Autoxidation Products Research Articles

Synthesis of sesquiterpene polyene hydroperoxides by regio- and stereoselective transposition reactions

Isomeric unsaturated alcohols ( 8), ( 16), ( 18) and ( 20), on exposure to hydrogen peroxide or water in the presence of an acid catalyst, undergo exceptionally regio- and stereoselective substitution reactions to form hydroperoxide ( 3) and alcohol ( 8) respectively. E Z isomeric ratios of transposition products indicate that the intermediate acyclic carbocations ( 21) and ( 22) are not interconverting under the reaction conditions. Practical syntheses of α-farnesene autoxidation products have been developed based on these transposition reactions.

Autoxidation of the furan fatty acid ester, methyl 9,12‐epoxyoctadeca‐9,11‐dienoate

AbstractThe objective of this study was to identify autoxidation products of methyl 9,12‐epoxyoctadeca‐9,11‐dienoate (F9,12). Previous work has shown that F9,12 is a product both of autoxidation and singlet oxygen oxidation of the methyl ester derivative of conjugated linoleic acid (CLA). F9,12, 95% pure, was synthesized from methyl ricinoleate. The synthetic F9,12 was heated at 50°C in sealed tubes containing air. Each tube contained 6 mg F9,12 and 1 mg methyl stearate as an internal standard. Samples were taken at 4.5, 7, 23, 46.5, 69.5, and 93 h. The oxidized F9,12 was dissolved in isooctane and analyzed by gas chromatography (GC), GC‐direct deposition‐Fourier transform infrared spectroscopy, and GC‐electron ionization mass spectrometry. CLA methyl ester was oxidized in a similar manner. Under these conditions, the half‐lives of CLA and F9,12 were 40 and 35 h, respectively. Oxidation products of F9,12 that were identified included: 5‐hexyl‐2‐furaldehyde (I), methyl 8‐oxooctanoate (II), methyl 13‐oxo‐9,12‐epoxytrideca‐9,11‐dienoate (III), methyl 8‐oxo‐9,12‐epoxy‐9,11‐octadecadienoate (IV), and methyl 13‐oxo‐9,12‐epoxy‐9,11‐octadecadienoate (V).

Characterization of model triacylglycerol (triolein, trilinolein and trilinolenin) autoxidation products via high-performance liquid chromatography coupled with atmospheric pressure chemical ionization mass spectrometry

Oxidation products from the autoxidation of three triacylglycerol standards have been analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to mass spectrometry via an atmospheric pressure chemical ionization (APCI) source. Triolein, trilinolein and trilinolenin were autoxidized in the dark at 50–60°C until the oxidation products represented approximately 30% of the starting material. These oxidation product mixtures were then analyzed using RP-HPLC–APCI-MS. Several classes of oxidation products were directly detected and identified. Monohydroperoxides were present in the largest amounts in the oxidation products mixtures. The hydroperoxides were found to provide several structurally useful fragments: epoxide intermediates were formed which then underwent further fragmentation, and other fragments were formed from concerted loss of the hydroperoxide group to form a site of unsaturation. Fragments formed by intra-annular cleavage of epoxide intermediates allowed identification of several hydroperoxide isomers. Bishydroperoxides were observed which underwent similar fragmentation pathways. Mono- and diepoxides were also formed by the autoxidation reaction. Two classes of epoxides were observed: those in which an epoxide formed in place of an existing double bond, and those in which an epoxide formed away from a double bond. Two distinct fragmentation mechanisms were observed for epoxides which were not formed across a double bond. Other oxidation products which were observed included hydroxy trilinolenin, epidioxy trilinolenin and hydroperoxy, epidioxy trilinolenin.

Formation of formaldehyde and peroxides by air oxidation of high purity polyoxyethylene surfactants.

Ethoxylated alcohols are non-ionic surfactants. The majority are used in household cleaners, laundry products, toiletries and in industrial and institutional cleaners. In previous studies, an ethoxylated non-ionic surfactant of technical quality showed allergenic activity in guinea pig experiments. Chemical analysis revealed a content of formaldehyde, a well-known contact allergen, and peroxides in the surfactant. Most cases of occupational contact dermatitis are considered to be of irritant origin, caused by contact with water and surfactants, but if allergenic autoxidation products can be formed, allergic contact dermatitis cannot be excluded. The sensitizing potential of a chemically defined high purity ethoxylated alcohol was investigated and oxidation under various storage and handling conditions was studied for this and a homologous product. The pure surfactant showed no significant allergenic activity on predictive testing in guinea pigs. When ethoxylated alcohols were stored in the refrigerator, their deterioration was limited. At room temperature, their content of peroxides and formaldehyde increased with time. Levels of formaldehyde above those capable of causing positive patch test reactions were found. Since such surfactants have wide applications, resulting exposure to formaldehyde could be more frequent than is generally realized, contributing to persistence of dermatitis in individuals allergic to formaldehyde.

