Photooxidation of Tryptophan Leading to 2‐Aminoacetophenone – A Possible Reason for the Untypical Aging Off‐flavor in Wine

2‐Aminoacetophenone (AAP) was recognized as the key compound for the so‐called untypical aging off‐flavor (UTA) in Vitis vinifera wines. In this study, it was shown that AAP can be formed by photooxidation of free and protein‐bound tryptophan (TRP) in combination with a subsequent storage in model wine. Solutions of TRP and lysozyme were exposed to artificial sunlight both in the presence and in the absence of the photosensitizer riboflavin. Aliquots of the irradiation batches were stored in model wine solutions containing tartaric acid, sulfite and ethanol in different combinations. AAP formation could be identified from both free and bound (lysozyme) TRP, while free TRP resulted in higher yields. The presence of riboflavin during irradiation generally favored the AAP formation. AAP formation increased with increasing irradiation times, but AAP was not detectable, if TRP was directly incubated in model wine. Not only the irradiation time but also the storage time of model wines favored the formation of AAP. Concerning the model wine composition, it became evident that the presence of tartaric acid resulted in the highest AAP formation during storage.     

The protective function of hydrogen sulfide for lysozyme against riboflavin-sensitized photo-oxidation

Hydrogen sulfide is the third endogenous signaling gasotransmitter, following nitric oxide and carbon monoxide. Recent studies showed that hydrogen sulfide could alleviate many diseases which were related to the oxidative damage of tissues. It reminded us that hydrogen sulfide might serve as an antioxidant to reduce oxidative pressure. This study showed that hydrogen sulfide protected lysozyme from photo-oxidation induced by riboflavin (RF). Laser flash photolysis was used to explore the mechanisms of antioxidant activity of hydrogen sulfide. The scavenging effects of hydrogen sulfide on the triplet state of riboflavin (3RF(*)) and radicals of tryptophan and tyrosine (TyrO· and TrpN·) were attributed to the protection of lysozyme from photo-oxidation. The results suggested that hydrogen sulfide could serve as an antioxidant in alleviation of oxidative pressure.     

Supramolecular Cationic Tetraruthenated Porphyrin and Light-Induced Decomposition of 2-Deoxyguanosine Predominantly Via a Singlet Oxygen-Mediated Mechanism

The tetraruthenated porphyrin, u.-[/wes0-5,1O,15,2O-tet-ra(pyridyl)porphyrin]tetrakis[ftis-(bipyridine)chloride ruthenium(II)] (TRP) is a supramolecular cationic species. The aim of the present investigation was to evaluate the photodynamic properties of TRP and Zn-TRP to damage DNA with emphasis on the mechanistic aspects. The ability for tetraruthenated porphyrin derivatives to induce photosensitization reactions has been determined using 2′-deoxyguanosine as a DNA model compound. The main photooxidation products of the targeted nucleoside were identified and classified according to their mechanisms of formation, involving either a radical pathway (type I) or a singlet oxygen-mediated mechanism (type II). Quantification of the different oxidation products provides a means to evaluate the relative contribution of type I and type II pathways associated with the oxidative photosensitization of 2′-deoxyguanosine by tetraruthenated porphyrin derivatives. Results indicate that ^x02 plays a major role in the mechanism of photooxidation mediated by these porphyrin derivatives. In addition an increase of the photosensitizing effect in the presence of zinc is observed. For each sensitizer, the ratio between type II and type I photoproducts has been calculated and compared to that of other known dyes such as methylene blue and riboflavin.     

Photochemical and Photobiotogical Studies with Acridine and Phenanthridine Hydroperoxides in Cell-free DNA

