalloxazine forms two structurally different anions, namely the alloxazinic monoanion and the isoalloxazinic monoanion; both simultaneously dissociate into the isoalloxazinic dianion at even higher pH values. The synchronous fluorescence spectra and total fluorescence spectra demonstrated their suitability to characterize and differentiate different fluorescent forms of 5-deazalloxazine, namely: the cation, the neutral form, two monoanions, and the dianion, in a wide pH range.

Time-resolved spectroscopy of the singlet excited state of betanin in aqueous and alcoholic solutions.

The photophysical properties of betanin in aqueous and alcoholic solutions were determined at room temperature using ultrafast UV-vis-NIR transient absorption spectroscopy (λexc = 535 nm). Its S1 → Sn (n > 1) absorption bands appear with maxima at about λ ∼ 450 and 1220 nm. The short betanin S1 state lifetime (6.4 ps in water) is mainly determined by the efficient S1 → S0 radiationless relaxation, probably requiring a strong change in geometry, since the S1 lifetime grows to 27 ps in the more viscous ethylene glycol. The fluorescence quantum yield is very low (Φf ∼ 0.0007 in water), therefore this deactivation path is of minor importance. Other processes, such as S1 → T1 intersystem crossing or photoproduct formation, are virtually absent, since full S0 ← S1 ground state recovery is observed within tens of picoseconds after photoexcitation. The observed fast light-to-heat conversion in the absence of triplet excited state formation supports the idea that betanin is a photoprotector in vivo.

Photophysics, excited-state double-proton transfer and hydrogen-bonding properties of 5-deazaalloxazines.

The photophysical properties of 5-deazaalloxazine and 1,3-dimethyl-5-deazaalloxazine were studied in different solvents. These compounds have higher values of fluorescence quantum yields and longer fluorescence lifetimes, compared to those obtained for their alloxazine analogs. Electronic structure and S0 -Si transitions were investigated using the ab initio methods [MP2, CIS(D), EOM-CCSD] with the correlation-consistent basis sets. Also the time-dependent density functional theory (TD-DFT) has been employed. The lowest singlet excited states of 5-deazaalloxazine and 1,3-dimethyl-5-deazaalloxazine are predicted to have the π, π* character, whereas similar alloxazines have two close-lying π, π* and n, π* transitions. Experimental steady-state and time-resolved spectral studies indicate formation of an isoalloxazinic excited state via excited-state double-proton transfer (ESDPT) catalyzed by an acetic acid molecule that forms a hydrogen bond complex with the 5-deazaalloxazine molecule. Solvatochromism of both 5-deazaalloxazine and its 1,3-dimethyl substituted derivative was analyzed using the Kamlet-Taft scale and four-parameter Catalán solvent scale. The most significant result of our studies is that the both scales show a strong influence of solvent acidity (hydrogen bond donating ability) on the emission properties of these compounds, indicating the importance of intermolecular solute-solvent hydrogen-bonding interactions in their excited state.

Spectroscopy and Photophysics of Monomethyl-Substituted Derivatives of 5-Deazaalloxazine and 10-Ethyl-5-Deaza-Isoalloxazine.

Steady-state and time-resolved spectra were used to describe the singlet and triplet states of 8-methyl-5-deazaalloxazine (8-Me-5-DAll), 9-methyl-5-deazaalloxazine (9-Me-5-DAll) and 10-ethyl-5-deaza-isoalloxazine (10-Et-5-DIAll). Solvatochromic properties were described using different polarity scales, including Δf and the four-parameter scale proposed by Catalán. The results indicate that the Catalán scale shows a strong influence of solvent acidity (hydrogen-bond donating ability) on the emission properties of 8-Me-5-DAll and 9-Me-5-DAll. These results indicate the importance of intermolecular solute-solvent hydrogen-bonding interactions in the excited state of these compounds. Contrary to deazaalloxazines, solvent acidity affects the absorption spectra of 10-Et-5-DIAll. Fluorescence lifetimes and quantum yields and also transient absorption spectra were determined for all of the compounds studied. Electronic structure and S(0)-S(i), S(0)-T(i ), T(1)-T(i) transitions energies and oscillator strengths were calculated using the TD-DFT methods. Theoretical calculations were compared to experimental data.

Influence of pH on photophysical properties of (E)-1-(4-chlorobenzyl)-4-(4-hydroxystyryl)pyridinium chloride.

