Molecular mechanism of the metal-independent production of hydroxyl radicals by thiourea dioxide and H2O2.

It is well-known that highly reactive hydroxyl radicals (HO•) can be produced by the classic Fenton system and our recently discovered haloquinone/H2O2 system, but rarely from thiol-derivatives. Here, we found, unexpectedly, that HO• can be generated from H2O2 and thiourea dioxide (TUO2), a widely used and environmentally friendly bleaching agent. A carbon-centered radical and sulfite were detected and identified as the transient intermediates, and urea and sulfate as the final products, with the complementary application of electron spin-trapping, oxygen-18 isotope labeling coupled with HPLC/MS analysis. Density functional theory calculations were conducted to further elucidate the detailed pathways for HO• production. Taken together, we proposed that the molecular mechanism for HO• generation by TUO2/H2O2: TUO2 tautomerizes from sulfinic acid into ketone isomer (TUO2-K) through proton transfer, then a nucleophilic addition of H2O2 on the S atom of TUO2-K, forming a S-hydroperoxide intermediate TUO2-OOH, which dissociates homolytically to produce HO•. Our findings represent the first experimental and computational study on an unprecedented new molecular mechanism of HO• production from simple thiol-derived sulfinic acids, which may have broad chemical, environmental, and biomedical significance for future research on the application of the well-known bleaching agent and its analogs.

Unusual enantioselective cytoplasm-to-nucleus translocation and photosensitization of the chiral Ru(II) cationic complex via simple ion-pairing with lipophilic weak acid counter-anions.

Targeted and enantioselective delivery of chiral diagnostic-probes and therapeutics into specific compartments inside cells is of utmost importance in the improvement of disease detection and treatment. The classical DNA 'light-switch' ruthenium(II)-polypyridyl complex, [Ru(DIP)2(dppz)]Cl2 (DIP = 4,7-diphenyl-1,10-phenanthroline, dppz = dipyridophenazine) has been shown to be accumulated only in the cytoplasm and membrane, but excluded from its intended nuclear DNA target. In this study, the cationic [Ru(DIP)2(dppz)]2+ is found to be redirected into live-cell nucleus in the presence of lipophilic 3,5-dichlorophenolate or flufenamate counter-anions via ion-pairing mechanism, while maintaining its original DNA recognition characteristics. Interestingly and unexpectedly, further studies show that only the Δ-enantiomer is selectively translocated into nucleus while the Λ-enantiomer remains trapped in cytoplasm, which is found to be mainly due to their differential enantioselective binding affinities with cytoplasmic proteins and nuclear DNA. More importantly, only the nucleus-relocalized Δ-enantiomer can induce obvious DNA damage and cell apoptosis upon prolonged visible-light irradiation. Thus, the use of Δ-enantiomer can significantly reduce the dosage needed for maximal treatment effect. This represents the first report of enantioselective targeting and photosensitization of classical Ru(II) complex via simple ion-pairing with suitable weak acid counter-anions, which opens new opportunities for more effective enantioselective cancer treatment.

Unprecedented enantio-selective live-cell mitochondrial DNA super-resolution imaging and photo-sensitizing by the chiral ruthenium polypyridyl DNA "light-switch".

Mitochondrial DNA (mtDNA) is known to play a critical role in cellular functions. However, the fluorescent probe enantio-selectively targeting live-cell mtDNA is rare. We recently found that the well-known DNA 'light-switch' [Ru(phen)2dppz]Cl2 can image nuclear DNA in live-cells with chlorophenolic counter-anions via forming lipophilic ion-pairing complex. Interestingly, after washing with fresh-medium, [Ru(phen)2dppz]Cl2 was found to re-localize from nucleus to mitochondria via ABC transporter proteins. Intriguingly, the two enantiomers of [Ru(phen)2dppz]Cl2 were found to bind enantio-selectively with mtDNA in live-cells not only by super-resolution optical microscopy techniques (SIM, STED), but also by biochemical methods (mitochondrial membrane staining with Tomo20-dronpa). Using [Ru(phen)2dppz]Cl2 as the new mtDNA probe, we further found that each mitochondrion containing 1-8 mtDNA molecules are distributed throughout the entire mitochondrial matrix, and there are more nucleoids near nucleus. More interestingly, we found enantio-selective apoptotic cell death was induced by the two enantiomers by prolonged visible light irradiation, and in-situ self-monitoring apoptosis process can be achieved by using the unique 'photo-triggered nuclear translocation' property of the Ru complex. This is the first report on enantio-selective targeting and super-resolution imaging of live-cell mtDNA by a chiral Ru complex via formation and dissociation of ion-pairing complex with suitable counter-anions.