Abstract— The acridine and phenanthridine hydroperoxides 3 and 7 were synthesized as photochemical hydroxyl radical sources for oxidative DNA damage studies. The generation of hydroxyl radicals upon UVA irradiation (Λ. = 350 nm) was verified by trapping experiments with 5,5-di-methyl-1-pyrroline N -oxide and benzene. The enzymatic assays of the damage in cell-free DNA from bacteriophage PM2 caused by the acridine and phenanthridine hydroperoxides 3 and 7 under near-UVA irradiation revealed a wide range of DNA modifications. Particularly, extensive single-strand break formation and DNA base modifications sensitive to formamidopyrimidine DNA glycosylase (Fpg protein) were observed. In the photooxida-tion of calf thymus DNA, up to 0.69±0.03% 8-oxo-7,8-dihydroguanine was formed by the hydroperoxides 3 and 7 on irradiation, whose yield was reduced up to 40% in the presence of the hydroxyl radical scavengers mannitol and fert-butanol. The acridine and phenanthridine hydroperoxides 3 and 7 also induce DNA damage through the type I photooxidation process, for which photoinduced electron transfer from 2'-deoxyguanosine to the singlet states of 3 and 7 was estimated by the Rehm-Weller equation to possess a negative Gibb's free energy of cα -5 kcal/ mol. Control experiments with the sensitizers acridine 1 and the acridine alcohol 4 in calf thymus and PM2 DNA confirmed the photosensitizing propensity of the UVA-ab-sorbing chromophores. The present study emphasizes that for the development of selective and efficient photochemical hydroxyl radical sources, chromophores with low photosensitizing ability must be chosen to avoid type I and type II photooxidation processes.     

Effect of visible light on selected enzymes, vitamins and amino acids.

In most cases, the presence of an endogenous photosensitizer is a requirement for visible light modification of biomolecules in animal tissues. Riboflavin (RF) is present in all aerobic cells and is a very efficient photosensitizer, presenting a complex photochemistry with a mixed type I-type II mechanisms. Visible light irradiation in the presence of RF diminished the enzymatic activity of horse radish peroxidase (HRP) only when this glyco-enzyme was deglycosilated. In contrast, the activity of catalase is inactivated via singlet oxygen, and that of lysozyme was efficiently inactivated by a mixed type I-type II mechanisms. The reactive species involved in the RF sensitized photoconversion of lysozyme and the aromatic amino acids tryptophan and tyrosine (both free in solution) is discussed. The role of ascorbate and the effect of RF photosensitized processes in biological complex systems, such as the ocular lens and tumoral cell in culture, is analyzed.     

Comparison of adsorption performances of metal–chelated polyamide hollow fibre membranes in lysozyme separation

Commercially available microporous polyamide hollow fibres are modified by acid hydrolysis to activate the reactive groups and subsequently binding of the ligand, i.e. Cibacron Blue F3GA. Then the Cibacron Blue F3GA-derived hollow fibres were loaded with different metal ions (i.e. Zn(II), Cu(II), Ni(II)) to form the metal chelate. The internal polymer matrix was characterised by scanning electron microscopy. The effects of pH, initial concentration of lysozyme, metal type and temperature on the adsorption of lysozyme to the metal–chelated hollow fibres were examined in a batch reactor. The non-specific adsorption of lysozyme onto the polyamide hollow fibres was 1.8 mg/g. Cibacron Blue F3GA immobilisation increased the lysozyme adsorption up to 62.3 mg/g. Metal–chelated hollow fibres showed a significant increase of the adsorption efficiency. Lysozyme adsorption capacities of Zn(II), Cu(II) and Ni(II)-chelated hollow fibres were different. The maximum capacities of Zn(II), Cu(II) or Ni(II)-chelated hollow fibres were 144.2, 75.2 and 68.6 mg/g, respectively. Significant amount of the adsorbed lysozyme (up to 97%) was eluted in 1 h in the elution medium containing 1.0 M NaSCN at pH 8.0 and 25 mM EDTA at pH 4.9. Repeated adsorption–desorption process showed that this novel metal–chelated polyamide hollow fibres are suitable for lysozyme adsorption.     

Binding of riboflavin to lysozyme promoted by peroxidase-generated triplet acetone

When riboflavin and lysozyme are added to the 2-methylpropanal/peroxidase/O₂ system, which generates triplet acetone, an adduct is formed to a small extent. The adduct can be separated by gel filtration and is similar to that prepared by irradiating riboflavin in the presence of lysozyme.     

Photosensitizer initiated attacks on DNA under dry conditions and their inhibition: a DNA archiving issue

Long-term aging of dry DNA is thought to be due to the attack of diverse cascades of reactive species with probably, no one single initiator of the cascades explaining all circumstances. Photosensitizer-initiated reactions from methylene blue and riboflavin were used to generate two model systems of reactive species around dry DNA in order to understand such systems and how to block them. Damage was assessed using plasmid DNA as a substrate with an in-situ microgel electrophoretic technique. Photodynamic methylene blue damage to DNA was very oxygen dependent but not that of riboflavin. This indicates that indirect type II pathways, probably via singlet oxygen were important for methylene blue but not for riboflavin. In both the absence and presence of oxygen, the DNA protection offered by dry caffeine and urate to both photodynamic agents indicated that most DNA attack was via electrophilic species. Overall, protection of dry archived DNA from spontaneously reactive species such as free radicals appears to be a real issue and, as expected, the predominant species in air appear to involve oxygen but not exclusively or necessarily so.     

