Fluorescent Coumarin Research Articles

Polyaromatic nanocapsules displaying aggregation-induced enhanced emissions in water.

V-shaped polyaromatic amphiphiles with phenanthrene or naphthalene rings spontaneously and quantitatively formed micelle-like nanocapsules in water at room temperature. In contrast to usual polyaromatic aggregates with weak fluorescent properties, the new capsules providing spherical polyaromatic shells with diameters of ∼2 nm show strong fluorescent emissions due to an aggregation-induced enhanced emission (AIEE) effect and moreover encapsulate a fluorescent coumarin dye to generate highly emissive host-guest composites.

Quantitative DNA interstrand cross-link formation by coumarin and thymine: structure determination, sequence effect, and fluorescence detection.

The coumarin analogues have been widely utilized in medicine, biology, biochemistry, and material sciences. Here, we report a detailed study on the reactivity of coumarins toward DNA. A series of coumarin analogues were synthesized and incorporated into oligodeoxynucleotides. A photoinduced [2 + 2] cycloaddition occurs between the coumarin moiety and the thymidine upon 350 nm irradiation forming both syn- and anti-cyclobutane adducts (17 and 18), which are photoreversible by 254/350 nm irradiation in DNA. Quantitative DNA interstrand cross-link (ICL) formation was observed with the coumarin moieties containing a flexible two-carbon or longer chain. DNA cross-linking by coumarins shows a kinetic preference when flanked by an A:T base pair as opposed to a G:C pair. An efficient photoinduced electron transfer between coumarin and dG slows down ICL formation. ICL formation quenches the fluorescence of coumarin, which, for the first time, enables fast, easy, and real-time monitoring of DNA cross-linking and photoreversibility via fluorescence spectroscopy. It can be used to detect the transversion mutation between pyrimidines and purines. Overall, this work provides new insights into the biochemical properties and possible toxicity of coumarins. A quantitative, fluorescence-detectable, and photoswitchable DNA cross-linking reaction of the coumarin moieties can potentially serve as mechanistic probes and tools for bioresearch without disrupting native biological environment.

High-efficiency fluorescent organic light-emitting diodes enabled by triplet-triplet annihilation and horizontal emitter orientation

A green organic light-emitting diode with the fluorescent emitter Coumarin 545T shows an external quantum efficiency ( ηEQE) of 6.9%, clearly exceeding the classical limit of 5% for fluorescent emitters. The analysis of the angular dependent photoluminescence spectrum of the emission layer reveals that 86% of the transition dipole moments are horizontally oriented. Furthermore, transient electroluminescence measurements demonstrate the presence of a delayed emission originating from triplet-triplet annihilation. A simulation based efficiency analysis reveals quantitatively the origin for the high ηEQE: a radiative exciton fraction higher than 25% and a light-outcoupling efficiency of nearly 30%.

Reduced fluorescence quenching of coumarin 102 at higher phenol mole fractions in cyclohexane–phenol and anisole–phenol solvent mixtures: role of competitive hydrogen bonding

Recently, we demonstrated that the fluorescence of coumarin 102 (C102) drastically quenches upon hydrogen bonding (H-bonding) with phenol in a non-polar solvent cyclohexane due to H-bond driven photoinduced electron transfer (PET) (J. Phys. Chem. A, 2013, 117, 3945–3953). However, the work was limited to low concentrations of phenol only, where predominately a 1 : 1 C102–phenol complex exists. Herein, we report an unusual fluorescence modulation of C102 over mole fractions of the H-bond donor (phenol) in the cyclohexane–phenol mixtures covering a higher range of mole fractions. We found that the fluorescence quantum yield (ϕf) and fluorescence lifetime (τf) of C102 first diminish steadily with increase in the concentration of phenol, but after a particular mole fraction (XPhOH = 0.013) both the quantities (ϕf and τf) increase upon further increase in the phenol content. The results can be attributed to the competitive nature of the C102–phenol and the phenol–phenol H-bonding. Since the PET quenching operates through the H-bond between the CO group of C102 and the HO– group of phenol, the H-bond may fail to attain an optimum geometry when the phenol is linked with other phenols via the H-bond. The variation of the C102–phenol H-bonding nature with the mole fraction of phenol was supplemented by FT-IR measurements. Similar unusual variation of the C102 fluorescence is observed in the phenol–anisole mixtures to a lesser extent, but is completely absent in the anisole–cyclohexane mixtures, where H-bonding is practically impossible.

