Chun-Hua Huang

Metal-independent decomposition of hydroperoxides by halogenated quinones: Detection and identification of a quinone ketoxy radical

We have shown recently that halogenated quinones could enhance the decomposition of hydroperoxides and formation of alkoxyl/hydroxyl radicals through a metal-independent mechanism. However, neither the proposed quinone enoxy radical intermediate, nor the major reaction products were unambiguously identified. In the present study, one of the major reaction products between 2,5-dichloro-1,4-benzoquinone (DCBQ) and t-butylhydroperoxide (t-BuOOH) was isolated and purified by semipreparative HPLC, and identified as 2-hydroxy-3-t-butoxy-5-chloro-1,4-benzoquinone [CBQ(OH)-O-t-Bu], which is the rearranged isomer of the postulated quinone-peroxide reaction intermediate. The formation of CBQ(OH)-O-t-Bu was found to be inhibited by the spin trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and concurrently, a new DMPO adduct with 1-chlorine isotope peak clusters at m/z 268 was observed. Further electron spin resonance (ESR) spin-trapping, 1H-NMR and HPLC/Fourier transform ion cyclotron resonance (FTICR) mass spectrometric studies with oxygen-17-labeled and unlabeled hydrogen peroxide strongly suggest that the radical trapped by DMPO is a carbon-centered quinone ketoxy radical, which is the spin isomer of the proposed oxygen-centered quinone enoxy radical. Analogous results were observed when DCBQ was substituted by other halogenated quinones. This study represents the first detection and identification of an unusual carbon-centered quinone ketoxy radical, which provides direct experimental evidence to further support and expand our previously proposed mechanism for metal-independent decomposition of hydroperoxides by halogenated quinones.

Unprecedented hydroxyl radical-dependent two-step chemiluminescence production by polyhalogenated quinoid carcinogens and H2O2

Most chemiluminescence (CL) reactions usually generate only one-step CL, which is rarely dependent on the highly reactive and biologically/environmentally important hydroxyl radicals (•OH). Here, we show that an unprecedented two-step CL can be produced by the carcinogenic tetrachloro-1,4-benzoquinone (also known as p-chloranil) and H2O2, which was found to be well-correlated to and directly dependent on its two-step metal-independent production of •OH. We proposed that •OH-dependent formation of quinone-dioxetane and electronically excited carbonyl species might be responsible for this unusual two-step CL production by tetrachloro-1,4-benzoquinone/H2O2. This is a unique report of a previously undefined two-step CL-producing system that is dependent on intrinsically formed •OH. These findings may have potential applications in detecting and quantifying •OH and the ubiquitous polyhalogenated aromatic carcinogens, which may have broad biological and environmental implications for future research on these types of important species.

Molecular Mechanism of Metal-Independent Decomposition of Organic Hydroperoxides by Halogenated Quinoid Carcinogens and the Potential Biological Implications

Halogenated quinones (XQ) are a class of carcinogenic intermediates and newly identified chlorination disinfection byproducts in drinking water. Organic hydroperoxides (ROOH) can be produced both by free radical reactions and enzymatic oxidation of polyunsaturated fatty acids. ROOH have been shown to decompose to alkoxyl radicals via catalysis by transition metal ions, which may initiate lipid peroxidation or transform further to the reactive aldehydes. However, it is not clear whether XQ react with ROOH in a similar manner to generate alkoxyl radicals metal-independently. By complementary applications of ESR spin-trapping, HPLC/high resolution mass spectrometric and other analytical methods, we found that 2,5-dichloro-1,4-benzoquinone (DCBQ) could significantly enhance the decomposition of a model ROOH tert-butylhydroperoxide, resulting in the formation of t-butoxyl radicals independent of transition metals. On the basis of the above findings, we detected and identified, for the first time, an unprecedented C-centered quinone ketoxy radical. Then, we extended our study to the more physiologically relevant endogenous ROOH 13-hydroperoxy-9,11-octadecadienoic acid and found that DCBQ could also markedly enhance its decomposition to generate the reactive lipid alkyl radicals and the genotoxic 4-hydroxy-2-nonenal (HNE). Similar results were observed with other XQ. In summary, these findings demonstrated that XQ can facilitate ROOH decomposition to produce reactive alkoxyl, quinone ketoxy, lipid alkyl radicals, and genotoxic HNE via a novel metal-independent mechanism, which may explain partly their potential genotoxicity and carcinogenicity.