The anti-cancer drug candidate CBL0137 induced necroptosis via forming left-handed Z-DNA and its binding protein ZBP1 in liver cells.

CBL0137, a promising small molecular anti-cancer drug candidate, has been found to effectively induce apoptosis via activating p53 and suppressing nuclear factor-kappa B (NF-κB). However, it is still not clear whether CBL0137 can induce necroptosis in liver cancer; and if so, what is the underlying molecular mechanism. Here we found that CBL0137 could significantly induce left-handed double helix structure Z-DNA formation in HepG2 cells as shown by Z-DNA specific antibody assay, which was further confirmed by observing the expression of Z-DNA binding protein 1 (ZBP1) and adenosine deaminase acting on RNA 1 (ADAR1). Interestingly, we found that caspase inhibition significantly promoted CBL0137-induced necroptosis, which was further supported with the increase of the late apoptosis and necrosis assessed by the flow cytometry. Furthermore, we found that CBL0137 can also induce the expression of the three necroptosis-related proteins: receptor interacting serine/threonine kinase 1 (RIPK1), receptor interacting serine/threonine kinase 3 (RIPK3), and mixed lineage kinase domain-like (MLKL). Taken together, it was assumed that CBL0137-indued necroptosis in liver cells was due to induction of Z-DNA and ZBP1, which activated RIPK1/RIPK3/MLKL pathway. This represents the first report on the induction of the Z-DNA-mediated necroptosis by CBL0137 in the liver cancer cells, which should provide new perspectives for CBL0137 treatment of liver cancer.

Molecular mechanism for the unusual enhancement of the second-step chemiluminescence production from the carcinogenic tetrabromohydroquinone and H2O2.

We have found recently that two-step intrinsic hydroxyl radical (·OH)-dependent chemiluminescence (CL) could be produced by carcinogenic tetrahaloquinone and H2O2. However, the first-step CL was too fast to clearly detect the stepwise generation of ·OH and CL, and to distinguish the exact dividing point between the first-step and second-step CL. Here we found that, extremely clear two-step intrinsic CL could be produced by the relative slow reaction of tetrabromohydroquinone (TBHQ) with H2O2, which was directly dependent on the two-step ·OH generation. Interestingly, the second-step, but not the first-step CL production of TBHQ/H2O2 (CRET donor) was markedly enhanced by fluorescein (a typical xanthene dye, CRET acceptor) through a unique chemiluminescence resonance energy transfer (CRET) process. The novel CRET system of TBHQ/H2O2/fluorescein was successfully applied for the sensitive detection of TBHQ with the detection limit as low as 2.5 µmol/L. These findings will help to develop more sensitive and highly efficient CL or CRET systems and specific CL sensor to detect the carcinogenic haloquinones, which may have broad environmental applications.

Mechanistic Investigation of H2 O2 -dependent Chemiluminescence from Tetrabromo-1,4-Benzoquinone.

As a H2 O2 -dependent bioluminescent substrate, tetrabromo-1,4-benzoquinone (TBBQ) was first isolated from acorn worm. The mechanism of chemiluminescence (CL) corresponding to the bioluminescence (BL) of acorn worm is largely unknown, let alone the mechanism of BL. In this article, we firstly studied the chemical and physical processes, and mechanism of H2 O2 -dependent CL from TBBQ by theoretical and experimental methods. The research results indicate: the CL process is initiated by a nucleophilic substitution reaction, which leads to the formation of an anionic dioxetane through five consecutive reactions; the anionic dioxetane decomposes to the first singlet excited state (S1 ) via a conical interaction of the potential energy surfaces (PESs) between the ground (S0 ) and S1 state; the anionic S1 -state changes to its neutral form by a proton transfer from the solvent and this neutral product is assigned as the actual luminophore. Moreover, the experimental detection of CL, . OH and the identifications of 2,3-dibromo maleic acid and 2-bromo malonic acid as the major final products provide direct evidence of the theoretically suggested mechanism. Finally, this study proves that the activity of the H2 O2 -dependent CL from TBBQ is significantly lower than the one from tetrachloro-1,4-benzoquinone (TCBQ), which is caused by the weaker electron withdrawing effect and the stronger heavy atomic effect of bromine.

