Harold W. Gardner

Oxygen radical chemistry of polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFA) are readily susceptible to autoxidation. A chain oxidation of PUFA is initiated by hydrogen abstraction from allylic or bis-allylic positions leading to oxygenation and subsequent formation of peroxyl radicals. In media of low hydrogen-donating capacity the peroxyl radical is free to react further by competitive pathways resulting in cyclic peroxides, double bond isomerization and formation of dimers and oligomers. In the presence of good hydrogen donators, such as alpha-tocopherol or PUFA themselves, the peroxyl radical abstracts hydrogen to furnish PUFA hydroperoxides. Given the proper conditions or catalysts, the hydroperoxides are prone to further transformations by free radical routes. Homolytic cleavage of the hydroperoxy group can afford either a peroxyl radical or an alkoxyl radical. The products of peroxyl radicals are identical to those obtained during autoxidation of PUFA; that is, it makes no difference whether the peroxyl radical is generated in the process of autoxidation or from a performed hydroperoxide. Of particular interest is the intramolecular rearrangement of peroxyl radicals to furnish cyclic peroxides and prostaglandin-like bicyclo endoperoxides. Other principal peroxyl radical reactions are the beta-scission of O2, intermolecular addition and self-combination. Alkoxyl radicals of PUFA, contrary to popular belief, do not significantly abstract hydrogens, but rather are channeled into epoxide formation through intramolecular rearrangement. Other significant reactions of PUFA alkoxyl radicals are beta-scission of the fatty chain and possibly the formation of ether-linked dimers and oligomers. Although homolytic reactions of PUFA hydroperoxides have received the most attention, hydroperoxides are also susceptible to heterolytic transformations, such as nucleophilic displacement and acid-catalyzed rearrangement.

Soybean lipoxygenase-1 enzymically forms both (9S)- and (13S)-hydroperoxides from linoleic acid by a pH-dependent mechanism.

Soybean lipoxygenase-1 produces a preponderance of two chiral products from linoleic acid, (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid and (9S)-(10E,12Z)-9-hydroperoxy-10,12-octadecadienoic acid. The former of these hydroperoxides was generated at all pH values, but in the presence of Tween 20, the latter product did not form at pH values above 8.5. As the pH decreased below 8.5, the proportion of (9S)-hydroperoxide increased linearly until at pH 6 it constituted about 25% of the chiral products attributed to enzymic action. Below pH 6, lipoxygenase activity was barely measurable, and the hydroperoxide product arose mainly from autoxidation and possibly non-enzymic oxygenation of the pentadienyl radical formed by the enzyme. The change in percent enzymically formed 9-hydroperoxide between pH 6.0 and 8.5 paralleled the pH plot of a sodium linoleate/linoleic acid titration. It was concluded that the (9S)-hydroperoxide is formed only from the nonionized carboxylic acid form of linoleic acid. Methyl esterification of linoleic acid blocked the formation of the (9S)-hydroperoxide by lipoxygenase-1, but not the (13S)-hydroperoxide. Since the hydroperoxydiene moieties of the (9S)- and (13S)-hydroperoxides are spatially identical when the molecules are arranged head to tail in opposite orientations, it is suggested that the carboxylic acid form of the substrate can arrange itself at the active site in either orientation, but the carboxylate anion can be positioned only in one orientation. These observations, as well as others in the literature, suggest and active-site model for soybean lipoxygenase-1.