Photosensitized DNA damage and its protection via a novel mechanism

UVA, which accounts for approximately 95% of solar UV radiation, can cause mutations and skin cancer. Based mainly on the results of our study, this paper summarizes the mechanisms of UVA-induced DNA damage in the presence of various photosensitizers, and also proposes a new mechanism for its chemoprevention. UVA radiation induces DNA damage at the 5'-G of 5'-GG-3' sequence in double-stranded DNA through Type I mechanism, which involves electron transfer from guanine to activated photosensitizers. Endogenous sensitizers such as riboflavin and pterin derivatives and an exogenous sensitizer nalidixic acid mediate DNA photodamage via this mechanism. The major Type II mechanism involves the generation of singlet oxygen from photoactivated sensitizers, including hematoporphyrin and a fluoroquinolone antibacterial lomefloxacin, resulting in damage to guanines without preference for consecutive guanines. UVA also produces superoxide anion radical by an electron transfer from photoexcited sensitizers to oxygen (minor Type II mechanism), and DNA damage is induced by reactive species generated through the interaction of hydrogen peroxide with metal ions. The involvement of these mechanisms in UVA carcinogenesis is discussed. In addition, we found that xanthone derivatives inhibited DNA damage caused by photoexcited riboflavin via the quenching of its excited triplet state. It is thus considered that naturally occurring quenchers including xanthone derivatives may act as novel chemopreventive agents against photocarcinogenesis.     

Protective effect of melatonin on photo-damage to lysozyme

The behavior of melatonin in the riboflavin-sensitized photo-oxidation of lysozyme was monitored. Melatonin was found to prevent aggregation of protein and the decrease of enzyme activity induced by photo-oxidation. Electron spin resonance experiments showed that photo-oxidation of lysozyme in the presence of riboflavin resulted in formation of protein radicals, and melatonin was highly effective in reducing the formation of protein radicals. Direct evidence of melatonin’s ability for quenching the triplet state of riboflavin and singlet oxygen was presented. A mechanism of the protective effect of melatonin on photo-oxidation of protein was proposed and the physiological relevance was discussed.     

Cholesterol-diaryl ketone stereoisomeric dyads as models for "clean" type I and type II photooxygenation mechanisms

Cholesterol (Ch) is a major target for oxidative degradation in cell membranes, a process which can occur by two mechanisms: Type I (via free radicals) and Type II (mediated by 1O2). In the present work, several dyads have been synthesized from beta- and alpha-Ch and ketoprofen (KP) or tiaprofenic acid (TPA). Upon irradiation under anaerobic conditions, KP-alpha-Ch dyads were efficiently photolyzed, via intramolecular hydrogen abstraction from C-7. By contrast, KP-beta-Ch, TPA-alpha-Ch, and TPA-beta-Ch remained unchanged after prolonged irradiation. The transient absorption spectra of KP-alpha-Ch were assigned to the short-lived biradicals resulting from intramolecular hydrogen abstraction. Interestingly, the spectra and lifetimes obtained for the TPA-derived dyads were very similar to those of the TPA triplet excited state. For the KP-alpha-Ch dyads, generation of singlet oxygen was expectedly negligible. Conversely, for TPA-alpha-Ch a Phi delta) value as high as 0.5 was determined. Thus, KP-based dyads are appropriate models for clean type I Ch oxidation, whereas the TPA derivatives are suitable systems for investigation of the purely type II process.     

Photooxidation of acyclovir with thermally generated triplet excited ketones. A comparison with type I and II photosensitizers

The antiviral drug acyclovir (Ac, 1) was treated with triplet excited ketones, which have been generated in thermal decomposition of 3-(hydroxymethyl)-3,4,4-trimethyl-1,2-dioxetane (HTMD), in the dark. Three major oxidation products were detected by means of spectroscopic measurements. The products were (2-hydroxyethoxy) methyl spiroiminodihydantoin (2), (2-hydroxyethoxy) methyl (amino)-2-imino-1,2-dihydroimidazole-5-one (3), and 2,2-diamino-4-[(2-hydroxyethoxy) methyl) amino)-5-[2H]-oxazolone (4). Equal amounts of type I and type II photooxidation products were found, as could be established by comparison with predominant type I (riboflavin) and type II (rose bengal) photosensitizers. The concentration and time profiles for the HTMD-induced oxidation of Ac were also determined. The participation of singlet oxygen in HTMD-induced oxidation was confirmed by the substantial D(2)O effect in the formation of spiroiminodihydantoin (2).     