The protonation/deprotonation equilibrium was investigated for N-p-chlorobenzyl-substituted (E)-4'-hydroxy stilbazolium halide, namely (E)-1-(4-chlorobenzyl)-4-(4-hydroxystyryl)pyridinium chloride (EPC). Absorption, emission and synchronous scanning spectra were used to explain the observed phenomena. The excited state lifetimes of the protonated and deprotonated forms of EPC were measured and discussed. Absorption spectra were used to determine its pK(a) value in the ground state. We conclude that the protonation/deprotonation equilibrium is not attained in the first excited state of EPC, for kinetic reasons. The quinoid and benzenoid structures of EPC in the ground and excited state are discussed in acidic and basic range of pH. Aqueous solutions of EPC were yellow at pH < 7 and red at pH > 7, and addition of alcohols (methanol or 2-propanol) enhanced this change. Therefore, quaternary stilbazolium salts were investigated for application as acid-base indicators.

Acid-base equilibriums of lumichrome and its 1-methyl, 3-methyl, and 1,3-dimethyl derivatives.

Lumichrome photophysical properties at different pH were characterized by UV-vis spectroscopy and steady-state and time-resolved fluorescence techniques, in four forms of protonation/deprotonation: neutral form, two monoanions, and dianion. The excited-state lifetimes of these forms of lumichrome were measured and discussed. The results were compared to those obtained for similar forms of alloxazine and/or isoalloxazine, and also to those of 1-methyl- and 3-methyllumichrome and 1,3-dimethyllumichrome. The absorption, emission, and synchronous spectra of lumichrome, 1-methyl- and 3-methyllumichrome, and 1,3-dimethyllumichrome at different pH were measured and used in discussion of fluorescence of neutral and deprotonated forms of lumichrome. The analysis of steady-state and time-resolved spectra and the DFT calculations both predict that the N(1) monoanion and the N(1,3) dianion of lumichrome have predominantly isoalloxazinic structures. Additionally, we confirmed that neutral lumichrome exists in its alloxazinic form only, in both the ground and the excited state. We also confirmed the existence and the alloxazinic structure of a second N(3) monoanion. The estimated values of pK(a) = 8.2 are for the equilibrium between neutral lumichrome and alloxazinic and isoalloxazinic monoanions, with proton dissociation from N(1)-H and N(3)-H groups proceeding at the almost the same pH, while the second value pK(a) = 11.4 refers to the formation of the isoalloxazinic dianion in the ground state.

Influence of water on photophysical properties of N-bromobenzyl- or nitrobenzyl derivatives of substituted 4-hydroxystilbazolium hemicyanines.

Absorption, steady-state and timed-resolved fluorescence spectra of nine N-p-bromobenzyl substituted (E)-4'-hydroxy-4-stilbazolium bromides and N-p-bromo- (or nitro-) benzyl substituted (E)-4'-hydroxy-3'methoxy-4-stilbazolium bromides, belonging to the hemicyanine class of compounds, were studied in dry and water-containing polar solvents and in water. All of the studied compounds displayed negative solvatochromism. In solvents with small amounts of water the solutions of each of the compounds change color to red, blue-green or blue, while in extra dry solvents they are all yellow. The new band causing the change in color is interpreted as belonging to the deprotonated form of the respective compound. The absorption and emission spectra of protonated and deprotonated forms of hemicyanines in solvents are presented in comparison with those of selected, isolated deprotonated forms (merocyanines) of the same compounds. The discrimination between the quinoid and zwitterionic deprotonated forms was achieved based on the absorption band location. Time-resolved fluorescence measurements in selected dry and water-containing solvents were also performed.

Photophysical properties of izomeric N-chlorobenzyl substituted (E)-2' (3'-or 4')-hydroxy-4-stilbazolium chlorides in alcohols.

Absorption and steady-state fluorescence spectra of nine N-p-(m- and o-) chlorobenzyl substituted (E)-2'-(3' and 4')-hydroxy-4-stilbazolium chlorides belonging to the hemicyanine class of compounds were studied in extra dry alcohols of different polarity. Derivatives with 2'-hydroxy or 4'-hydroxy substituent in the benzene moiety of stilbazol molecule displayed negative solvatochromizm. On the other hand, the excited state decay of compounds with a 3'-hydroxy group in the benzene moiety was dominated by non-radiative processes in protic solvents. Solutions of each of the compounds are yellow in extra dry solvents, red in solvents with small amount of water and yellow again if more water is added. The absorbance and steady-state fluorescence methods were used to explain the protonation/deprotonation processes for N-p-chlorobenzyl-(E)-4'-hydroxy-4-stilbazolium chloride and its zwitterionic or quinoid form in 2-propanol responsible for these phenomena.

Two isomorphous azastilbene derivatives: 1-(2-chlorobenzyl)-4-[(E)-2-(4-hydroxyphenyl)ethenyl]pyridinium chloride and 1-(2-bromobenzyl)-4-[(E)-2-(4-hydroxyphenyl)ethenyl]pyridinium bromide.