O3-induced formation of bioactive lipids: estimated surface concentrations and lining layer effects.

Recent evidence suggests that inhaled ozone (O3) does not induce toxicity via direct epithelial interactions. Reactions with epithelial lining fluid (ELF) constituents limit cellular contact and generate products, including lipid ozonation products, postulated to initiate pathophysiological cascades. To delineate specific aspects of lipid ozonation product formation and to estimate in situ surface concentrations, we studied the O3 absorption characteristics of ELF constituent mixtures and measured hexanal, heptanal, and nonanal yields as a function of ascorbic acid (AH2) concentration. Exposures of isolated rat lungs, bronchoalveolar lavage fluid (BALF) and egg phosphatidylcholine (PC) liposomes were conducted. 1) O3 absorption by AH2, uric acid, and albumin exceeded that by egg PC and glutathione. O3 reaction with egg PC occurred when AH2 concentrations were reduced. 2) Aldehydes were produced in low yield during lung and BALF exposures in a time- and O3 concentration-dependent manner. 3) Diminishing BALF AH2 content lowered O3 uptake but increased aldehyde yields. Conversely, AH2 addition to egg PC increased O3 uptake but reduced aldehyde yields. Estimations of bioactive ozonation and autoxidation product accumulation within the ELF suggested possible nanomolar to low micromolar concentrations. The use of reaction products as metrics of O3 exposure may have intrinsic sensitivity and specificity limitations. Moreover, due to the heterogenous nature of O3 reactions within the ELF, dose-response relationships may not be linear with respect to O3 absorption.

Functional and structural successions in arbitrary samples of heterotrophic bacteria during aerobic treatments of lignite-carbonization wastewater inin situenclosures

In situ mesocosm experiments were performed in Lake Schwelvollert (located in the district of Weissenfels, Saxony-Anhalt, Germany), an anaerobic lignite-carbonization effluent lake containing phenolic compounds and their autoxidation products (anthropogenic humic matter). In the aeration enclosure, the anaerobic Schwelvollert wastewater was aerated and in the flocculation enclosure, it was flocculated to precipitate the oxygen-trapping anthropogenic humic matter to enhance the input of oxygen by diffusion. To gain an insight into the metabolic state of the aerobic heterotrophic microbiota during the treatments, arbitrary samples of bacterial isolates were taken from a general agar medium and tested for their abilities to cleave predominant phenolic contaminants by a procedure called the isolate sample assay. In this way, successions of degradation potentials were observed in both mesocosms, with degradation abilities for meta- and para-alkylated phenols appearing before degradation abilities for ortho-substituted phenols as a common phenomenon. To examine the structure of samples, the respective isolates were characterized using the Biolog GN MicroPlate system, the random amplified polymorphic DNA nucleic acid (RAPD) fingerprinting technique, and amplified ribosomal DNA restriction analysis (ARDRA). Although similar functional patterns occurred in both mesocosms, the compositions and diversities of the respective bacterial communities varied significantly, even at different depths from the same enclosure, with members of the Pseudomonas RNA group I being predominant.

THE PREPARATION OF α-FARNESENE HYDROPEROXIDES FOR THE STUDY OF SUPERFICIAL SCALD

The short and efficient synthesis of four α-farnesene autoxidation products is described. Geraniol was converted to the conjugated trienyl hydroperoxide (3) via the known trienol (2). Trienyl hydroperoxide (3) was efficiently cyclised in the presence of oxygen and an intiator to afford the very labile endoperoxy hydroperoxide (5), a route which mimics α-farnesene autoxidation. Reduction of (5) gave the endoperoxy alcohol (4).

Role of oxygen derived radicals for vascular dysfunction in the diabetic heart: prevention by alpha-tocopherol?

