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Yasuhisa Asano - One of the best experts on this subject based on the ideXlab platform.
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a promiscuous fatty acid ω hydroxylase cyp94a90 is likely to be involved in biosynthesis of a floral nitro compound in loquat eriobotrya japonica
New Phytologist, 2021Co-Authors: Takuya Yamaguchi, Yasuhisa Asano, Yumi Matsui, Naoki Kitaoka, Yasumasa Kuwahara, Hideyuki Matsuura, Yukari Sunohara, Hiroshi MatsumotoAbstract:Nitro groups are often associated with synthetically manufactured compounds such as medicines and explosives, and rarely with natural products. Loquat emits a nitro compound, (2-nitroethyl)benzene, as a flower scent. The nitro compound exhibits fungistatic activity and is biosynthesised from l-phenylalanine via (E/Z)-phenylacetAldoxime. Although Aldoxime-producing CYP79s have been intensively studied, it is unclear what enzymes form nitro groups from Aldoximes either in plants or in other organisms. Here, we report the identification of two cytochrome P450s that are likely to be involved in (2-nitroethyl)benzene biosynthesis in loquat through differential gene expression analysis using RNA-seq and functional identification using yeast and tobacco. CYP79D80 and CYP94A90 catalysed the formation of (E/Z)-phenylacetAldoxime from l-phenylalanine and (2-nitroethyl)benzene from the Aldoxime, respectively. Expression profiles of CYP79D80 and CYP94A90 were correlated with the emission of (2-nitroethyl)benzene from loquat flowers. CYP94A90 also functioned as a fatty acid ω-hydroxylase as do other CYP94A fatty acid ω-hydroxylases. The CYP94As tested from other plants were all found to catalyse the formation of (2-nitroethyl)benzene from (E/Z)-phenylacetAldoxime. CYP79D80 and CYP94A90 are likely to operate in concert to biosynthesise (2-nitroethyl)benzene in loquat. CYP94A90 and other CYP94As are 'promiscuous fatty acid ω-hydroxylases', catalysing the formation of nitro groups from Aldoximes, and are widely distributed in dicot plants.
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recent progress on discovery and research of Aldoxime dehydratases
Green Synthesis and Catalysis, 2021Co-Authors: Zhongqiang Wang, Ke Chen, Kan Ding, Yongzheng Chen, Yasuhisa AsanoAbstract:Abstract Since Asano and Kato first reported in 1998 an Aldoxime dehydratase from Bacillus sp. strain OxB-1 catalyzes the degradation of Aldoximes to their corresponding nitriles, six Aldoxime dehydratases have been isolated and characterized by this group. The potential applications of these enzymes in organic synthesis represent promising green methods for the preparation of pharmaceuticals and industrial chemicals. This review focuses on recent progress made in studies of these selected six Aldoxime dehydratases.
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cyanide free synthesis of an aromatic nitrile from a biorenewable based Aldoxime development and application of a recombinant Aldoxime dehydratase as a biocatalyst
Biocatalysis and Biotransformation, 2019Co-Authors: Ji Eun Choi, Harald Groger, Suguru Shinoda, Risa Inoue, Daijun Zheng, Yasuhisa AsanoAbstract:An Escherichia coli host microorganism was used for the production of the Aldoxime dehydratase from Rhodococcus sp. YH3-3 (OxdYH3-3), which showed good activity toward aromatic Aldoximes. Biotransf...