Homolytic decomposition of linoleic acid hydroperoxide: Identification of fatty acid products

An isomeric mixture of linoleic acid hydroperoxides, 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid (79%) and 9-hydroperoxy-cis-12,trans-10-octadecadienoic acid (21%), was decomposed homolytically by Fe(II) in an ethanol-water solution. In one series of experiments, the hydroperoxides were decomposed by catalytic concentrations of Fe(II). The 10−5 M Fe(III) used to initiate the decomposition was kept reduced as Fe(II) by a high concentration of cysteine added to the reaction in molar excess of the hydroperoxides. Nine different monomeric (no detectable dimeric) fatty acids were identified from the reaction. Analyses of these fatty acids revealed that they were mixtures of positional isomers identified as follows: (I) 13-oxo-trans,trans-(andcis,trans-) 9,11-octadecadienoic and 9-oxo-trans,trans- (andcis,trans-) 10,12-octadecadienoic acids; (II) 13-oxo-trans-9,10-epoxy-trans-11-octadecenoic and 9-oxo-trans-12, 13-epoxy-trans-10-octadecenoic acids; (III) 13-oxo-cis-9,10-epoxy-trans-11-octadecenoic and 9-oxo-cis-12, 13-epoxy-trans-10-octadecenoic acids; (IV) 13-hydroxy-9,11-octadecadienoic and 9-hydroxy-10,12-octadecadienoic acids; (V) 11-hydroxy-trans-12, 13-epoxy-cis-9-octadecenoic and 11-hydroxy-trans-9, 10-epoxy-cis-12-octadecenoic acids; (VI) 11-hydroxy-trans-12, 13-epoxy-trans-9-octadecenoic and 11-hydroxy-trans-9,10-epoxy-trans-12-octadecenoic acids; (VII) 13-oxo-9-hydroxy-trans-10-octadecenoic acids; (VIII) isomeric mixtures of 9, 12, 13-dihydroxyethoxy-trans-10-octadecenoic and 9, 10, 13-dihydroxyethoxy-trans-11-octadecenoic acids; and (IX) 9, 12, 13-trihydroxy-trans-10-octadecenoic and 9, 10, 13-trihydroxy-trans-11-octadecenoic acids. In another experiment, equimolar amounts of Fe(II) and hydroperoxide were reacted in the absence of cysteine. A large proportion of dimeric fatty acids and a smaller amount of monomeric fatty acids resulted. The monomeric fatty acids were examined by gas liquid chromatography-mass spectroscopy. Spectra indicated that the monomers were largely similar to those produced by the Fe(III)-cysteine reaction.

Isolation of a pure isomer of linoleic acid hydroperoxide

A mixture of positional isomers of linoleic acid hydroperoxide was produced from the oxidation of linoleic acid by lipoxygenase from corn or soybean. Chromatography on a column of silicic acid separated 13-hydroperoxy-11,9-octadecadienoic acid in 99+% purity from the mixture obtained by soybean lipoxygenase oxidation of linoleic acid. Attempts at isolation of pure 9-hydroperoxy-10,12-octadecadienoic acid from hydroperoxides obtained by corn lipoxygenase oxygenation of linoleic acid were partially successful with isolation of the 9-hydroperoxide in 97% purity.

Degradation of linoleic acid hydroperoxides by a cysteine FeCl3 catalyst as a model for similar biochemical reactions: II. Specificity in formation of fatty acid epoxides☆

1. The degradation of linoleic acid hydroperoxide by cysteine and FeCl3 resulted in formation of a number of oxygenated fatty acids, among which isomeric epoxyoxooctadecenoic and epoxyhydroxyoctadecenoic acids were major products. Pure isomeric hydroperoxides, either 13-L(S)-hydroperoxy-cis-9,trans-11-octadecadienoic acid or 9-D(S)-hydroperoxy-trans-10,cis-12-octadecadienoic acid, were transformed into either 12,13-epoxides or 9,10-epoxides, respectively. 2. From 13-L(S)-hydroperoxy-cis-9,trans-11-octadecadienoic acid, the epoxides were identified as trans-12,13-epoxy-9-oxo-trans-10-octadecenoic acid, trans-12,13-epoxy-9-hydroxy-trans-10-octadecenoic acid, cis-12,13-epoxy-9-oxo-trans-10-octadecenoic acid, trans-12,13-epoxy-erythro-11-hydroxy-cis(trans)-9-octadecenoic acid and trans-12,13-epoxy-threo-11-hydroxy-cis(trans)-9-octadecenoic acid. 3. The 12,13-epoxides were found to be optically active, indicating that the chiral center of the 13-L(S)-hydroperoxy carbon was retained. 4. Although many epoxy fatty acids previously have been identified as linoleic acid hydroperoxide products, this study reports a more complete structural analysis of the various epoxides and allows an assessment of the mechanisms of their formation from hydroperoxides.

