Copper-catalyzed aerobic aliphatic C-H oxygenation with hydroperoxides.
ABSTRACT: We report herein Cu-catalyzed aerobic oxygenation of aliphatic C-H bonds with hydroperoxides, which proceeds by 1,5-H radical shift of putative oxygen-centered radicals (O-radicals) derived from hydroperoxides followed by trapping of the resulting carbon-centered radicals with molecular oxygen.
Project description:Hydroxyl radical footprinting is a technique for studying protein structure and binding that entails oxidizing a protein system of interest with diffusing hydroxyl radicals, and then measuring the amount of oxidation of each amino acid. One important issue in hydroxyl radical footprinting is limiting amino acid oxidation by secondary oxidants to prevent uncontrolled oxidation, which can cause amino acids to appear more solvent accessible than they really are. Previous work suggested that hydrogen peroxide was the major secondary oxidant of concern in hydroxyl radical footprinting experiments; however, even after elimination of all hydrogen peroxide, some secondary oxidation was still detected. Evidence is presented for the formation of peptidyl hydroperoxides as the most abundant product upon oxidation of aliphatic amino acids. Both reverse phase liquid chromatography and catalase treatment were shown to be ineffective at eliminating peptidyl hydroperoxides. The ability of these peptidyl hydroperoxides to directly oxidize methionine is demonstrated, suggesting the value of methionine amide as an in situ protectant. Hydroxyl radical footprinting protocols require the use of an organic sulfide or similar peroxide scavenger in addition to removal of hydrogen peroxide to successfully eradicate all secondary oxidizing species and prevent uncontrolled oxidation of sulfur-containing residues.
Project description:Exposure of amino acids, peptides and proteins to radicals, in the presence of oxygen, gives high yields of hydroperoxides. These materials are readily decomposed by transition metal ions to give further radicals. We hypothesized that hydroperoxide formation on nuclear proteins, and subsequent decomposition of these hydroperoxides to radicals, might result in oxidative damage to associated DNA. We demonstrate here that exposure of histone H1 and model compounds to gamma-radiation in the presence of oxygen gives hydroperoxides in a dose-dependent manner. These hydroperoxides decompose to oxygen- and carbon-centred radicals (detected by electron paramagnetic resonance spectroscopy) on exposure to Cu(+) and other transition metal ions. These hydroperoxide-derived radicals react readily with pyrimidine DNA bases and nucleosides to give adduct species (i.e. protein-DNA base cross-links). Product analysis has demonstrated that radicals from histone H1-hydroperoxides, and other protein and amino acid hydroperoxides, can also oxidize both free 2'-deoxyguanosine and intact calf thymus DNA to give the mutagenic oxidized base 7, 8-dihydro-8-oxo-2'-deoxyguanosine (8-hydroxy-2'-deoxyguanosine, 8-oxodG). The yield of 7,8-dihydro-8-oxo-2'-deoxyguanosine is proportional to the initial protein-hydroperoxide concentration, and corresponds (for histone H1-hydroperoxide, 280 microM) to approx. 1. 4% conversion for free 2'-deoxyguanosine (200 microM), and 0.14% for 2'-deoxyguanosine in DNA (70 microgram/ml). Evidence has also been obtained with DNA for reaction at cytosine and thymine, but not adenine; the lack of damage to the latter may result from damage transfer to 2'-deoxyguanosine residues. These studies demonstrate that initial radical-induced damage to nuclear proteins can give rise to subsequent DNA damage; the latter includes both DNA-protein cross-links and formation of oxidized DNA bases.