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Approaching Bulk Chemical Nitriles from Alkenes: A Hydrogen Cyanide-Free Approach through a Combination of Hydroformylation and Biocatalysis
2019Co-Authors: Carmen Plass, Yasuhisa Asano, Tobias Betke, Keiko Oike, Alessa Hinzmann, Michael Terhorst, Waldemar Brauer, Hilmi Yavuzer, Andreas J. Vorholt, Harald GrögerAbstract:A current challenge in catalysis is the development of methodologies for the production of bulk chemicals needed at levels of tens and hundreds of thousands of tons per year with the requirement to be produced at very low costs often being in the single-digit US dollar range. At the same time, such methodologies should address challenges raised by current manufacturing processes. Within this research area, a cyanide-free approach toward aliphatic nitriles used as industrial chemicals was developed starting from readily accessible n-alkenes as starting materials available in bulk quantities. This chemoenzymatic process concept is exemplified for the synthesis of nonanenitrile (as an n-/iso-mixture) and runs in water at low to moderate temperatures without the need for any types of cyanide sources. The process is based on a combination of a metal-catalyzed hydroformylation as the world-leading production technology for alkyl aldehydes with an emerging enzyme technology, namely, the recently developed transformation of Aldoximes into nitriles through dehydration by means of Aldoxime dehydratases. As a missing link, an efficient Aldoxime formation with subsequent removal of remaining traces of hydroxylamine as an enzyme-deactivating component was found, which enabled the merging of these three steps, hydroformylation, Aldoxime formation, and enzymatic dehydration, toward a nitrile synthesis without the need for purification of intermediates
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biocatalytic synthesis of nitriles through dehydration of Aldoximes the substrate scope of Aldoxime dehydratases
ChemBioChem, 2018Co-Authors: Yasuo Kato, Yasuhisa Asano, Tobias Betke, Jun Higuchi, Philipp Rommelmann, Keiko Oike, Taiji Nomura, Harald GrogerAbstract:Nitriles, which are mostly needed and produced by the chemical industry, play a major role in various industry segments, ranging from high-volume, low-price sectors, such as polymers, to low-volume, high-price sectors, such as chiral pharma drugs. A common industrial technology for nitrile production is ammoxidation as a gas-phase reaction at high temperature. Further popular approaches are substitution or addition reactions with hydrogen cyanide or derivatives thereof. A major drawback, however, is the very high toxicity of cyanide. Recently, as a synthetic alternative, a novel enzymatic approach towards nitriles has been developed with Aldoxime dehydratases, which are capable of converting an Aldoxime in one step through dehydration into nitriles. Because the Aldoxime substrates are easily accessible, this route is of high interest for synthetic purposes. However, whenever a novel method is developed for organic synthesis, it raises the question of substrate scope as one of the key criteria for application as a "synthetic platform technology". Thus, the scope of this review is to give an overview of the current state of the substrate scope of this enzymatic method for synthesizing nitriles with Aldoxime dehydratases. As a recently emerging enzyme class, a range of substrates has already been studied so far, comprising nonchiral and chiral Aldoximes. This enzyme class of Aldoxime dehydratases shows a broad substrate tolerance and accepts aliphatic and aromatic Aldoximes, as well as arylaliphatic Aldoximes. Furthermore, Aldoximes with a stereogenic center are also recognized and high enantioselectivities are found for 2-arylpropylAldoximes, in particular. It is further noteworthy that the enantiopreference depends on the E and Z isomers. Thus, opposite enantiomers are accessible from the same racemic aldehyde and the same enzyme.
Ben-zhan Zhu - One of the best experts on this subject based on the ideXlab platform.
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an unusual double radical homolysis mechanism for the unexpected activation of the Aldoxime nerve agent antidotes by polyhalogenated quinoid carcinogens under normal physiological conditions
Free Radical Biology and Medicine, 2019Co-Authors: Lin-na Xie, Chun-hua Huang, Balaraman Kalyanaraman, Jie Shao, Ben-zhan ZhuAbstract: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.
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a new detoxification mechanism for Aldoxime therapeutic antidotes for chemical warfare nerve agents
SCIENTIA SINICA Chimica, 2018Co-Authors: Furong Ren, Lin-na Xie, Chun-hua Huang, Xuan Xiao, Jie Shao, Ben-zhan ZhuAbstract:Pyridinium Aldoximes are best-known as therapeutic antidotes for organophosphorus chemical warfare nerve-agents and pesticides. Polyhalogenated quinones are a class of carcinogenic intermediates and newly identified chlorination disinfection byproducts in drinking water. However, it is not clear what is the exact chemical mechanism underlying such detoxication. Here we showed that pralidoxime, one of the representatives of pyridinium Aldoximes, can markedly enhance the dechlorination and hydroxylation of the highly reactive and toxic tetrachloro-1,4-benzoquinone (also called chloranil) in two-consecutive steps to produce the much less toxic 2,5-dichloro-3,6-dihydroxy-1,4-benzoquonine (chloranilic acid), with rate acceleration of up to 180000-times. In contrast, no enhancing effect was observed when the hydroxylamine group was blocked via methylation to form O -methylated pralidoxime. The major reaction product from pralidoxime was characterized as its corresponding nitrile (2-cyano-1-methylpyridinium chloride). Along with oxygen-18 isotope-labeling studies, we proposed that nucleophilic substitution coupled with an unprecedented double Beckmann fragmentation reaction was responsible for this dramatic enhancement of the detoxification reaction. This paper reviewed the unprecedented double Beckmann fragmentation reaction. Our findings may have broad biological and environmental implications for future research on the Aldoxime therapeutic agents and carcinogenic polyhalogenated quinones, which are two important classes of compounds of major biomedical and environmental interest.