Directed-Evolution Analysis of Human Cytochrome P450 2A6 for Enhanced Enzymatic Catalysis

Cytochrome P450 2A6 (P450 2A6) is the major enzyme responsible for the oxidation of coumarin, nicotine, and tobacco-specific nitrosamines in human liver. In this study, the catalytic turnover of coumarin oxidation was improved by directed-evolution analysis of P450 2A6 enzyme. A random mutant library was constructed using error-prone polymerase chain reaction (PCR) of the open reading frame of the P450 2A6 gene and individual mutant clones were screened for improved catalytic activity in analysis of fluorescent coumarin 7-hydroxylation. Four consecutive rounds of random mutagenesis and screening were performed and catalytically enhanced mutants were selected in each round of screening. The selected mutants showed the sequentially accumulated mutations of amino acid residues of P450 2A6: B1 (F209S), C1 (F209S, S369G), D1 (F209S, S369G, E277K), and E1 (F209S, S369G, E277K, A10V). E1 mutants displayed approximately 13-fold increased activity based on fluorescent coumarin hydroxylation assays at bacterial whole cell level. Steady-state kinetic parameters for coumarin 7-hydroxylation and nicotine oxidation were measured in purified mutant enzymes and indicated catalytic turnover numbers (kcat) of selected mutants were enhanced up to sevenfold greater than wild-type P450 2A6. However, all mutants displayed elevated Km values and therefore catalytic efficiencies (kcat/Km) were not improved. The increase in Km values was partially attributed to reduction in substrate binding affinities measured in the analysis of substrate binding titration. The structural analysis of P450 2A6 indicates that F209S mutation is sufficient to affect direct interaction of substrate at the active site.

A thiazole coumarin (TC) turn-on fluorescence probe for AT-base pair detection and multipurpose applications in different biological systems.

Sequence-specific recognition of DNA by small turn-on fluorescence probes is a promising tool for bioimaging, bioanalytical and biomedical applications. Here, the authors report a novel cell-permeable and red fluorescent hemicyanine-based thiazole coumarin (TC) probe for DNA recognition, nuclear staining and cell cycle analysis. TC exhibited strong fluorescence enhancement in the presence of DNA containing AT-base pairs, but did not fluoresce with GC sequences, single-stranded DNA, RNA and proteins. The fluorescence staining of HeLa S3 and HEK 293 cells by TC followed by DNase and RNase digestion studies depicted the selective staining of DNA in the nucleus over the cytoplasmic region. Fluorescence-activated cell sorting (FACS) analysis by flow cytometry demonstrated the potential application of TC in cell cycle analysis in HEK 293 cells. Metaphase chromosome and malaria parasite DNA imaging studies further confirmed the in vivo diagnostic and therapeutic applications of probe TC. Probe TC may find multiple applications in fluorescence spectroscopy, diagnostics, bioimaging and molecular and cell biology.

New Gold(I) Organometallic Compounds with Biological Activity in Cancer Cells

AbstractInvited for the cover of this issue is the group of Angela Casini at the Department of Pharmacokinetics, Toxicology and Targeting, Groningen Research Institute of Pharmacy, The Netherlands. The cover image shows the uptake of gold N‐heterocyclic (NHC) complexes, bearing a fluorescent coumarin moiety, in cancer cells and the resulting anticancer effects.

Coumarin 6 as a fluorescent model drug: How to identify properties of lipid colloidal drug delivery systems via fluorescence spectroscopy?