EFFECTS OF QUENCHING AGENTS ON THE PHOTODYNAMICALLY-INDUCED CHEMOTACTIC RESPONSE OF THE COLORLESS FLAGELLATE Polytomella magna

Abstract In the presence of the photosensitizer riboflavin at high fluence rates a photoproduct, most probably H₂O₂, is formed which causes negative phototaxis in the colorless flagellate Polytomella magna . The aim of this study was to find out whether H₂O₂ is produced in a type I or II reaction. As has been shown, ¹O₂ quenchers either do not influence the photodynamic action of riboflavin (furfuryl ethanol, DPBF, l -histidine, crocetin) or show slight quenching effects only at very high concentrations ≧ 10⁻² M (DABCO, DMF, imidazole). D₂O is toxic to P. magna even in 1:1 and 1:2 mixtures with H₂O. On the other hand, the quenching effect of 1,4-benzoquinone, highly indicative for the type I pathway, is more than two orders of magnitude stronger than the one of the above mentioned ¹O₂ quenchers. The results suggest that H₂O₂ is produced in a type I reaction. Superoxide does not seem to be involved since superoxide dismutase does not diminish the photodynamic effect of riboflavin.     

Supramolecular Cationic Tetraruthenated Porphyrin Induces Single-Strand Breaks and 8-Oxo-7,8-dihydro-2'-deoxyguanosine Formation in DNA in the Presence of Light

Abstract— The aim of this investigation is the evaluation of DNA interaction of with tetraruthenated porphyrin (TRP) and of DNA damage in the presence of light. Direct-fluorescence and electronic absorption measurements after incubation of DNA with TRP indicate strong binding between pBR322 DNA or calf thymus DNA with the modified porphyrin. Exposure of pBR322 DNA to TRP (up to 3 μ M ) and light leads to single-strand break formation as determined by the conversion of the supercoiled form (form I) of the plasmid into the nicked circular form (form II). Oxidative DNA base damage was evaluated by the detection of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) after irradiation of calf thymus DNA in the presence of the TRP. The data demonstrated a dose and time dependence with each type of DNA damage. These data indicate (1) a specificity of the binding mode and (2) type I and II photoinduced mechanisms leading to strand scission activity and 8-oxodGuo formation. Accordingly, singlet molecular oxygen formation, after TRP excitation, was confirmed by near-infrared emission. From these investigations a potential application of TRP in photodynamic therapy is proposed.     

Studies on the Photodimerization of Isoeugenol

Under the irradiation of the ultraviolet light, Isoeugenol (I) undergoes dimerization in acetone to give diisoeugenol (II) and dehydrodiisoeugenol (III). The structures are determined by the comparison of the mixed m. p., I. R. spectra and T. L. C. with synthesized authentic samples. Also the photoreaction mechanism is discussed in detail. The reaction represents a new type and as an exception of photochemical process for the C=C double bond in conjugation with aromatic ring that generally dimerizes to produce cyclobutane type derivatives.     

Infrared characterization of polymorphism in polybutene-1

The transformations of the polymorphic forms of polybutene-1 (I,II,III) were studied by infrared spectroscopy. Attenuated total reflectance spectra demonstrate that the II → I transformation occurs initially and most rapidly on the film surfaces. Electron irradiation experiments showed the II → I conversion can be suppressed by irradiation. The degree of suppression was dependent on the irradiation dose. Comparison of spectra on irradiation in air and vacuum indicate that radiation-produced radicals are scavenged by oxygen, preventing intermolecular crosslinking and allowing normal expansion of the helix during transformation. In addition, remolding an irradiated sample of II caused immediate conversion to I rather than its return to II as was noted when unirradiated samples are remolded. These observations are related to certain conformational changes in the molecular structure.     