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an unexpected double radical homolysis mechanism for the reactions between the pyridinium Aldoxime nerve agent antidotes and polyhalogenated quinones under normal physiological conditions
Free Radical Biology and Medicine, 2018Co-Authors: Ben-zhan ZhuAbstract: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 remains not clear why pralidoxime (2-PAM) cannot provide full protection against TCBQ-induced biological damage 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 2-PAM isotope-labeling studies. The end product of iminyl radical was isolated and identified as its corresponding reactive and toxic aldehyde. We proposed that the reaction of 2-PAM and TCBQ might be via the following two competing mechanisms: double Beckmann fragmentation vs double radical homolysis. 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 the potential side-toxic effects induced by 2-PAM and other pyridinium Aldoxime nerve-agent antidotes.
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Unusual Double Beckmann Fragmentation Reaction under Physiological Conditions
2017Co-Authors: Lin-na Xie, Chun-hua Huang, Jun-ge Zhu, Chen Shen, Bo Shao, Hui-ying Gao, Balaraman Kalyanaraman, Ben-zhan ZhuAbstract: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
Isabel Diaz - One of the best experts on this subject based on the ideXlab platform.
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stripping voltammetric determination of pyridine 2 Aldoxime methochloride at the iron iii doped zeolite modified glassy carbon electrode
Analyst, 2012Co-Authors: Solomon Mehretie, Jose Losada, Merid Tessema, Shimelis Admassie, Theodros Solomon, Joaquin Perezpariente, Isabel DiazAbstract:An iron(III) doped zeolite modified glassy carbon electrode was constructed for the determination of pyridine-2-Aldoxime methochloride. X-ray diffraction and chemical analysis were utilized to determine the optimum pH and chemical content for doping zeolite. Cyclic voltammetry was used to characterize the modified electrode and study the kinetics of the acid treated and untreated modified electrode. Acid treatment of the modified electrode showed a better electrochemical behavior compared to the untreated iron(III) doped zeolite modified electrode. Square wave anodic stripping voltammetry was employed to investigate the working pH and preconcentration time. The analytical performance of the modified electrode was evaluated, and a linear anodic stripping response for pyridine-2-Aldoxime methochloride in the concentration range of 0.5-100.0 μM with a detection limit of 1.61 × 10(-7) M was obtained. Finally, the developed method was successfully applied for the determination of pyridine-2-Aldoxime methochloride in a biological sample.
Michihiko Kobayashi - One of the best experts on this subject based on the ideXlab platform.
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new function of Aldoxime dehydratase redox catalysis and the formation of an expected product
PLOS ONE, 2017Co-Authors: Masatoshi Yamada, Yoshiteru Hashimoto, Takuto Kumano, Seiya Tsujimura, Michihiko KobayashiAbstract:In general, hemoproteins are capable of catalyzing redox reactions. Aldoxime dehydratase (OxdA), which is a unique heme-containing enzyme, catalyzes the dehydration of Aldoximes to the corresponding nitriles. Its reaction is a rare example of heme directly activating an organic substrate, unlike the utilization of H2O2 or O2 as a mediator of catalysis by other heme-containing enzymes. While it is unknown whether OxdA catalyzes redox reactions or not, we here for the first time detected catalase activity (which is one of the redox activities) of wild-type OxdA, OxdA(WT). Furthermore, we constructed a His320 → Asp mutant of OxdA [OxdA(H320D)], and found it exhibits catalase activity. Determination of the kinetic parameters of OxdA(WT) and OxdA(H320D) revealed that their Km values for H2O2 were similar to each other, but the kcat value of OxdA(H320D) was 30 times higher than that of OxdA(WT). Next, we examined another redox activity and found it was the peroxidase activity of OxdAs. While both OxdA(WT) and OxdA(H320D) showed the activity, the activity of OxdA(H320D) was dozens of times higher than that of OxdA(WT). These findings demonstrated that the H320D mutation enhances the peroxidase activity of OxdA. OxdAs (WT and H320D) were found to catalyze another redox reaction, a peroxygenase reaction. During this reaction of OxdA(H320D) with 1-methoxynaphthalene as a substrate, surprisingly, the reaction mixture changed to a color different from that with OxdA(WT), which was due to the known product, Russig's blue. We purified and identified the new product as 1-methoxy-2-naphthalenol, which has never been reported as a product of the peroxygenase reaction, to the best of our knowledge. These findings indicated that the H320D mutation not only enhanced redox activities, but also significantly altered the hydroxylation site of the substrate.