Degradation of linoleic acid hydroperoxides by a cysteine . FeCl3 catalyst as a model for similar biochemical reactions. I. Study of oxygen requirement, catalyst and effect of pH.

Abstract 1. 1. A redox reaction between cysteine and linoleic acid hydroperoxide was catalyzed by FeCl3 at pH values under 6. This reaction was assayed by the rate of O2 uptake, and resulted in formation of mainly oxodiene, epoxyhydroxyene and epoxyoxoene fatty acids as well as cystine and oxides of cysteine. 2. 2. The rate of O2 uptake was dependent upon the relative concentrations of FeCl3, linoleic acid hydroperoxide and cysteine. Rate dependence on FeCl3 concentration yielded a sigmoid curve, but rate dependence on concentrations of either cysteine or linoleic acid hydroperoxide was best described by a Lineweaver-Burk double reciprocal plot. The possible participation of a linoleic acid hydroperoxide-cysteine-FeCl3 complex is discussed. 3. 3. About one-half mol of O2 was absorbed per mol of linoleic acid hydroperoxide, whereas O2 uptake per mol of cysteine amounted to one-fourth mol. A mechanism is proposed to account for the O2 uptake stoichiometry. 4. 4. In the presence of O2, the reaction products were oxooctadecadienoic acid and fatty acids derived from addition of O2 to an epoxyallylic radical (secondary hydroperoxidation). In the absence of O2, the products were oxooctadecadienoic acid and cysteine-fatty acid adducts. 5. 5. Either Fe(II) or Fe(III) salts were equally effective catalysts, but CN− or EDTA complexes of iron were ineffective catalysts. 6. 6. At pH values over 7 hydroxyoctadecadienoic acid became the only product formed from linoleic acid hydroperoxide. Hydroxydiene fatty acid formed in the presence or absence of either FeCl3 or O2. Only cysteine and alkaline pH values were essential to hydroxydiene formation. 7. 7. At pH values over 7, O2− was formed in the presence of O2, as ascertained by Superoxide dismutase-inhibitable O2 uptake and nitroblue tetrazolium reduction. Rationale is presented that O2 was not involved in product formation from linoleic acid hydroperoxide. 8. 8. The reaction and products of linoleic acid hydroperoxide-cysteine-FeCl3 complex served as a model for the degradation of linoleic acid hydroperoxide by certain biological preparations.

Formation oftrans-12,13-epoxy-9-hydroperoxy-trans-10-octadecenoic acid from 13-L-hydroperoxy-cis-9,trans-11-octadecadienoic acid catalyzed by either a soybean extract or cysteine-FeC13

A soybean extract or an ethanolic solution of cysteine and ferric chloride catalyzed the conversion of 13-L-hydroperoxy-cis-9,trans-11-octadecadienoic acid to numerous products among which wastrans-12,13-epoxy-9-hydroperoxy-trans-10-octadecenoic acid. When this fatty acid was treated further with the cysteine-ferric chloride solution, 9-hydroxy-12,13-epoxy-10-octadecenoic and 9-oxo-12,13-epoxy-10-octadecenoic acids were formed. Thus,trans-12,13-epoxy-9-hydroperoxy-trans-10-octadecenoic acid probably is an intermediate in the formation of the latter two compounds. Additionally, theerythro andthreo isomers oftrans-12,13-epoxy-11-hydroperoxy-cis-9-octadecenoic acid tenatatively were identified as products.