Photodegradation of a polyamidehydroxyurethane type polymer in aqueous solution. The role of some dyes in the degradation

The influence of some dyes on the photodegradation of a polyamidehydroxyurethane type polymer in aqueous solution has been studied. It has been found that, among the dyes used, only riboflavin sensitizes and accelerates the degradation of the polymer. It is proposed that under ultraviolet irradiation riboflavin undergoes photoreduction to lumichrome, which sensitizes the photodegradation of the polymer. It is also possible that the mechanism of photodegradation involves the participation of singlet oxygen.     

Photochemical and Photobiological Studies of a Furonaphthopyranone as a Benzo-spaced Psoralen Analog in Cell-free and Cellular DNA

Abstract— Photobiological activities of the benzo-spaced psoralen analog furonaphthopyranone 3 have been investigated in cell-free and cellular DNA. The molecular geometry parameters of 3 suggest that it should not form interstrand crosslinks with DNA. With cell-free DNA no evidence for crosslinking but also not for monoadduct formation was obtained; rather, the unnatural furocoumarin 3 induces oxidative DNA modifications under near-UVA irradiation. The enzymatic assay of the photosensitized damage in cell-free PM2 DNA revealed the significant formation of lesions sensitive to formamidopyrimidine DNA glyco-sylase (Fpg protein). In the photooxidation of calf thymus DNA by the furonaphthopyranone 3, 0.29±0.02% 8-oxo-7,8-dihydroguanine (8-oxoGua) was observed. With 2'-deoxyguanosine (dGuo), the guanidine-releasing photooxidation products oxazolone and oxoimidazolidine were formed predominately, while 8-oxodGuo and 4-HO-8-oxodGuo were obtained in minor amounts. The lack of a significant D₂O effect in the photooxidation of DNA and dGuo reveals that singlet oxygen (type II process) plays a minor role; control experiments with tert -butanol and mannitol confirm the absence of hydroxyl radicals as oxidizing species. The furonaphthopyranone 3 (E_red= -1.93±0.03V) should act in its singlet-excited state as electron acceptor for the photooxidation of dGuo (δG_ET ca – kcal/mol), which corroborates photoinduced electron transfer (type I) as a major DNA-oxidizing mechanism. A comet assay in Chinese hamster ovary (CHO) AS52 cells demonstrated that the psoralen analog 3 damages cellular DNA upon near-UVA irradiation; however, no photosensitized mutagenicity was observed in CHO AS52 cell cultures     

Photodynamic action of benzo[a]pyrene in K562 cells

Benzo[a]pyrene (BaP) is ubiquitously distributed in the environment, being considered the most phototoxic element among polycyclic aromatic hydrocarbon (PAHs). In presence of oxygen, PAHs can act as a photosensitizer by means of promoting photo-oxidation of biological molecules (photodynamic action, PDA). Thus, the present study analyzed the photodynamic action of BaP under UVA irradiation (BaP + UVA) and its oxidative effects on K562 cells. The evaluation of BaP kinetics showed that the highest intracellular concentration occurred after 18 h of incubation. Cell viability, reactive oxygen species (ROS) generation, lipid peroxidation, DNA damage (breaks and DNA-protein cross-link [DNAPC]) were assessed after exposure to BaP, UVA and BaP plus UVA irradiation (BaP + UVA). Cell viability was decreased just after exposure to BaP + UVA. Lipid peroxidation and DNA breaks increased after BaP + UVA exposure, while the DNAPC increased after BaP, UVA and BaP + UVA exposure, suggesting that BaP + UVA effects were a consequence of both type II (producing mainly singlet oxygen) and type I (producing others ROS) mechanisms of PDA.     

Thermal and Photochemical Isomerization of Tetraaryl Tetrakis(trifluoromethyl)

The isomerization of tetraaryl tetrakis(trifluoromethyl)[4]radialenes was studied. When type II (all-Z) isomers of 5,6,7,8-tetraaryl-5,6,7,8-tetrakis(trifluoromethyl)[4]radialenes were heated in tetralin at 170-200 degrees C, isomerization occurred to give mixtures of four [4]radialenes in a ratio of ca. I:II:III:IV = 1:10:5:1. However, when the isomeric mixtures were heated in the solid state at the same temperature, selective isomerization took place to give type II isomers in good selectivity (>91%). Upon irradiation with light, the type II isomers first isomerized to mixtures of the four [4]radialene isomers (I:II:III:IV = 2:2:48:48) and then rearranged to cyclobuta[b]naphthalenes via a 6pi-electrocyclic reaction followed by 1,3-hydrogen migration.