The evidence that the generation of reactive oxygen intermediates (ROI) plays an important role for the increased cardiovascular risk in diabetes is summarised. In addition to the well known parameters of oxidative stress as lipid hydroperoxides and thiobarbituric acid substances (TBARS), recent observations indicate that isoprostanes which can be taken as a more specific parameter of oxidative stress, are generated in higher amounts by diabetic patients. This increased formation of isoprostanes can be inhibited by an installment of a close metabolic control or the supplementation with tocopherol. The cause for the elevated oxidative stress is not yet fully understood, however the autoxidation of glucose, the formation of advanced glycation endproducts and the activation of NADPH-oxidase seem to be relevant processes. Since ROI are able to quench nitric oxide and to inhibit the synthesis of prostacyclin, the antithrombotic, vasodilating and antiatherosclerotic properties of endothelium are impaired in diabetes. Additionally, the balance of endothelial mediators released by endothelium is shifted to angiotensin II and endothelin, compounds which enhance the proliferation of smooth muscle cells and may limit the coronary reserve of myocardium. The activation of the transcription factor NF-kappa B by glucose and its autoxidative products is regarded as a key event in the transformation of the vasculature in diabetes. Epidemiological observations and very recent clinical studies underlie the impact of ROI for the development of cardiovascular complications in diabetes and suggest that an antioxidative treatment might be helpful to reduce the cardiac risk in diabetes.

Irreversible inhibition of mitochondrial complex I by 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxyli c acid (DHBT-1): a putative nigral endotoxin of relevance to Parkinson's disease.

Based on a number of lines of evidence, we have proposed recently that a very early step in the pathogenesis of idiopathic Parkinson's disease might be elevated translocation of L-cysteine into neuromelanin-pigmented dopaminergic cell bodies in the substantia nigra. In vitro studies suggest that such an influx of L-cysteine would divert the neuromelanin pathway by scavenging dopamine-o-quinone, the proximate autoxidation product of dopamine, to give 5-S-cysteinyldopamine, which is oxidized further to 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1) and other cysteinyldopamines and dihydrobenzothiazines. In this study, it is demonstrated that DHBT-1 inhibits ADP-stimulated oxidation of malate and pyruvate (state 3 or complex I respiration) when incubated with intact rat brain mitochondria with an IC50 of approximatelly 0.80 mM. Incubation of DHBT-1 with freeze-thawed rat brain mitochondria in both the presence and absence of KCN and/or NADH causes an irreversible, time-dependent decrease of NADH-coenzyme Q1 reductase activity. Significantly lower concentrations of DHBT-1 are necessary to cause this effect when mitochondrial membranes are incubated in the absence of KCN and NADH. The irreversible inhibition of mitochondrial complex I caused by DHBT-1 under the latter conditions could be blocked only partially by glutathione, ascorbic acid, superoxide dismutase, or catalase. Together, these results suggest that DHBT-1 can cross the outer mitochondrial membrane and irreversibly inhibit complex I by a mechanism that is not primarily related to oxygen radical-mediated damage. Formation of DHBT-1 requires only dopamine, L-cysteine, and an oxidizing environment, conditions that may well exist in the cytoplasm of neuromelanin-pigmented dopaminergic neurons in the parkinsonian substantia nigra. The results of this study raise the possibility that DHBT-1 might be an endotoxin formed specifically in pigmented dopaminergic neurons that can contribute to irreversible damage to mitochondrial complex I and substantia nigra cell death in Parkinson's disease.

Autoxidation of synthetic isomers of triacylglycerol containing eicosapentaenoic acid

AbstractSeveral triacylglycerols (TAG) that contained eicosapentaenoic acid (EPA) were chemically synthesized and stored at 25°C to assess the influence of TAG structure on oxidative stability and formation of oxidation products. Oxidative stability was evaluated by oxygen consumption during storage of the TAG. Autoxidation products of TAG were analyzed by high‐performance liquid chromatography (HPLC) and liquid chromatography‐mass spectrometry (LC‐MS). Results showed that a 2:1 (mole/mole) mixture of trieicosapentaenoylglycerol (EEE) and tripalmitoylglycerol (PPP) was most susceptible to autoxidation. The oxidative stability of TAG that contained EPA and palmitic acid was negatively correlated with the moles of EPA in a single TAG molecule. When TAG with one EPA and two other fatty acids were oxidized, chainlength of constituent fatty acids hardly affected the oxidative stability of EPA‐containing TAG molecules, except for stearic acid. HPLC and LC‐MS analyses showed that monohydroperoxides were major oxidation products regardless of type of TAG. Bis‐ and tris‐hydroperoxides were formed during autoxidation of EEE and dieicos‐apentaenoylpalmitoylglycerol. Monohydroperoxy epidioxides were found in all autoxidized TAG. These observations suggested that TAG structure affected the oxidation of TAG with highly unsaturated fatty acids.