A Combined Experimental and Computational Investigation on the Unusual Molecular Mechanism of the Lossen Rearrangement Reaction Activated by Carcinogenic Halogenated Quinones

The classic Lossen rearrangement is a well-known reaction describing the transformation of an O-activated hydroxamic acid into the corresponding isocyanate. In this study, we found that chlorinated benzoquinones (CnBQ) serve as a new class of agents for the activation of benzohydroxamic acid (BHA), leading to Lossen rearrangement. Compared to the classic one, this new kind of CnBQ-activated Lossen rearrangement has the following unique characteristics: (1) The stability of CnBQ-activated BHA intermediates was found to depend not only on the degree but also on the position of Cl-substitution on CnBQs, which can be divided into two subgroups. (2) It is the relative energy of the anionic CnBQ–BHA intermediates that determine the rate of this CnBQ-activated rearrangement, which is the rate-limiting step, and the Cl or H ortho to the reaction site at CnBQ is crucial for the stability of the anionic intermediates. (3) A pKa–activation energy correlation was observed, which can explain why the correlation exists between the rate of the rearrangement and the acidity of the conjugate acid of the anionic leaving group, the hydroxlated quinones. These findings may have broad implications for future research on halogenated quinoid carcinogens and hydroxamate biomedical agents.

Molecular mechanism of metal-independent decomposition of lipid hydroperoxide 13-HPODE by halogenated quinoid carcinogens

Halogenated quinones are a class of carcinogenic intermediates and newly identified chlorination disinfection by-products in drinking water. 13-Hydroperoxy-9,11-octadecadienoic acid (13-HPODE) is the most extensively studied endogenous lipid hydroperoxide. Although it is well known that the decomposition of 13-HPODE can be catalyzed by transition metal ions, it is not clear whether halogenated quinones could enhance its decomposition independent of metal ions and, if so, what the unique characteristics and similarities are. Here we show that 2,5-dichloro-1,4-benzoquinone (DCBQ) could markedly enhance the decomposition of 13-HPODE and formation of reactive lipid alkyl radicals such as pentyl and 7-carboxyheptyl radicals, and the genotoxic 4-hydroxy-2-nonenal (HNE), through the complementary application of ESR spin trapping, HPLC-MS, and GC-MS methods. Interestingly, two chloroquinone-lipid alkoxyl conjugates were also detected and identified from the reaction between DCBQ and 13-HPODE. Analogous results were observed with other halogenated quinones. This represents the first report that halogenated quinoid carcinogens can enhance the decomposition of the endogenous lipid hydroperoxide 13-HPODE and formation of reactive lipid alkyl radicals and genotoxic HNE via a novel metal-independent nucleophilic substitution coupled with homolytic decomposition mechanism, which may partly explain their potential genotoxicity and carcinogenicity.

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.

Why Does 2,3,5,6-Tetrachlorophenol Generate the Strongest Intrinsic Chemiluminescence among All Nineteen Chlorophenolic Persistent Organic Pollutants during Environmentally-friendly Advanced Oxidation Process?

We found recently that intrinsic chemiluminescence (CL) could be produced by all 19 chlorophenolic persistent organic pollutants during environmentally-friendly advanced oxidation processes. Interestingly and unexpectedly, the strongest CL was produced not by the most-highly chlorinated pentachlorophenol (PCP), but rather by the less chlorinated 2,3,5,6-tetrachlorophenol (2,3,5,6-TeCP), one of the three tetrachlorophenol (TeCPs) isomers. However, it remains unclear what is the underlying molecular mechanism. Here we show that not only chlorinated quinoid intermediates, but more interestingly, semiquinone radicals were produced during the degradation of the three TeCPs and PCP by Fenton reagents, and the type and yield of which were found to be well correlated with CL generation. We propose that hydroxyl radical-dependent formation of more tetrachlorinated quinoids, quinone-dioxetanes and electronically excited carbonyl species might be responsible for the exceptionally strong CL production by 2,3,5,6-TeCP as compared to PCP and its two isomers. This is the first report showing the critical role of quinoid intermediates and semiquinone radicals in CL generation from polychlorinated phenols and Fenton system. These new findings may have broad chemical and environmental implications for future studies on remediation of other halogenated persistent aromatic pollutants by advanced oxidation processes.

Mechanism of synergistic DNA damage induced by the hydroquinone metabolite of brominated phenolic environmental pollutants and Cu(II): Formation of DNA-Cu complex and site-specific production of hydroxyl radicals

ABSTRACT 2,6‐Dibromohydroquinone (2,6‐DBrHQ) has been identified as an reactive metabolite of many brominated phenolic environmental pollutants such as tetrabromobisphenol‐A (TBBPA), bromoxynil and 2,4,6‐tribromophenol, and was also found as one of disinfection byproducts in drinking water. In this study, we found that the combination of 2,6‐DBrHQ and Cu(II) together could induce synergistic DNA damage as measured by double strand breakage in plasmid DNA and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodG) formation, while either of them alone has no effect. 2,6‐DBrHQ/Cu(II)‐induced DNA damage could be inhibited by the Cu(I)‐specific chelating agent bathocuproine disulfonate and catalase, but not by superoxide dismutase, nor by the typical hydroxyl radical (•OH) scavengers such as DMSO and mannitol. Interestingly, we found that Cu(II)/Cu(I) could be combined with DNA to form DNA‐Cu(II)/Cu(I) complex by complementary application of low temperature direct ESR, circular dichroism, cyclic voltammetry and oxygen consumption methods; and the highly reactive •OH were produced synergistically by DNA‐bound‐Cu(I) with H2O2 produced by the redox reactions between 2,6‐DBrHQ and Cu(II), which then immediately attack DNA in a site‐specific manner as demonstrated by both fluorescent method and by ESR spin‐trapping studies. Further DNA sequencing investigations provided more direct evidence that 2,6‐DBrHQ/Cu(II) caused preferential cleavage at guanine, thymine and cytosine residues. Based on these data, we proposed that the synergistic DNA damage induced by 2,6‐DBrHQ/Cu(II) might be due to the synergistic and site‐specific production of •OH near the binding site of copper and DNA. Our findings may have broad biological and environmental implications for future research on the carcinogenic polyhalogenated phenolic compounds. HIGHLIGHTSOH was found to be responsible for 2,6‐DBrHQ/Cu(II)‐induced DNA damage.First systematic investigation of the interactions between Cu(II)/Cu(I) and DNA.2,6‐DBrHQ/Cu(II) induce DNA damage via a site‐specific mechanism.Site‐specifically generated •OH attacks adjacent DNA sites, mainly at T, C and G.