Mechanistic Study on Oxidative DNA Damage and Modifications by Haloquinoid Carcinogenic Intermediates and Disinfection Byproducts.

Haloquinones (XQs) are a group of carcinogenic intermediates of the haloaromatic environmental pollutants and newly identified chlorination disinfection byproducts (DBPs) in drinking water. The highly reactive hydroxyl radicals/alkoxyl radicals and quinone enoxy/ketoxy radicals were found to arise in XQs and H2O2 or organic hydroperoxides system, independent of transition-metal ions. However, it was not clear whether these haloquinoid carcinogens and hydroperoxides can cause oxidative DNA damage and modifications, and if so, what are the underlying molecular mechanisms. We found that 8-oxodeoxyguanosine (8-oxodG), DNA strand breaks, and three methyl oxidation products could arise when DNA was treated with tetrachloro-1,4-benzoquinone and H2O2 via a metal-independent and intercalation-enhanced oxidation mechanism. Similar effects were observed with other XQs, which are generally more efficient than the typical Fenton system. We further extended our studies from isolated DNA to genomic DNA in living cells. We also found that potent oxidation of DNA to the more mutagenic imidazolone dIz could be induced by XQs and organic hydroperoxides such as t-butylhydroperoxide or the physiologically relevant hydroperoxide 13S-hydroperoxy-9Z,11E-octadecadienoic acid via an unprecedented quinone-enoxy radical-mediated mechanism. These findings should provide new perspectives to explain the potential genotoxicity, mutagenesis, and carcinogenicity for the ubiquitous haloquinoid carcinogenic intermediates and DBPs.

Structure-Activity Relationship Investigation on Reaction Mechanism between Chlorinated Quinoid Carcinogens and Clinically-Used Aldoxime Nerve-Agent Antidote under Physiological Condition.

Pyridinium aldoximes are best-known therapeutic antidotes used for clinical treatment of poisonings by organophosphorus nerve-agents and pesticides. Recently, we found that pralidoxime (2-PAM, a currently clinically used nerve-agent antidote) could also detoxify tetrachloro-1,4-benzoquinone (TCBQ), which is a carcinogenic quinoid metabolite of the widely used wood preservative pentachlorophenol under normal physiological conditions, via an unusually mild and facile Beckmann fragmentation mechanism accompanied by radical homolysis. However, it is not clear whether the less-chlorinated benzoquinones (CnBQs, n ≤ 3) act similarly; if so, what is the structure-activity relationship? In this study, we found that (1) The stability of reaction intermediates produced by different CnBQs and 2-PAM was dependent not only on the position but also the degree of Cl-substitution on CnBQs, which can be divided into TCBQ- and DCBQ (dichloro-1,4-benzoquinone)-subgroup; (2) The pKa value of hydroxlated quinones (Cn-1BQ-OHs, the hydrolysis products of CnBQs), determined the stability of corresponding intermediates, that is, the decomposition rate of the intermediates depended on the acidity of Cn-1BQ-OHs; (3) The pKa value of the corresponding Cn-1BQ-OHs could also determine the reaction ratio of Beckmann fragmentation to radical homolysis in CnBQs/2-PAM. These new findings on the structure-activity relationship of the halogenated quinoid carcinogens detoxified by pyridinium aldoxime therapeutic agents via Beckmann fragmentation and radical homolysis reaction may have broad implications on future biomedical and environmental research.

Mechanistic Study on Chemiluminescence of Chloranilic Acid by Co(II)-Mediated Fenton-like System.

Reacting with H2O2, tetrachloro-1,4-benzoquinone (TCBQ) produces chemiluminescence (CL), but chloranilic acid (CA), the dihydroxylation product of TCBQ, does not. However, an unprecedented strong CL generates from CA/H2O2 in the presence of Co(II). Why? We performed quantum chemical calculations on the entire reaction process of CA/H2O2 and CA/H2O2/Co(II) systems. The computational results indicate: for CA/H2O2 system, the reason leading to non-CL as: there is no free •OH produced by CA/H2O2, which prevents the subsequent reaction from taking place; for CA/H2O2/Co(II) system, the chemical process resulting in the CL as: First, a neutral dioxetane is formed via six sequential reactions. Then, the neutral dioxetane decomposes to generate a neutral excited-state (S1) product via a gradually reversible charge transfer initiated luminescence mechanism. A conical intersection of the ground and the S1-state potential energy surfaces facilitates the production of the S1-state product. Ultimately, the neutral S1-state product emits light as a practical light emitter. The key component for forming dioxetane and the following CL is the intrinsically generated •OH, which is roaming around at the region of C2 atoms of the CA moiety, instead of being free. The quantum chemical calculations supported the experimental observation and conclusion by providing the mechanistic explanation in detail.