A Novel Synthesis and the Autoxidation of 2,4,6,8‐Tetraphenylbarbaralane

AbstractAddition to the diphenyldiketone 11 of the reagent prepared from rigorously dried cerium(III) chloride and phenyllithium yields the tetraphenyldiol endo,endo‐13 after extended periods of time. The configuration of this diol, which was previously assigned the (incorrect) configuration exo,exo‐13, is established by an X‐ray diffraction analysis. Dehydration of endo,endo‐13 with sulphuric acid in acetic acid affords a mixture of the oxatwistane 16 and the tetraphenyldiene 17 (3:1). Only the latter is obtained from endo,endo‐13 by the action of boron trifluoride–diethyl ether in dichloromethane solution. While attempts at allylic bromination of 17 with N‐bromosuccinimide in conventional solvents inevitably lead to complete decomposition, use of cyclohexane as solvent allows to obtain solutions of allylic dibromides that can be cyclised with the zinc‐copper couple to afford tetraphenylbarbaralane 9 in 58% yield based on 17. Thus, 9 is now available from the diphenyldiketone 11 in only three steps with an overall yield of 42%. – The autoxidation of 9, studied in various solvents, yields mixtures of products of which the epoxyendoperoxides 20 and 21, the unsaturated ketone 22, and a labile, probably oligomeric or polymeric product C are isolated in pure form. The structures 21 and 22 are elucidated by X‐ray diffraction analysis and independent synthesis, respectively. On warming, 20 rearranges into 21. Traces of acid convert the unknown autoxidation product A instantaneously into the unsaturated ketone 22. – Rate studies show that the autoxidation of 9 starts after a short initiation period. 2,6‐Di‐tert‐butyl‐4‐methylphenol exhibits powerful inhibitory effects. These results demonstrate the free radical nature of the autoxidation of 9. – The results are interpreted in terms of a mechanistic scheme involving initiation of the radical chain by addition of triplet oxygen to 9 to generate the diradical 28 followed by a radical chain 1:1 copolymerisation of 9 and oxygen to produce diradical 29. Eventually, endo addition of oxygen to 29 gives rise to the formation of the epoxyendoperoxides 20 and 21. Intramolecular hydrogen abstraction from the methylene group (C9) is accompanied by cleavage of a bridgehead bond (C1–C2) and the adjacent O–O bond to afford the extremely labile product A (perhaps 32 or 33) which undergoes acid‐catalysed elimination to furnish ketone 22.

Isomerization, But Not Oxidation, Is Suppressed by a Single Point Mutation, E361Q, in the Reaction Catalyzed by Cholesterol Oxidase

The putative active site base of cholesterol oxidase from Streptomyces has been removed by site-directed mutagenesis and the mutant enzyme characterized. When glutamate-361 is mutated to a glutamine, the isomerization chemistry catalyzed by cholesterol oxidase is suppressed and the intermediate cholest-5-ene-3-one is isolated. The specific activity for oxidation is 20-fold slower than the wild-type reaction, though the specific activity for isomerization is 10 000-fold slower. Furthermore, incubation of cholest-5-ene-3-one with the E361Q cholesterol oxidase resulted in the production of cholest-4-ene-6β-hydroperoxy-3-one (6%), cholest-4-ene-3,6-dione (32%), cholest-4-ene-6β-ol-3-one (36%), and cholest-4-ene-6α-hydroperoxy-3-one/cholest-4-ene-6α-ol-3-one (13%), in addition to cholest-4-ene-3-one (13%). Measurement of reaction stoichiometry eliminated the possibility that H2O2 or the C4a-hydroperoxy flavin was the oxygenation agent. It is proposed that cholest-4-ene-6-hydroperoxy-3-one is the product of radical chain autoxidation and that cholest-4-ene-3,6-dione and cholest-4-ene-6-ol-3-one are decomposition products of the hydroperoxy steroid radical. The characterization of the E361Q mutant chemistry has illuminated the importance of intermediate sequestration in enzyme catalysis. The mutant enzyme will be used to obtain information about the structure of the enzyme in the presence of the reaction intermediate. Moreover, the altered activity of E361Q cholesterol oxidase will facilitate its application in studies of cell membranes.

Autoxidation of pyrrolylurethanes

Oxygen sensitive pyrrolylurethanes have been prepared and their autoxidation products isolated and characterized by 1H- and 13C-NMR.