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crystal structure of Aldoxime dehydratase and its catalytic mechanism involved in carbon nitrogen triple bond synthesis
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Junpei Nomura, Kenichi Oinuma, Yoshiteru Hashimoto, Takehiro Ohta, Hiroshi Hashimoto, Koichi Wada, Yoshinori Naruta, Michihiko KobayashiAbstract:Aldoxime dehydratase (OxdA), which is a unique heme protein, catalyzes the dehydration of an Aldoxime to a nitrile even in the presence of water in the reaction mixture. Unlike the utilization of H2O2 or O2 as a mediator of catalysis by other heme-containing enzymes (e.g., P450), OxdA is notable for the direct binding of a substrate to the heme iron. Here, we determined the crystal structure of OxdA. We then constructed OxdA mutants in which each of the polar amino acids lying within ∼6 A of the iron atom of the heme was converted to alanine. Among the purified mutant OxdAs, S219A had completely lost and R178A exhibited a reduction in the activity. Together with this finding, the crystal structural analysis of OxdA and spectroscopic and electrostatic potential analyses of the wild-type and mutant OxdAs suggest that S219 plays a key role in the catalysis, forming a hydrogen bond with the substrate. Based on the spatial arrangement of the OxdA active site and the results of a series of mutagenesis experiments, we propose the detailed catalytic mechanism of general Aldoxime dehydratases: (i) S219 stabilizes the hydroxy group of the substrate to increase its basicity; (ii) H320 acts as an acid-base catalyst; and (iii) R178 stabilizes the heme, and would donate a proton to and accept one from H320.
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discovery of a reaction intermediate of aliphatic Aldoxime dehydratase involving heme as an active center
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Kazunobu Konishi, Kenichi Oinuma, Yoshiteru Hashimoto, Takehiro Ohta, Teizo Kitagawa, Michihiko KobayashiAbstract:Recently, we discovered an intriguing hemoprotein [aliphatic Aldoxime dehydratase (OxdA)] that catalyzes the dehydration of aliphatic Aldoximes [R–CH=N–OH] to the corresponding nitriles [R–C≡N] in the industrial Pseudomonas chlororaphis B23 strain. Unlike the utilization of H2O2 or O2 as a mediator of the catalysis by other heme-containing enzymes (e.g., P450), OxdA is notable for the direct binding of a substrate to the heme iron, experimental evidence of which was obtained here by means of resonance Raman (RR) analysis with an isotope technique. We found that the addition of a large amount of butyrAldoxime (final concentration, 200 mM) to ferrous OxdA with a low enzyme concentration (final concentration, 5 μM) yields a long-lived OxdA–substrate complex (named OS-II), whose UV-vis spectrum is different from the corresponding spectra of the OxdA–substrate complex I and CO-bound, ferrous, and ferric forms of OxdA. Intriguingly, the RR analysis demonstrated that OS-II includes a highly oxidized heme with strong bonding between a substrate and the heme iron, as judged from the heme oxidation state marker ν4 band at 1,379 cm–1 and the 15N-isotope-substituted butyrAldoxime sensitive band at 857 cm–1 in the RR spectra. It is noteworthy that OS-II has a highly oxidized heme like the ferryl-oxo heme species (e.g., compound II) formed by some general hemoproteins, although the function of OxdA is different from those (transport of electrons, transport of oxygen, sensing of oxygen or carbon monoxide, and catalysis of redox reactions) of general hemoproteins.
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identification of crucial histidines involved in carbon nitrogen triple bond synthesis by Aldoxime dehydratase
Journal of Biological Chemistry, 2004Co-Authors: Kazunobu Konishi, Kenichi Oinuma, Yoshiteru Hashimoto, Hiroki Higashibata, Kyoko Ishida, Takehiro Ohta, Teizo Kitagawa, Michihiko KobayashiAbstract:Aldoxime dehydratase (OxdA), which is a novel heme protein, catalyzes the dehydration of an Aldoxime to a nitrile even in the presence of water in the reaction mixture. The combination of site-directed mutagenesis of OxdA (mutation of all conserved histidines in the Aldoxime dehydratase superfamily), estimation of the heme contents and specific activities of the mutants, and CD and resonance Raman spectroscopic analyses led to the identification of the proximal and distal histidines in this unique enzyme. The heme contents and CD spectra in the far-UV region of all mutants except for the H299A one were almost identical to those of the wild-type OxdA, whereas the H299A mutant lost the ability of binding heme, demonstrating that His(299) is the proximal histidine. On the other hand, substitution of alanine for His(320) did not affect the overall structure of OxdA but caused loss of its ability of carbon-nitrogen triple bond synthesis and a lower shift of the Fe-C stretching band in the resonance Raman spectrum for the CO-bound form. Furthermore, the pH dependence of the wild-type OxdA closely followed the His protonation curves observed for other proteins. These findings suggest that His(320) is located in the distal heme pocket of OxdA and would donate a proton to the substrate in the Aldoxime dehydration mechanism.