In the development of colloidal lipid drug delivery systems (DDS), the localization of the drug within the DDS, the protective potential of the DDS as well as the compatibility of the drug with the excipients are of special interest. In this study, Coumarin 6 (C6), a fluorescent dye frequently used to facilitate the traceability of DDS in vitro, is demonstrated as a tool to gain insight into the aforementioned properties by fluorescence spectroscopy. Both ways of loading C6 to solid colloidal DDS (pre‐loading to the lipid matrix or post‐loading to the ready DDS after its processing) led to a localization of C6 in the interfacial layer between solid lipid nanoparticles (SLN) and aqueous medium. In the case of pre‐loading, C6 is homogenously distributed in emulsion droplets and expelled from the lipid matrix due to crystallization. Nanoemulsions distributed C6 in the liquid lipid matrix itself and cause a hypsochromic shift of fluorescence spectra. Larger surface areas of emulsion droplets due to smaller droplet sizes displayed more interaction of C6 with the aqueous phase as a bathochromic shoulder in fluorescence spectra. Measured fluorescence intensity was linearly dependent on light scattering of colloidal DDS of the same excipients. A simple method to evaluate the protective properties of DDS was presented and displayed that (i) nanoemulsions provide more protection compared with SLN and (ii) higher phospholipid concentration improve the protection within SLN.Practical applications: In the development of colloidal DDS, it is important to identify the localization and distribution of a (model) drug in compartments within the disperse system. The simple approach by fluorescence spectroscopy presented in this study supplies information about the localization of drug substances with properties comparable with the fluorescent probe used. The results assist formulation development as a quick screening tool to elucidate (i) the localization of the probe, (ii) the microenvironment of the probe (e.g., differentiation of the physical state of the disperse phase), and (iii) the protection of the probe against damaging agents from the aqueous phase.The fluorescent dye Coumarin 6 was applied as model drug in colloidal lipid drug delivery systems. Fluorescence spectroscopy proved able to identify the localization of the model compound, the physical state of the dispersed phase, and the protective potential of the DDS, especially dependent on phospholipid concentration.

An appraisal of the Suzuki cross-coupling reaction for the synthesis of novel fluorescent coumarin derivatives

We report the chemical design and development of 3-aryl-substituted 7-alkoxy-4-methylcoumarins with enhanced fluorogenic properties. The 3-aryl substituents are installed via an optimized Suzuki–Miyaura cross-coupling (SMC) reaction between a 7-alkoxy-3-bromo-4-methylcoumarin and aryl boronic MIDA esters using Pd(OAc)2/XPhos in a catalytic system with K2CO3 in aqueous THF. Under these conditions, an exocyclic ester functionality is found to be unaffected. Subsequent saponification revealed a carboxylic acid functionality that is suitable for conjugation reactions. Evaluation of their fluorescence properties indicated that the installed 3-heteroaryl substituent, particularly benzofuran-2-yl, resulted in a significant red shift of both the excitation and emission wavelengths.

Fluorescent coumarin derivatives with viscosity sensitive emission--synthesis, photophysical properties and computational studies.

New derivatives of (benzo[d]azolyl)-benzo[f]chromenone were synthesized from the intermediate 3-(1,3-benzazol-2-yl)naphthalen-2-ol, obtained from 3-hydroxynaphthalene-2-carboxylic acid and 2-amino phenol in the presence of PCl3 in chlorobenzene at 130-135 °C. The compounds were characterized by FT-IR, (1) H NMR, mass spectroscopy and elemental analysis. The synthesized compounds are fluorescent which absorb in the range of 296 to 332 nm while emit in the range of 368 to 404 nm. The experimental absorption and emission wavelengths for the compounds 5 and 6 are in good agreement with those predicted using the Time-Dependent Density Functional Theory (TD-DFT) [B3LYP/6-31G(d)]. The largest wavelength difference between the experimental and computed absorption maxima was 29 nm (tetrahedrofuran) for compound 5 while for emission it was 61 nm (dichloromethane) for compound 7. The emission intensities of all the compounds decrease continuously as the viscosity of the microenvironment increases. The compounds are thermally stable up to a temperature range of 300 to 350 °C.

Energy transfer from the singlet levels of diketones and dyes to lanthanide ions in nanoparticles consisting of their diketonate complexes