Characterization of glutathione conjugates of pyrrolylated amino acids and peptides by liquid chromatography-mass spectrometry and tandem mass spectrometry with electrospray ionization.

High-performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ES-MS) and tandem mass spectrometry (MS-MS) was used to identify the products formed upon reaction of lysine-containing peptides with the neurotoxicant 2,5-hexanedione (2,5-HD). In addition, secondary autoxidative reaction products of the resultant alkylpyrroles with the biological thiol, glutathione, were characterized. ES mass spectra of the HPLC-separated conjugates showed intense [M+H]+ ions as well as several ions formed by amide and C-S bond cleavage. The glutathione conjugates of pyrrolylated amino acids and peptides were analyzed by ES ionization and MS-MS, and product-ion spectra showed fragmentation pathways typical of glutathione conjugates. ES-MS-MS analysis of a synthetic nonapeptide modeling a sequence found in neurofilament proteins showed pyrrole formation after incubation with 2,5-HD, and sequence ions were used to assign the position of the pyrrole adduct. Subsequent reaction of the pyrrolylated peptide with reduced glutathione was evidenced by a shift in m/z of the sequence ions of the reaction products with or without prior methylation. The results demonstrate the utility of ES-MS and ES-MS-MS in the characterization of xenobiotic-modified peptides and confirm that stable pyrrole-thiol conjugates are formed by the reaction of biological thiols with pyrrolylated peptides.

高度不飽和リン脂質の自動酸化挙動（第３報） 高度不飽和アシル基をもつリン脂質と油脂の自動酸化挙動の比較

トリアシルグリセリン (TG) とリン脂質 (PL) を含むサケ卵油が, TGのみからなるサケ油に比較して酸化に対して非常に安定であること, またこのサケ卵油の高い安定性はその脂質組成とトコフェロールやアスタキサンチンの含有量に依存することが前報において明らかになったため, 高度不飽和TG, 高度不飽和PL及びこれらの3 : 1混合試料の自動酸化挙動について検討した。サケ卵油から分画したPL, TG及びこれらの3 : 1混合試料をトコフェロール添加系または無添加系において30℃暗所にて自動酸化させた。これらの試料の自動酸化挙動は過酸化物価 (PV), カルボニル価 (CV) 及び脂肪酸組成により追跡した。TGではPVが上昇した後にCVが上昇したのに対し, PLでは酸化の初期段階においてCVがPVと同時に上昇し始めた。PLとTGの自動酸化挙動の差は, PLでは主な酸化一次生成物であるヒドロペルオキシドの安定性が悪いこと, あるいは脂質ペルオキシラジカルから二次生成物であるカルボニル化合物への経路が異なることによると思われた。これらの試料にトコフェロールを0.1%添加するといずれにおいても酸化が抑制され, ラジカル捕捉剤としてのトコフェロールの酸化防止作用はPLやTGにおいてよりもTGとPLの混合試料において, より顕著に観察された。これらの結果から, TGにPLとトコフェロールが共存することは高度不飽和TGを自動酸化条件下で安定化させるために非常に有効であることが示唆された。

Autoxidation of Intermediary Mesoionic 1,3-Oxazolium-5-olates Generated from Cyclic N-Acyl .ALPHA.-Amino Acids.

Mesoionic 1, 3-oxazolium-5-olates (munchnones) react fairly rapidly with oxygen to give the autoxidation products when the C-4 substituent is aromatic. The autoxidation occurred in the munchnones generated from N-acyl tetrahydroisoquinoline-1-carboxylic acids or tetrahydro-β-carboline-1-carboxylic acids. The mechanism was elucidated by 18O labeling experiments and involved a series of well-precedented autoxidative processes including oxygenation, cyclization of the resulting peroxy anion, and oxidative cleavage.

Synthesis and Spin Trapping Applications of 2,2-Dimethyl-d6-4-methyl-2H-imidazole-1-oxide-1-15N

A new spin trap, 2,2-dimethyl-d6-4-methyl-2H-imida-zole-1-oxide-1- 15N (ITMIO), was synthesized and characterized. Hyperfine splitting (HFS) constants of spin adduct ESR spectra of this compound with oxygen-centered, carbon-centered, thiyl and sulfite-derived radicals were determined and compared with the data of the unsubstituted compound. The increase in ESR spectral intensity and the accompanying decrease of the spectral linewidth result in resolution of the HFS due to interaction with α-protons of alkyl radicals trapped by ITMIO. Trapping of the formate radical in deoxygenated aqueous solution revealed a very low spectral linewidth (ABpp = 0.028 mT) of the corresponding adduct. A strong dependence of the ESR spectra on pH was observed when the autoxidation product of sulfite, SO3-, was trapped. The pKa was found to be 5.8 ± 0.3. In comparison to other nitrones, application of this spin trap provides more detailed information on the structure of the species trapped, especially for carbon-centered radicals.