Sulfur-centered hemi-bond radicals as active intermediates in S-DNA phosphorothioate oxidation.

Phosphorothioate (PS) modifications naturally appear in bacteria and archaea genome and are widely used as antisense strategy in gene therapy. But the chemical effects of PS introduction as a redox active site into DNA (S-DNA) is still poorly understood. Herein, we perform time-resolved spectroscopy to examine the underlying mechanisms and dynamics of the PS oxidation by potent radicals in free model, in dinucleotide, and in S-oligomer. The crucial sulphur-centered hemi-bonded intermediates -P-S∴S-P- were observed and found to play critical roles leading to the stable adducts of -P-S-S-P-, which are backbone DNA lesion products. Moreover, the oxidation of the PS moiety in dinucleotides d[GPSG], d[APSA], d[GPSA], d[APSG] and in S-oligomers was monitored in real-time, showing that PS oxidation can compete with adenine but not with guanine. Significantly, hole transfer process from A+• to PS and concomitant -P-S∴S-P- formation was observed, demonstrating the base-to-backbone hole transfer unique to S-DNA, which is different from the normally adopted backbone-to-base hole transfer in native DNA. These findings reveal the distinct backbone lesion pathway brought by the PS modification and also imply an alternative -P-S∴S-P-/-P-S-S-P- pathway accounting for the interesting protective role of PS as an oxidation sacrifice in bacterial genome.

Targeted live-cell nuclear delivery of the DNA 'light-switching' Ru(II) complex via ion-pairing with chlorophenolate counter-anions: the critical role of binding stability and lipophilicity of the ion-pairing complexes.

We have found recently that nuclear uptake of the cell-impermeable DNA light-switching Ru(II)-polypyridyl cationic complexes such as [Ru(bpy)2(dppz)]Cl2 was remarkably enhanced by pentachlorophenol (PCP), by forming ion-pairing complexes via a passive diffusion mechanism. However, it is not clear whether the enhanced nuclear uptake of [Ru(bpy)2(dppz)]2+ is only limited to PCP, or it is a general phenomenon for other highly chlorinated phenols (HCPs); and if so, what are the major physicochemical factors in determining nuclear uptake? Here, we found that the nuclear uptake of [Ru(bpy)2(dppz)]2+ can also be facilitated by other two groups of HCPs including three tetrachlorophenol (TeCP) and six trichlorophenol (TCP) isomers. Interestingly and unexpectedly, 2,3,4,5-TeCP was found to be the most effective one for nuclear delivery of [Ru(bpy)2(dppz)]2+, which is even better than the most-highly chlorinated PCP, and much better than its two other TeCP isomers. Further studies showed that the nuclear uptake of [Ru(bpy)2(dppz)]2+ was positively correlated with the binding stability, but to our surprise, inversely correlated with the lipophilicity of the ion-pairing complexes formed between [Ru(bpy)2(dppz)]Cl2 and HCPs. These findings should provide new perspectives for future investigations on using ion-pairing as an effective method for delivering other bio-active metal complexes into their intended cellular targets.

Detecting and Quantifying Polyhaloaromatic Environmental Pollutants by Chemiluminescence-Based Analytical Method.