Project description:Proteins damaged by free-radical-generating systems in the presence of oxygen yield relatively long-lived protein hydroperoxides. These hydroperoxides have been shown by e.p.r. spectroscopy to be readily degraded to reactive free radicals on reaction with iron(II) complexes. Comparison of the observed spectra with those obtained with free amino acid hydroperoxides had allowed identification of some of the protein-derived radical species (including a number of carbon-centred radicals, alkoxyl radicals and a species believed to be the CO2 radical anion) and the elucidation of novel fragmentation and rearrangement processes involving amino acid side chains. In particular, degradation of hydroperoxide functions on the side chain of glutamic acid is shown to result in decarboxylation at the side-chain carboxy group via the formation of the CO2 radical anion; the generation of an identical radical from hydroperoxide groups on proteins suggests that a similar process occurs with these molecules. In a number of cases these fragmentation and rearrangement reactions give rise to further reactive free radicals (R., O2-./HO2., CO2-.) which may act as chain-carrying species in protein oxidations. These studies suggest that protein hydroperoxides are capable of initiating further radical chain reactions both intra- and inter-molecularly, and provide information on some of the fundamental mechanisms of protein alteration and side-chain fragmentation.
Project description: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, (1)H-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.
Project description:Spin trapping using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has been used to detect and distinguish between the carbon-centred, alkoxyl, and peroxyl radicals produced during the photolytic decomposition of hydroperoxides. Photolysis of tert-butyl and cumene hydroperoxides, and peroxidized fatty acids, in toluene, with low levels of u.v. light, is shown to lead to the initial production of alkoxyl radicals by homolysis of the oxygen-oxygen bond. Subsequent reaction of these radicals with excess hydroperoxide leads, by hydrogen abstraction, to the production of peroxyl radicals that can be detected as their corresponding adducts with the spin trap. Subsequent breakdown of these adducts produces alkoxyl radicals and a further species that is believed to be the oxidized spin-trap radical 5,5-dimethyl-1-pyrrolidone-2-oxyl. No evidence was obtained at low hydroperoxide concentrations, with either the cumene or lipid alkoxyl radicals, for the occurrence of beta-scission reactions; the production of low levels of carbon-centred radicals is believed to be due to the alternative reactions of hydrogen abstraction, ring closure, and/or 1,2 hydrogen shifts. Analogous experiments with 3,3,5,5-tetramethyl-1-pyrroline N-oxide (TMPO) led only to the trapping of alkoxyl radicals with no evidence for peroxyl radical adducts, this is presumably due to a decreased rate of radical addition because of increased steric hindrance.
Project description:The site selectivities of intermolecular, aliphatic C-H bond functionalizations are central to the value of these transformations. While the scope of these reactions continues to expand, the site selectivities remain largely dictated by the inherent reactivity of the substrate C-H bonds. Herein, we introduce reagent-dictated site selectivity to intermolecular aliphatic C-H functionalizations using nitrogen-centered amidyl radicals. Simple modifications of the amide lead to high levels of site selectivity in intermolecular C-H functionalizations across a range of simple and complex substrates. DFT calculations demonstrate that the steric demand of the reacting nitrogen-centered radical is heavily affected by the substitution pattern of the starting amide. Optimization of transition state structures consistently indicated higher reagent-dictated steric selectivities using more hindered amides, consistent with experimental results.
Project description:Masked alcohols are particularly appealing as directing groups because of the ubiquity of hydroxy groups in organic small molecules. Herein, we disclose a general strategy for aliphatic ?-C(sp3 )-H functionalization guided by a masked alcohol. Specifically, we determine that sulfamate ester derived nitrogen-centered radicals mediate 1,6-hydrogen-atom transfer (HAT) processes to guide ?-C(sp3 )-H chlorination. This reaction proceeds through a light-initiated radical chain-propagation process and is capable of installing chlorine atoms at primary, secondary, and tertiary centers.