Autoxidation Reaction Mechanism for l-Ascorbic Acid in Methanol without Metal Ion Catalysis.

The autoxidation reaction of l-ascorbic acid (ASA) in methanol without metal ion catalysis was studied. Besides l-threonolactone (THL) and oxalic acid (OXA), methyl l-threonate, and threonic acid were identified as initial autoxidation products of ASA, which were the C(2)-C(3) fission product via the C(2) oxygen adduct of ASA. This pathway is different from the one via dehydro-l-ASA (DASA), which has long been believed to be the only oxidation pathway of ASA. It was confirmed that this reaction also occurred in both water and other polar solvents, including methanol. It was clarified that mono-dissociated ASA was more reactive than the non-dissociated ASA in this pathway, and that the main reaction products formed from these two forms of ASA were also somewhat different. Determination of the amount of remaining ASA and the yields of THL and OXA, C(2)-C(3) fission products, and of DASA were carried out doing the autoxidation of ASA under various reaction conditions.

Volatile Cuticular Components Involved in Superficial Scald

Autoxidation products alpha-farnesene of have been implicated in superficial scald induction for apple (Malus domestica cv. Cortland Apple) fruit. We suspect the apple cuticle acts as a sink where α-farnesene can accumulate and eventually autoxidize into hydroperoxides, conjugated trienes, 6-methyl-5-hepten-2-one (ketone), and other compounds. These oxidized byproducts may diffuse back into the peel, thereby initiating the scald process. Cortland apples were stored at 0.8°C. Volatile cuticular components were analyzed at 2-week intervals by gas chromatography–mass spectroscopy. Only two scald-related volatiles were found, 6-methyl-5-hepten-2-one and α-farnesene. The identification of these compounds may allow the determination of cuticular involvement in superficial scald, as well as a possible correlation between the volatiles and apple scald development. α-farnesene concentrations initially increased and was followed by a decline, possibly due to its autoxidation.

Formation of Nitrating and Chlorinating Species by Reaction of Nitrite with Hypochlorous Acid: A NOVEL MECHANISM FOR NITRIC OXIDE-MEDIATED PROTEIN MODIFICATION

Detection of 3-nitrotyrosine has served as an in vivo marker for the production of the cytotoxic species peroxynitrite (ONOO-). We show here that reaction of nitrite (NO2-), the autoxidation product of nitric oxide (.NO), with hypochlorous acid (HOCl) forms reactive intermediate species that are also capable of nitrating phenolic substrates such as tyrosine and 4-hydroxyphenylacetic acid, with maximum yields obtained at physiological pH. Monitoring the reaction of NO2- with HOCl by continuous flow photodiode array spectrophotometry indicates the formation of a transient species with spectral characteristics similar to those of nitryl chloride (Cl-NO2). Reaction of synthetic Cl-NO2 with N-acetyl-L-tyrosine results in the formation of 3-chlorotyrosine and 3-nitrotyrosine in ratios that are similar to those obtained by the NO2-/HOCl reaction (4:1). Tyrosine residues in bovine serum albumin are also nitrated and chlorinated by NO2-/HOCl and synthetic Cl-NO2. The reaction of N-acetyl-L-tyrosine with NO2-/HOCl or authentic Cl-NO2 also produces dityrosine, suggesting that free radical intermediates are involved in the reaction mechanism. Our data indicate that while chlorination reactions of Cl-NO2 are mediated by direct electrophilic addition to the aromatic ring, a free radical mechanism appears to be operative in nitrations mediated by NO2-/HOCl or Cl-NO2, probably involving the combination of nitrogen dioxide (.NO2) and tyrosyl radical. We propose that NO2- reacts with HOCl by Cl+ transfer to form both cis- and trans-chlorine nitrite (Cl-ONO) and Cl-NO2 as intermediates that modify tyrosine by either direct reaction or after decomposition to reactive free and solvent-caged Cl. and .NO2 as reactive species. Formation of Cl-NO2 and/or Cl-ONO in vivo may represent previously unrecognized mediators of inflammation-mediated protein modification and tissue injury, and offers an additional mechanism of tyrosine nitration independent of ONOO-.