Polyhaloaromatic compounds (XAr) are ubiquitous and recalcitrant in the environment. They are potentially carcinogenic to organisms and may induce serious risks to the ecosystem, raising increasing public concern. Therefore, it is important to detect and quantify these ubiquitous XAr in the environment, and to monitor their degradation kinetics during the treatment of these recalcitrant pollutants. We have previously found that unprecedented intrinsic chemiluminescence (CL) can be produced by a haloquinones/H2O2 system, a newly-found ●OH-generating system different from the classic Fenton system. Recently, we found that the degradation of priority pollutant pentachlorophenol by the classic Fe(II)-Fenton system could produce intrinsic CL, which was mainly dependent on the generation of chloroquinone intermediates. Analogous effects were observed for all nineteen chlorophenols, other halophenols and several classes of XAr, and a novel, rapid and sensitive CL-based analytical method was developed to detect these XAr and monitor their degradation kinetics. Interestingly, for those XAr with halohydroxyl quinoid structure, a Co(II)-mediated Fenton-like system could induce a stronger CL emission and higher degradation, probably due to site-specific generation of highly-effective ●OH. These findings may have broad chemical and environmental implications for future studies, which would be helpful for developing new analytical methods and technologies to investigate those ubiquitous XAr.

Unusual Two-Step Claisen-type Rearrangement Reaction under Physiological Conditions.

N-aryl hydroxamic acids, which are best known for their metal-chelating properties in chemical and biomedical research, have been found to markedly detoxify carcinogenic halogenated quinones. However, the exact chemical mechanism underlying such detoxication remains unclear. Here, we show that a very fast reaction took place between N-phenylbenzohydroxamic acid (N-PhBHA) and 2,5-dichloro-1,4-benzoquinone (DCBQ), forming an unexpected new carbon-carbon bonding phenyl-quinone product with high yield. In contrast, no reaction was observed with O-benzoyl N-PhBHA. Analogous results were observed for other N-aryl hydroxamic acids and halogenated quinones, which have an ortho-hydrogen adjacent to the reaction site (DCBQ-type). Interestingly, no free radical intermediates could be detected by both ESR spin-trapping and radical-scavenging methods during the reaction process. Taken together, we proposed that nucleophilic substitution followed by an unusual two-step Claisen-type rearrangement reaction was responsible for the formation of a new C-C bonding compound and the detoxication reaction. This represents the first report of an unusually mild and facile two-step Claisen-type rearrangement, which could take place under normal physiological conditions.

First Direct and Unequivocal Electron Spin Resonance Spin-Trapping Evidence for pH-Dependent Production of Hydroxyl Radicals from Sulfate Radicals.

Recently, the sulfate radical (SO4•-) has been found to exhibit broad application prospects in various research fields such as chemical, biomedical, and environmental sciences. It has been suggested that SO4•- could be transformed into a more reactive hydroxyl radical (•OH); however, no direct and unequivocal experimental evidence has been reported yet. In this study, using an electron spin resonance (ESR) secondary radical spin-trapping method coupled with the classic spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and the typical •OH-scavenging agent dimethyl sulfoxide (DMSO), we found that •OH can be produced from three SO4•--generating systems from weakly acidic (pH = 5.5) to alkaline conditions (optimal at pH = 13.0), while SO4•- is the predominant radical species at pH < 5.5. A comparative study with three typical •OH-generating systems strongly supports the above conclusion. This is the first direct and unequivocal ESR spin-trapping evidence for •OH formation from SO4•- over a wide pH range, which is of great significance to understand and study the mechanism of many SO4•--related reactions and processes. This study also provides an effective and direct method for unequivocally distinguishing •OH from SO4•-.

Potent Oxidation of DNA by Haloquinoid Disinfection Byproducts to the More Mutagenic Imidazolone dIz via an Unprecedented Haloquinone-Enoxy Radical-Mediated Mechanism.

Halogenated quinones are a class of carcinogenic intermediates and newly identified chlorination disinfection byproducts in drinking water. We found recently that halogenated quinones could enhance the decomposition of hydroperoxides independent of transition-metal ions and formation of the novel quinone enoxy/ketoxy radicals. Here, we show that the major oxidation product was 2-amino-5-[(2-deoxy-ß-d-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (dIz) when the nucleoside 2'-deoxyguanosine (dG) was treated with tetrachloro-1,4-benzoquinone (TCBQ) and t-butyl hydroperoxide (t-BuOOH). The formation of dIz was markedly inhibited by typical radical spin-trapping agents. Interestingly and unexpectedly, we found that the generated quinone enoxy radical played a critical role in dIz formation. Using [15N5]-8-oxodG, dIz was found to be produced either directly from dG or through the transient formation of 8-oxodG. Based on these data, we proposed that the production of dIz might be through an unusual haloquinone-enoxy radical-mediated mechanism. Analogous results were observed in the oxidation of ctDNA by TCBQ/t-BuOOH and when t-BuOOH was substituted by the endogenously generated physiologically relevant hydroperoxide 13S-hydroperoxy-9Z,11E-octadecadienoic acid. This is the first report that halogenated quinoid carcinogens and hydroperoxides can induce potent oxidation of dG to the more mutagenic product dIz via an unprecedented quinone-enoxy radical-mediated mechanism, which may partly explain their potential carcinogenicity.