The energy transfer from the S1 levels of p-phenylbenzoyltrifluoroacetone (PhBTA) and dyes to different Ln3+ ions is studied in nanoparticles (NPs) composed of complexes of this diketone with Ln3+ and 1,10-phenanthroline (phen) and doped with dye molecules. The quenching rate constants in the NPs consisting from complexes of Pr3+, Nd3+, Sm3+, Eu3+, Ho3+, Er3+, and Tm3+ are determined from the data on the quenching of sensitized (cofluorescence) and ordinary fluorescence of coumarin 30 (C30) and rhodamine 6G (R6G). The quenching rate constants vary from ≤5 × 1011 to 1013 s−1 for the fluorescence quenching of PhBTA by different Ln3+ ions, while the quenching of dye fluorescence occurs at rates of the order of 109 s−1. In the case of complexes with the Pr3+ ions, the fluorescence quenching of PhBTA in NPs composed of its complexes is accompanied by sensitized luminescence of Pr3+. The quenching observed is due to a nonradiative energy transfer from the S1 states of ligands and dyes to these ions. It is shown that in NPs composed of complexes with Eu3+, Yb3+, and Sm3+ the cofluorescence of C30 is quenched via the electron-transfer mechanism. The study of quenching of cofluorescence and fluorescence of dyes in NPs composed of mixed complexes of La3+ and Nd3+ (Ho3+) shows that the observed quenching of fluorescence and cofluorescence is governed mainly by the quenching of the S1 state of dyes when the Nd3+ (Ho3+) content does not exceed 5–10% and by the quenching of the S1 state of a ligand when the Nd3+ (Ho3+) content exceeds 50%. It is assumed that the high rate constant of energy transfer from the S1 level of ligands to ions Pr3+, Nd3+, Ho3+, Er3+, and Tm3+ in NPs composed of beta-diketonate complexes is caused by exchange interactions.

New Gold(I) Organometallic Compounds with Biological Activity in Cancer Cells

AbstractN‐Heterocyclic carbene gold(I) complexes bearing a fluorescent coumarin ligand were synthesized and characterized by various techniques. The compounds were examined for their antiproliferative effects in normal and tumor cells in vitro; they demonstrated moderate activity and a certain degree of selectivity. The compounds were also shown to efficiently inhibit the selenoenzyme thioredoxin reductase (TrxR), whereas they were poorly effective towards the glutathione reductase (GR) and glutathione peroxidase enzymes. Notably, {3‐[(7‐methoxy‐2‐oxo‐2H‐chromen‐4‐yl)methyl]‐1‐methylimidazol‐2‐ylidene}(tetra‐O‐acetyl‐1‐thio‐β‐D‐glucopyranosido)gold(I) (3) showed a pronounced inhibition of TrxR also in cell extracts, and it appeared to activate GR. Mechanistic information on the system derived from biotin‐conjugated iodoacetamide assays showed selective metal binding to selenocysteine residues. Preliminary confocal fluorescence microscopy experiments proved that 3 enters tumor cells, where it reaches the nuclear compartment.

Ligand-Dependent Dynamics of the Active-Site Lid in Bacterial Dimethylarginine Dimethylaminohydrolase

The dimethylarginine dimethylaminohydrolase (DDAH) enzyme family has been the subject of substantial investigation as a potential therapeutic target for the regulation of vascular tension. DDAH enzymes catalyze the conversion of asymmetric Nη,Nη-dimethylarginine (ADMA) to l-citrulline. Here the influence of substrate and product binding on the dynamic flexibility of DDAH from Pseudomonas aeruginosa (PaDDAH) has been assessed. A combination of heteronuclear NMR spectroscopy, static and time-resolved fluorescence measurements, and atomistic molecular dynamics simulations was employed. A monodisperse monomeric variant of the wild-type enzyme binds the reaction product l-citrulline with a low millimolar dissociation constant. A second variant, engineered to be catalytically inactive by substitution of the nucleophilic Cys249 residue with serine, can still convert the substrate ADMA to products very slowly. This PaDDAH variant also binds l-citrulline, but with a low micromolar dissociation constant. NMR and molecular dynamics simulations indicate that the active site “lid”, formed by residues Gly17-Asp27, exhibits a high degree of internal motion on the picosecond-to-nanosecond time scale. This suggests that the lid is open in the apo state and allows substrate access to the active site that is otherwise buried. l-Citrulline binding to both protein variants is accompanied by an ordering of the lid. Modification of PaDDAH with a coumarin fluorescence reporter allowed measurement of the kinetic mechanism of the PaDDAH reaction. A combination of NMR and kinetic data shows that the catalytic turnover of the enzyme is not limited by release of the l-citrulline product. The potential to develop the coumarin–PaDDAH adduct as an l-citrulline sensor is discussed.

Solvation of Coumarin 480 within nano-confining environments: structure and dynamics.