Project description:The chemistry underlying superoxide toxicity is not fully understood. A potential mechanism for superoxide-mediated injury involves addition to tyrosyl radicals, to give peptide or protein hydroperoxides. The rate constant for the reaction of tyrosyl radicals with superoxide is higher than for dimerization, but the efficiency of superoxide addition to peptides depends on the position of the Tyr residue. We have examined the requirements for superoxide addition and structurally characterized the products for a range of tyrosyl peptides exposed to a peroxidase/O(2)(.) system. These included enkephalins as examples of the numerous proteins and physiological peptides with N-terminal tyrosines. The importance of amino groups in promoting hydroperoxide formation and effect of methionine residues on the reaction were investigated. When tyrosine was N-terminal, the major products were hydroperoxides that had undergone cyclization through conjugate addition of the terminal amine. With non-N-terminal tyrosine, electron transfer from O(2)(.) to the peptide radical prevailed. Peptides containing methionine revealed a novel and efficient intramolecular oxygen transfer mechanism from an initial tyrosine hydroperoxide to give a dioxygenated derivative with one oxygen on the tyrosine and the other forming methionine sulfoxide. Exogenous amines promoted hydroperoxide formation on tyrosyl peptides lacking a terminal amine, without forming an adduct. These findings, plus the high hydroperoxide yields with N-terminal tyrosine, can be explained by a mechanism in which hydrogen bonding of O(2)(.) to the amine increases is oxidizing potential and alters its reactivity. If this amine effect occurred more generally, it could increase the biological reactivity of O(2)(.) and have major implications.
Project description:The Mn(III)-initiated aerobic oxidation of heterocyclic 1,3-dicarbonyl compounds, such as 4-alkyl-1,2-diphenylpyrazolidine-3,5-diones, 1,3-dialkylpyrrolidine-2,4-diones, 3-alkyl-1,5-dimethylbarbituric acids, and 3-butyl-4-hydroxy-2-quinolinone gave excellent to good yields of the corresponding hydroperoxides, which were gradually degraded by exposure to the metal initiator after the reaction to afford the corresponding alcohols. The synthesis of 30 heterocyclic 1,3-dicarbonyl compounds, the corresponding hydroperoxides and the 10 alcohols, their characterization, and the limitations of the procedure are described. In addition, the mechanism of the hydroperoxidation and the redox decomposition of the hydroperoxides are discussed.
Project description:The in vivo metabolism of plasma lipids generates lipid hydroperoxides that, upon one-electron reduction, give rise to a wide spectrum of genotoxic unsaturated aldehydes and epoxides. These metabolites react with cellular DNA to form a variety of pre-mutagenic DNA lesions. The mechanisms of action of the radical precursors of these genotoxic electrophiles are poorly understood. In this work we investigated the nature of DNA products formed by a one-electron reduction of (13S)-hydroperoxy-(9Z,11E)-octadecadienoic acid (13S-HPODE), a typical lipid molecule, and the reactions of the free radicals thus generated with neutral guanine radicals, G(-H)(*). A novel approach was devised to generate these intermediates in solution. The two-photon-induced ionization of 2-aminopurine (2AP) within the 2'-deoxyoligonucleotide 5'-d(CC[2AP]TCGCTACC) by intense nanosecond 308 nm excimer laser pulses was employed to simultaneously generate hydrated electrons and radical cations 2AP(*+). The latter radicals either in cationic or neutral forms, rapidly oxidize the nearby G base to form G(-H)(*). In deoxygenated buffer solutions (pH 7.5), the hydrated electrons rapidly reduce 13S-HPODE and the highly unstable alkoxyl radicals formed undergo a prompt beta-scission to pentyl radicals that readily combine with G(-H)(*). Two novel guanine products in these oligonucleotides, 8-pentyl- and N(2)-pentylguanine, were identified. It is shown that the DNA secondary structure significantly affects the ratio of 8-pentyl- and N(2)-pentylguanine lesions that changes from 0.9:1 in single-stranded, to 1:0.2 in double-stranded oligonucleotides. The alkylation of guanine by alkyl radicals derived from lipid hydroperoxides might contribute to the genotoxic modification of cellular DNA under hypoxic conditions. Thus, further research is warranted on the detection of pentylguanine lesions and other alkylguanines in vivo.