Mechanism of unprecedented hydroxyl radical production and site-specific oxidative DNA damage by photoactivation of the classic arylhydroxamic acid carcinogens.

The carcinogenicity of N-hydroxy-2-acetamidofluorene (N-OHAAF), the major genotoxic metabolite of the classic model aromatic amine (AA) carcinogen 2-acetylaminofluorene, has been attributed mainly to the formation of DNA adducts via arylnitrenium upon enzymatic activation. Here, we show, unexpectedly, that exposure of N-OHAAF to UV or sunlight irradiation can not only induce the formation of the well-known covalent DNA adducts, but, more interestingly, simultaneous generation of oxidative DNA damage was also observed as measured by the formation of DNA single-/double-strand breaks (SSBs/DSBs) and 8-oxo-2'-deoxyguanosine (8-oxodG), which were partly inhibited by the typical hydroxyl radical (•OH) scavengers. Electron spin resonance spin-trapping and fluorescent studies unequivocally confirmed that the highly reactive •OH was generated from photolysis of N-OHAAF. Further DNA sequencing investigations suggest that photoactivation of N-OHAAF caused preferential cleavage at guanine, thymine and cytosine sites. More importantly, the formation of 8-oxodG and DSBs were also observed when fibroblast Balb/c-3T3 cells were co-exposed to N-OHAAF/UV irradiation as measured by double immunofluorescence staining. Taken together, we propose that both •OH and amidyl radicals can be readily produced via N-OH homolysis in N-OHAAF by photoirradiation, which can induce both oxidative and covalent DNA damage. This represents the first report of •OH production and site-specific DNA damage via photoactivation of the genotoxic hydroxamic acid intermediate, which provides a new free radical perspective to better understand the molecular mechanism for the carcinogenicity of AAs.

Reactive Nitrogen Species Are Also Involved in the Transformation of Micropollutants by the UV/Monochloramine Process.

The UV/monochloramine (NH2Cl) process is an emerging advanced oxidation process (AOP) in water treatment via radicals produced from the UV photolysis of NH2Cl. This study investigated the degradation of micropollutants by the UV/NH2Cl AOP, with ibuprofen (IBP) and naproxen (NPX) selected as representative micropollutants. Hydroxyl radical (HO•) and chlorine atom (Cl•) were identified in the process, and unexpectedly, we found that reactive nitrogen species (RNS) also played important roles in the transformation of micropollutants. The electron paramagnetic resonance (EPR) analysis proved the production of •NO as well as HO•. The concentrations of HO•, Cl•, and •NO in UV/NH2Cl remained constant at pH 6.0-8.6, resulting in the slightly changed UV fluence-based pseudo-first-order rate constants (k') of IBP and NPX, which were about 1.65 × 10-3 and 2.54 × 10-3 cm2/mJ, respectively. For IBP, the relative contribution of RNS to k' was 27.8% at pH 7 and 50 µM NH2Cl, which was higher than that of Cl• (6.5%) but lower than that of HO• (58.7%). For NPX, the relative contribution of RNS to k' was 13.6%, which was lower than both Cl• (23.2%) and HO• (46.9%). The concentrations of HO•, Cl•, and •NO increased with the increasing NH2Cl dosage. Water matrix components of natural organic matter (NOM) and bicarbonate can scavenge HO•, Cl•, and RNS. The presence of 5 mg/L NOM decreased the k' of IBP and NPX by 66.9 and 57.6%, respectively, while 2 mM bicarbonate decreased the k' of IBP by 57.4% but increased the k' of NPX by 10.5% due to the contribution of CO3•- to NPX degradation. Products containing nitroso-, hydroxyl-, and chlorine-groups were detected during the degradation of IBP and NPX by UV/NH2Cl, indicating the role of nitrogen oxide radical (•NO) as well as HO• and Cl•. Trichloronitromethane formation was strongly enhanced in the UV/NH2Cl-treated samples, further indicating the important roles of RNS in this process. This study first demonstrates the involvement of RNS in the transformation of micropollutants in UV/NH2Cl.