Equilibrium and dynamical characteristics pertaining to the solvation of the fluorescent probe Coumarin 480 within different confining environments are investigated using molecular dynamics simulations. Three kinds of confining systems are examined: (i) the cetyltrimethylammonium bromide (CTAB)/isooctane/1-hexanol/water; cationic inverse micelle (IM) (ii) a CTAB/water direct micelle (DM), and (iii) a silica-surfactant nanocomposite, comprising a cylindrical silica pore (SP) containing small amounts of water and CTAB species adsorbed at the pore walls. The solvation structures in the three environments differ at a qualitative level: an exchange between bulk- and interface-like solvation states was found in the IM, whereas in the DM, the solvation states of the probe are characterized by its embedding at the interface, trapped among the surfactant heads and tails. Within the SP structure, the coumarin exhibits alternations between internal and interfacial solvation states that occur on a ∼20 ns time scale and operate via 90° rotations of its molecular plane. The solvation responses of the environment following a vertical excitation of the probe are also investigated. Solvation times resulted between 2 and 1000 times longer than those found in bulk water, with a fast-to-slow trend IM→DM→SP, which can be interpreted in terms of the solvation structures that prevail in each case.

The reduction of anti-cancer drug antagonism by the spatial protection of drugs with PLA–TPGS nanoparticles

Docetaxel (DCL) and tamoxifen (TAM) individually are potent drugs in the fight against breast cancer. However when used in combination, they become antagonistic because of differential metabolism of both drugs. We reasoned that by spatially protecting them from metabolizing enzymes with poly (lactide)-d-α-tocopheryl polyethylene glycol succinate (PLA–TPGS) nanoparticles (NPs), we might reduce this drug antagonism. We now report that the drug antagonism between DCL and TAM in MCF7 cell line, was significantly reduced when co-delivered in PLA–TPGS NPs. In addition, this effect of NPs attenuated at high drug concentrations. To investigate the role of NPs in the reduction of drug antagonism, we quantified cellular uptake of the fluorescent model drug coumarin 6 (C6) encapsulated in a rigorous permutation of drugs-nanoparticles ratios. NPs carrying C6 exhibited enhanced cellular uptake over their free C6 counterparts at correspondingly low drug concentrations. This led us to conclude that the reduction of drug antagonism by NPs is correlated to cellular uptake and being in NPs therefore protects both drugs until they are released intracellular for therapeutic anti-cancer effect.

Synthesis, characterization and spectroscopic properties of water soluble coumarins substituted with oligomeric alkoxy functions

Novel water soluble robust fluorescent coumarins substituted with oligomeric alkoxy functions were synthesized by incorporating the Blaise reaction in the key step. Mono-methylated oligomeric polyethylene glycols were subjected to a three step protocol, namely (i) Michael addition to acrylonitrile, (ii) Blaise reaction with ethyl bromoacetate and (iii) condensation with 4-N,N-diethylamino-2-hydroxybenzaldehyde to give fluorescent water soluble coumarins. Water solubility of the coumarins increased with the number of oxygen atoms in the side chain. However, even the most water soluble coumarin in this series can be readily extracted out of water with organic solvents like dichloromethane or ethyl acetate. Both absorption and emission spectra, recorded in four solvents, namely, hexane (non-polar), ethyl acetate (moderately polar), methanol (polar protic) and water (highly polar and protic) displayed a bathochromic shift of the absorption (Δλmax ≈ 25 nm) and emission (Δλmax ≈ 57 nm) bands with increasing solvent polarity. The Δλmax of emission is more pronounced than the Δλmax of absorption, which indicates intramolecular charge-transfer (ICT) is less in the ground state compared to the excited state. Emission spectra recorded in these four solvents showed that fluorescent intensity is maximum in ethyl acetate.

Photophysical behavior of a new cholesterol attached coumarin derivative and fluorescence spectroscopic studies on its interaction with bile salt systems and lipid bilayer membranes

A new fluorescent-cholesterol (Cum-Chl) molecule has been synthesized by attaching 3-acetyl-7-(diethylamino)-2H-chromen-2-one (Cum) to cholesterol via cholesterol bound β-keto ester. The β-keto ester was synthesized from the corresponding nitrile by applying the Blaise reaction. The molecule forms H-aggregates in solutions. The efficiency of aggregation is high in water. The H-aggregates are non-fluorescent in non-aqueous solvents but fluorescent in water. Aqueous bile salt media suppress the formation of H-aggregates, this effect being more pronounced for sodium deoxycholate (NaDC) which is more hydrophobic. With increasing bile salt concentration, the enhancement of monomeric fluorescence intensity of Cum-Chl generally follows the progressive miceller aggregation of bile salts. Incorporation of Cum-Chl into the dimyristoylphosphatidylcholine (DMPC) lipid bilayer membrane results in a significant enhancement of monomeric fluorescence intensity. The variation of fluorescence intensity is also sensitive to the thermotropic phase transition of the bilayer. It is seen that in such aqueous micro- and nanoscale organized media like bile salts and lipid bilayer membranes the monomer-to-aggregate fluorescence intensity ratio reflects the state of organization of the medium.