Characterization of a C-5,13-Cleaving Enzyme of 13(S)-Hydroperoxide of Linolenic Acid by Soybean Seed

An activity was found in mature soybean seeds (Glycine max L. cv Century) that cleaved 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13S-HPOT) into 13-oxo-9(Z),11(E)-tridecadienoic acid and two isomeric pentenols, 2(Z)-penten-1-ol and 1-penten-3-ol. Isomeric pentene dimers were also produced and were presumably derived from the combination of two pentene radicals. 13(S)-Hydroperoxy-9(Z),11(E)-octadecadienoic acid (13S-HPOD) was, by contrast, a poor substrate. Activity with 13S-HPOT increased 24-fold under anaerobic conditions reminiscent of a similar anaerobic promoted reaction of 13S-HPOD catalyzed by lipoxygenase (LOX) in the presence of linoleic acid. However, prior to ion-exchange chromatography, cleavage activity did not require linoleic acid. After separation by gel filtration followed by ion-exchange chromatography, cleavage activity was lost but reappeared in the presence of either linoleic acid or dithiothreitol. Under these conditions cleavage activity was coincident with the activity of types 1 and 2 LOX. LOX inhibitors suppressed the cleavage reaction in a manner similar to inhibition of LOX activity. Heat-generated alkoxyl radicals derived from either 13S-HPOT or 13S-HPOD afforded similar products and yields of 13-oxo-9(Z),11(E)-tridecadienoic acid compared to the enzymic reaction. The product 1-penten-3-ol may be the precursor of the “raw-bean” volatile ethylvinylketone.

10(S)-Hydroxy-8(E)-octadecenoic acid, an intermediate in the conversion of oleic acid to 7,10-dihydroxy-8(E)-octadecenoic acid

The new microbial isolate Pseudomonas aeruginosa (PR3) has been reported to produce from oleic acid a new compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD), with 10-hydroxy-8-octadecenoic acid (HOD) being a probable intermediate. The production of DOD involves the introduction of two hydroxyl groups at carbon numbers 7 and 10, and a rearrangement of the double bond from carbons 9–10 to 8–9. It has been shown that the 8–9 unsaturation of HOD was possibly in the cis configuration. Now we report that the rearranged double bond of HOD is trans rather than cis, as determined by spectral data. Also, it was found that the 10-hydroxyl was in the S-configuration as determined by gas chromatographic separation of R- and S-isomers after preparation of the (−)-menthoxycarbonyl derivative of the hydroxyl group followed by oxidative cleavage of the double bond and methyl esterification. This latter result coincides with our recent finding that the main final product, DOD, is in the 7(S),10(S)-dihydroxy configuration. In addition, a minor isomer of HOD (about 3%) with the 10(R)-hydroxyl configuration was also detected. From the data obtained herein, we concluded that 10(S)-hydroxy-8(E)-octadecenoic acid is the probable intermediate in the bioconversion of oleic acid to 7(S),10(S)-dihydroxy-8(E)-octadecenoic acid by PR3.

Lipoxygenase as a versatile biocatalyst

This review of lipoxygenase and lipoxygenase pathway enzymes focuses on the potential for the efficient production of useful compounds. Although the existence of lipoxygenase has been inown for many years, only recently has there been progress toward understanding the conditions required to improve yields and immobilize its activity. Maintaining a high O2 tension is necessary to obtain good yeilds of hydroperoxides; whereas, partial anaerobic conditions can lead to hydroperoxide decomposition. Fatty hydroperoxides, obtained from lipoxygenase action, can serve as precursors for further transformation by either enzymes or chemical reactions. Well over one-hundred products from lipoxygenase-generated hydroperoxides of linoleic acid alone have been described. Examples will be given of the formation of fatty acids with epoxide, hydroxy, ketone, cyclic, and multiple functional groups. The cleavage of fatty hydroperoxides into short-chain aldehydes and alcohols also will be described. Many of the products have biological activity, suggesting a significant physiological function for lipoxygenase.