Unusual Double Beckmann Fragmentation Reaction under Physiological Conditions.

Pyridinium aldoximes, which are best-known as therapeutic antidotes for organophosphorus chemical warfare nerve-agents and pesticides, have been found to markedly detoxify polyhalogenated quinones, which are a class of carcinogenic intermediates and recently identified disinfection byproducts in drinking water. However, the exact chemical mechanism underlying this detoxication remains unclear. Here we demonstrate that pralidoxime can remarkably facilitate the dechlorination/hydroxylation of the highly toxic tetrachloro-1,4-benzoquinone in two-consecutive steps to generate the much less toxic 2,5-dichloro-3,6-dihydroxy-1,4-benzoquonine, with rate enhancements of up to 180 000-times. On the contrary, no accelerating effect was noticed with O-methylated pralidoxime. The major reaction product from pralidoxime was identified as its corresponding nitrile (2-cyano-1-methylpyridinium chloride). Along with oxygen-18 isotope-labeling studies, a reaction mechanism was proposed in which nucleophilic substitution coupled with an unprecedented double Beckmann fragmentation reaction was responsible for the dramatic enhancement in the detoxification process. This represents the first report of an unusually mild and facile Beckmann-type fragmentation that can occur under normal physiological conditions in two-consecutive steps. The study may have broad biomedical and environmental significance for future investigations of aldoxime therapeutic agents and carcinogenic polyhalogenated quinones.

Mechanism of Intrinsic Chemiluminescence Production from the Degradation of Persistent Chlorinated Phenols by the Fenton System: A Structure-Activity Relationship Study and the Critical Role of Quinoid and Semiquinone Radical Intermediates.

We found recently that intrinsic chemiluminescence (CL) could be produced by all 19 chlorophenolic persistent organic pollutants during environmentally friendly advanced oxidation processes. However, the underlying mechanism for the structure-activity relationship (SAR, i.e., the chemical structures and the CL generation) remains unclear. In this study, we found that, for all 19 chlorophenol congeners tested, the CL increased with an increasing number of chlorine atoms in general; and for chlorophenol isomers (such as the 6 trichlorophenols), the CL decreased in the order of meta- > ortho-/para-Cl-substituents with respect to the -OH group of chlorophenols. Further studies showed that not only chlorinated quinoid intermediates but also, more interestingly, chlorinated semiquinone radicals were produced during the degradation of trichlorophenols by the Fenton reagent; and the type and yield of which were determined by the directing effects, hydrogen bonding, and steric hindrance effect of the OH- and/or Cl-substitution groups. More importantly, a good correlation was observed between the formation of these quinoid intermediates and CL generation, which could fully explain the above SAR findings. This represents the first report on the structure-activity relationship study and the critical role of quinoid and semiquinone radical intermediates, which may have broad chemical and environmental implications for future studies on remediation of other halogenated persistent organic pollutants by advanced oxidation processes.

The Unexpected and Exceptionally Facile Chemical Modification of the Phenolic Hydroxyl Group of Tyrosine by Polyhalogenated Quinones under Physiological Conditions.

The phenolic hydroxyl group of tyrosine residue plays a crucial role in the structure and function of many proteins. However, little study has been reported about its modification by chemical agents under physiological conditions. In this study, we found, unexpectedly, that the phenolic hydroxyl group of tyrosine can be rapidly and efficiently modified by tetrafluoro-1,4-benzoquinone and other polyhalogenated quinones, which are the major genotoxic and carcinogenic quinoid metabolites of polyhalogenated aromatic compounds. The modification was found to be mainly due to the formation of a variety of fluoroquinone-O-tyrosine conjugates and their hydroxylated derivatives via nucleophilic substitution pathway. Analogous modifications were observed for tyrosine-containing peptides. Further studies showed that the blockade of the reactive phenolic hydroxyl group of tyrosine in the substrate peptide, even by very low concentration of tetrafluoro-1,4-benzoquinone, can prevent the kinase catalyzed tyrosine phosphorylation. This is the first report showing the exceptionally facile chemical modification of the phenolic hydroxyl group of tyrosine by polyhalogenated quinones under normal physiological conditions, which may have potential biological and toxicological implications.