The crystal structures of the two title (E)-stilbazolium halogenates, C(20)H(17)ClNO(+)·Cl(-) and C(20)H(17)BrNO(+)·Br(-), are isomorphous, with an isostructurality index of 0.985. The azastyryl fragments are almost planar, with dihedral angles between the benzene and pyridine rings of ca 4.5°. The rings of the benzyl groups are, in turn, almost perpendicular to the azastyryl planes, with dihedral angles larger than 80°. The cations and anions are connected by O-H...X(-) (X = halogen) hydrogen bonds. The halide anions are `sandwiched' between the charged pyridinium rings of neighbouring molecules, and weak C-H...O hydrogen bonds and C-H...X and C-H...π interactions also contribute to the crystal structures.

Electron ionization and electrospray ionization mass spectrometric study of a series of isomeric N-chloro(or bromo)benzyl-substituted (E)-2'(3'- or 4')-hydroxy-4-stilbazole halides.

The electron ionization (EI) mass spectra and electrospray ionization (ESI) mass spectra of a series of isomeric N-chlorobenzyl- and N-bromobenzyl-substituted (E)-2'(3' or 4')-hydroxy-4-stilbazole chlorides and bromides (1-12) were recorded. The fragmentation pathways of all of the compounds and the characteristic fragment ions formed by EI-MS were studied by means of B/E and B(2)/E constant linked-scanning techniques. The formation of ions originating from preionization reactions, characteristic of quaternary halides under EI-MS conditions, such as the elimination of chloro- or bromobenzyl halides, dehydrohalogenation or substitution reactions, is explained. As soft ionization methods cause no such degradation reactions, the ESI-MS spectra of the studied compounds were also obtained for comparison. We thus demonstrated the applicability of EI-MS even in cases when preionization takes place, as long as such secondary processes are properly accounted for.

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 B(2)/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.

Isomorphism in 1-(2-halidobenzyl)-4-[(E)-2-(3-hydroxyphenyl)ethenyl]pyridinium halide hemihydrates (halide = Cl, Br).

The crystal structures of two (E)-stilbazolium salts, namely 1-(2-chlorobenzyl)-4-[(E)-2-(3-hydroxyphenyl)ethenyl]pyridinium chloride hemihydrate, C(20)H(17)ClNO(+) x Cl(-) x 0.5 H(2)O, (I), and 1-(2-bromobenzyl)-4-[(E)-2-(3-hydroxyphenyl)ethenyl]pyridinium bromide hemihydrate, C(20)H(17)BrNO(+) x Br(-) x 0.5 H(2)O, (II), are isomorphous; the isostructurality index is 99.3%. In both salts, the azastyryl fragments are almost planar, while the rings of the benzyl groups are almost perpendicular to the azastyryl planes. The building blocks of the structures are twofold symmetric hydrogen-bonded systems of two cations, two halide anions and one water molecule, which lies on a twofold axis. In the crystal structure, these blocks are connected by means of weaker interactions, viz. van der Waals, weak hydrogen bonding and stacking. This study illustrates the robustness of certain supramolecular motifs created by a spectrum of intermolecular interactions in generating these isomorphous crystal structures.

Electron ionisation and electrospray ionisation mass spectrometric study of a series of isomeric methyl-, dimethyl- and trimethylalloxazines.

Electron ionisation (EI) mass spectra and electrospray ionisation (ESI) mass spectra at different cone voltages of a series of isomeric methyl- and dimethylalloxazines are discussed, and compared with those of lumichrome, and 1- and 3-methyllumichrome. Examination of ESI mass spectra taken at a higher cone voltage and the use of isotope-labelled methanol allow us to discuss the fragmentation pathways of [M+H]+ and [M-H](-) ions. The fragmentation pathways of all of the compounds and the characteristic fragment ions formed in EI-MS are compared with published data. The influence of methyl and dimethyl substituents in the benzene ring on the fragmentation pathways leading to the loss of 43 and 45 Da upon both electron and electrospray ionisation is described.

Letter: electron impact mass spectrometry study of a series of substituted 5- aminoalkylmethyl-cytosines and their 1-N-(o-, m- and p-)bromobenzyl-substituted derivatives.

The electron-impact mass spectra of 5-aminoalkylmethyl-substituted cytosine and of their 1-N- o-(m- or p-)bromobenzyl-substituted derivatives are discussed. The influence of 5-aminoalkyl and 1-N-bromobenzyl substituents on the mode of mass fragmentation occurring upon electron impact ionization was demonstrated. The fragmentation pathways of all compounds and the characteristic ions in these spectra originating from the McLafferty rearrangement, RDA reactions and simple cleavages are presented.