Feruloyl-CoA 6'-Hydroxylase1-dependent coumarins mediate iron acquisition from alkaline substrates in Arabidopsis.

Although iron (Fe) is one of the most abundant elements in the earth's crust, its low solubility in soils restricts Fe uptake by plants. Most plant species acquire Fe by acidifying the rhizosphere and reducing ferric to ferrous Fe prior to membrane transport. However, it is unclear how these plants access Fe in the rhizosphere and cope with high soil pH. In a mutant screening, we identified 2-oxoglutarate-dependent dioxygenase Feruloyl-CoA 6'-Hydroxylase1 (F6'H1) to be essential for tolerance of Arabidopsis (Arabidopsis thaliana) to high pH-induced Fe deficiency. Under Fe deficiency, F6'H1 is required for the biosynthesis of fluorescent coumarins that are released into the rhizosphere, some of which possess Fe(III)-mobilizing capacity and prevent f6'h1 mutant plants from Fe deficiency-induced chlorosis. Scopoletin was the most prominent coumarin found in Fe-deficient root exudates but failed to mobilize Fe(III), while esculetin, i.e. 6,7-dihydroxycoumarin, occurred in lower amounts but was effective in Fe(III) mobilization. Our results indicate that Fe-deficient Arabidopsis plants release Fe(III)-chelating coumarins as part of the strategy I-type Fe acquisition machinery.

New Continuous Fluorometric Assay for Bacterial Transglycosylase Using Förster Resonance Energy Transfer

The emergence of antibiotic resistance has prompted scientists to search for new antibiotics. Transglycosylase (TGase) is an attractive target for new antibiotic discovery due to its location on the outer membrane of bacteria and its essential role in peptidoglycan synthesis. Though there have been a few molecules identified as TGase inhibitors in the past thirty years, none of them have been developed into antibiotics for humans. The slow pace of development is perhaps due to the lack of continuous, quantitative, and high-throughput assay available for the enzyme. Herein, we report a new continuous fluorescent assay based on Förster resonance energy transfer, using lipid II analogues with a dimethylamino-azobenzenesulfonyl quencher in the lipid chain and a coumarin fluorophore in the peptide chain. During the process of transglycosylation, the quencher-appended polyprenol is released and the fluorescence of coumarin can be detected. Using this system, the substrate specificity and affinity of lipid II analogues bearing various numbers and configurations of isoprene units were investigated. Moreover, the inhibition constants of moenomycin and two previously identified small molecules were also determined. In addition, a high-throughput screening using the new assay was conducted to identify potent TGase inhibitors from a 120,000 compound library. This new continuous fluorescent assay not only provides an efficient and convenient way to study TGase activities, but also enables the high-throughput screening of potential TGase inhibitors for antibiotic discovery.

OH Radical Formation at Distinct Faces of Rutile TiO2 Crystal in the Procedure of Photoelectrochemical Water Oxidation

It has been believed that the photogenerated OH radicals are major active species which cause photocatalytic oxidation of water. To investigate the actual contribution of OH radicals to the photocatalytic O2 generation, the amount of the OH radicals was measured for the three kinds of rutile TiO2 electrodes having (100), (110), and (001) crystalline surfaces. The current efficiencies for O2 generation measured with an oxygen sensor were almost 100% for all electrodes. However, the current efficiencies for OH radical formation estimated by means of a coumarin fluorescence probe method were less than 1%. Thus, it was experimentally elucidated that the contribution of OH radicals to the O2 production is negligibly small. The amount of OH radical production decreased in the order of (100) > (110) > (001), along with the increase in the efficiency of the O2 production. A plausible mechanism of OH radical formation as a byproduct in the O2 generation process was proposed.