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heme environment in Aldoxime dehydratase involved in carbon nitrogen triple bond synthesis
FEBS Letters, 2004Co-Authors: Kenichi Oinuma, Yoshiteru Hashimoto, Kazunobu Konishi, Hiroki Higashibata, Takehiro Ohta, Teizo Kitagawa, Michihiko KobayashiAbstract:Resonance Raman spectra have been measured to characterize the heme environment in Aldoxime dehydratase (OxdA), a novel hemoprotein, which catalyzes the dehydration of Aldoxime into nitrile. The spectra showed that the ferric heme in the enzyme is six-coordinate low spin, whereas the ferrous heme is five-coordinate high spin. We assign a prominent vibration that occurs at 226 cm−1 in the ferrous enzyme to the Fe-proximal histidine stretching vibration. In the CO-bound form of OxdA, the correlation between the Fe–CO stretching (512 cm−1) and C–O stretching (1950 cm−1) frequencies also supports our assignment of proximal histidine coordination.
Lin-na Xie - One of the best experts on this subject based on the ideXlab platform.
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structure activity relationship investigation on reaction mechanism between chlorinated quinoid carcinogens and clinically used Aldoxime nerve agent antidote under physiological condition
Chemical Research in Toxicology, 2021Co-Authors: Lin-na Xie, Chun-hua Huang, Li Qin, Guoqiang Shan, Zhisheng Liu, Dong Cao, Fanglan Geng, Li MaoAbstract: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.
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an unusual double radical homolysis mechanism for the unexpected activation of the Aldoxime nerve agent antidotes by polyhalogenated quinoid carcinogens under normal physiological conditions
Free Radical Biology and Medicine, 2019Co-Authors: Lin-na Xie, Chun-hua Huang, Balaraman Kalyanaraman, Jie Shao, Ben-zhan ZhuAbstract: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.
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a new detoxification mechanism for Aldoxime therapeutic antidotes for chemical warfare nerve agents
SCIENTIA SINICA Chimica, 2018Co-Authors: Furong Ren, Lin-na Xie, Chun-hua Huang, Xuan Xiao, Jie Shao, Ben-zhan ZhuAbstract:Pyridinium Aldoximes are best-known as therapeutic antidotes for organophosphorus chemical warfare nerve-agents and pesticides. Polyhalogenated quinones are a class of carcinogenic intermediates and newly identified chlorination disinfection byproducts in drinking water. However, it is not clear what is the exact chemical mechanism underlying such detoxication. Here we showed that pralidoxime, one of the representatives of pyridinium Aldoximes, can markedly enhance the dechlorination and hydroxylation of the highly reactive and toxic tetrachloro-1,4-benzoquinone (also called chloranil) in two-consecutive steps to produce the much less toxic 2,5-dichloro-3,6-dihydroxy-1,4-benzoquonine (chloranilic acid), with rate acceleration of up to 180000-times. In contrast, no enhancing effect was observed when the hydroxylamine group was blocked via methylation to form O -methylated pralidoxime. The major reaction product from pralidoxime was characterized as its corresponding nitrile (2-cyano-1-methylpyridinium chloride). Along with oxygen-18 isotope-labeling studies, we proposed that nucleophilic substitution coupled with an unprecedented double Beckmann fragmentation reaction was responsible for this dramatic enhancement of the detoxification reaction. This paper reviewed the unprecedented double Beckmann fragmentation reaction. Our findings may have broad biological and environmental implications for future research on the Aldoxime therapeutic agents and carcinogenic polyhalogenated quinones, which are two important classes of compounds of major biomedical and environmental interest.
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Unusual Double Beckmann Fragmentation Reaction under Physiological Conditions
2017Co-Authors: Lin-na Xie, Chun-hua Huang, Jun-ge Zhu, Chen Shen, Bo Shao, Hui-ying Gao, Balaraman Kalyanaraman, Ben-zhan ZhuAbstract: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