An Exceptionally Facile Two-Step Structural Isomerization and Detoxication via a Water-Assisted Double Lossen Rearrangement

N-hydroxyphthalimide (NHPI), which is best known as an organocatalyst for efficient C-H activation, has been found to be oxidized by quinoid compounds to its corresponding catalytically active nitroxide-radical. Here, we found that NHPI can be isomerized into isatoic anhydride by an unusually facile two-step method using tetrachloro-1,4-benzoquinone (TCBQ, p-chloranil), accompanied by a two-step hydrolytic dechlorination of highly toxic TCBQ into the much less toxic dihydroxylation product, 2,5-dichloro-3,6-dihydroxy-1,4-benzoquinone (chloranilic acid). Interestingly, through the complementary application of oxygen-18 isotope-labeling, HPLC combined with electrospray ionization quadrupole time-of-flight and high resolution Fourier transform ion cyclotron resonance mass spectrometric studies, we determined that water was the source and origin of oxygen for isatoic anhydride. Based on these data, we proposed that nucleophilic attack with a subsequent water-assisted Lossen rearrangement coupled with rapid intramolecular addition and cyclization in two consecutive steps was responsible for this unusual structural isomerization of NHPI and concurrent hydroxylation/detoxication of TCBQ. This is the first report of an exceptionally facile double-isomerization of NHPI via an unprecedented water-assisted double-Lossen rearrangement under normal physiological conditions. Our findings may have broad implications for future research on hydroxamic acids and polyhalogenated quinoid carcinogens, two important classes of compounds of major chemical and biological interest.

An Unusual Double Radical Homolysis Mechanism for the Unexpected Activation of the Aldoxime Nerve-Agent Antidotes by Polyhalogenated Quinoid Carcinogens under Normal Physiological Conditions

ABSTRACT We have recently shown that the pyridinium aldoximes, best‐known as therapeutic antidotes for chemical warfare nerve‐agents, could markedly detoxify the carcinogenic tetrachloro‐1,4‐benzoquinone (TCBQ) via an unusual double Beckmann fragmentation mechanism. However, it is still not clear why pralidoxime (2‐PAM) cannot provide full protection against TCBQ‐induced biological damages even when 2‐PAM was in excess. Here we show, unexpectedly, that TCBQ can also activate pralidoxime to generate a reactive iminyl radical intermediate in two‐consecutive steps, which was detected and unequivocally characterized by the complementary application of ESR spin‐trapping, HPLC/MS and nitrogen‐15 isotope‐labeling studies. The same iminyl radical was observed when TCBQ was substituted by other halogenated quinones. The end product of iminyl radical was isolated and identified as its corresponding reactive and toxic aldehyde. Based on these data, we proposed that the reaction of 2‐PAM and TCBQ might be through the following two competing pathways: a nucleophilic attack of 2‐PAM on TCBQ forms an unstable transient intermediate, which can decompose not only heterolytically to form 2‐CMP via double Beckmann fragmentation, but also homolytically leading to the formation of a reactive iminyl radical in double‐steps, which then via H abstraction and further hydrolyzation to form its corresponding more toxic aldehyde. Analogous radical homolysis mechanism was observed with other halogenated quinones and pyridinium aldoximes. This study represents the first detection and identification of reactive iminyl radical intermediates produced under normal physiological conditions, which provides direct experimental evidence to explain only the partial protection by 2‐PAM against TCBQ‐induced biological damages, and also the potential side‐toxic effects induced by 2‐PAM and other pyridinium aldoxime nerve‐agent antidotes. Graphic abstract Figure. No Caption available. HighlightsTCBQ activated 2‐PAM to generate reactive iminyl radicals in two‐consecutive steps.Analogous radical homolysis mechanism was observed with other haloquinones & aldoximes.This is the 1st detection of iminyl radical under normal physiological condition.The finding can explain partial protection by 2‐PAM against TCBQ‐induced damages.It may also explain some of the side‐toxic effects induced by pyridinium aldoximes.