Determination of stereochemistry in the fatty acid hydroperoxide products of lipoxygenase catalysis

ANALYTICALBIOCHEMISTRY

158,316-321(1986)

Determination

of Stereochemistry in the Fatty Acid Hydroperoxide Products of Lipoxygenase Catalysis’ JON C. ANDRE AND MAX

Department

of Chemistry,

Bowman-Oddy

Laboratories,

0. FUNKY

The University

of Toledo, Toledo, Ohio 43606

Received May 2, 1986 High-performance liquid chromatography has been found to be an effective method for the determination of absolute configuration in the products of the lipoxygenase-catalyzed oxygenation of polyunsaturated fatty acids. Methyl esters of fatty acid hydroperoxides that had been reduced to the corresponding alcohols were converted into (+)-ol-methoxy-a-tritluoromethylphenylacetic acid esters. Enantiomeric alcohols were converted into diastereomeric esters that were readily resolved by normal-phase HPLC. The instrumental requirements for the technique are an isocratic HPLC and a uv absorbance monitor. The method was found to be effective in the determination of stereochemistry in the products derived from the action of plant lipoxygenases on linoleic acid. In addition, the chromatography of the derivatives obtained from lipoxygenase catalysis on arachidonic acid was found to be effective for the assignment of stereochemistry in those products. A comparison of the chromatography of these derivatives with that for the corresponding menthyloxycarbonyl derivatives demonstrated the superiority of this approach for the resolution of the diastereomeric pairs. The technique was applied to the determination of stereochemistry in the products derived from soybean lipoxygenase isoenzymes under a variety of experimental conditions. 0 1986 Academic Press. Inc. KEY WORDS: oxygenases; lipoxygenase; HPLC, lipids; stereochemistry; absolute configuration; enzyme specificity.

In recent years, it has become apparent that the lipoxygenase-mediated metabolism of arachidonic acid results in the formation of a number of compounds with important biological properties. For example, the reaction catalyzed by lipoxygenase is an inaugural event in the biosynthesis ofthe leukotrienes (1). The enzyme causes molecular oxygen to be incorporated into certain naturally occurring polyunsaturated fatty acids as illustrated in Eq. [ 11. MC,,”

moxK!enase-l

•~co,, HO0

111 Catalysis generally occurs in a regiospecific fashion with individual enzymes acting at spe’ The research described in this report was supported financially by the National Institutes of Health and the De Arce Memorial Endowment for Medical Research and Development. * National Institutes of Health Research Career Development Award Recipient. 0003-2697/86 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

316

cific positions on the polyunsaturated fatty acid. Enzymatic oxygenation at individual carbon atoms can occur by both stereospecific and stereorandom processes. The products of the mammalian enzymes have generally been identified as single enantiomers. For example, the products generated by the platelet arachidonic acid 12-lipoxygenase and the leukocyte 5lipoxygenase have the S stereochemistry (2,3). This is in contrast to the nonenzymatic process which occurs in a random fashion with regard to stereochemistry. In the plant lipoxygenase realm, the extensively studied type- 1 enzyme from soybeans produces the 13-hydroperoxide of S absolute configuration from linoleic acid whereas the enzymes from corn, tomato, and potato yield the 9-isomer of S stereochemistry (4-7). An unusual feature of the type-2 isoenzyme from soybeans is that it produces both 9 and 13 isomers from linoleic acid as well as a mixture of stereoisomers at each position (8). Clearly, determination of

STEREOCHEMISTRY

OF LIPOXYGENASE

stereochemistry in the products of lipoxygenase catalysis is an important factor in the overall characterization of the process and, in some cases, a method for distinguishing between the enzymatic and nonenzymatic oxygenation. There are currently several approaches to the determination of stereochemistry in lipoxygenase products available. The measurement of optical rotation and comparison with standards continues to be useful in cases where sufficiently large quantities of the material can be obtained for an accurate determination. However, by far the most frequently used method is that developed by Hamberg which involves the formation of diastereomeric derivatives of the alcohol corresponding to the hydroperoxide product and the separation of these isomers by gas chromatography (9). The derivatization procedure is carried out in two steps beginning with the methyl ester of the alcohol. First, the diastereomeric menthyloxycarbonyl derivatives are formed and these compounds are subjected to oxidative ozonolysis. A similar procedure was reported more recently ( 10). It has also been demonstrated that the stereochemistry of some lipoxygenase products can be determined by high-performance liquid chromatography (HPLC) of the menthyloxycarbonyl derivatives without further derivatization (11). This technique, however, apparently only applies to certain lipoxygenase products, since resolution was not reported for several positional isomers. The chromatographic resolution of diastereomeric derivatives of lipoxygenase products has been successfully applied to the production of pure enantiomers in synthetically useful quantities. Corey et al. employed the isocyanate derived from dehydroabietylamine to resolve the diastereomeric urethanes of (6E,SZ, 11 Z, 142)-S hydroxy-6,8,11,14-eicosatetraenoic acid using column chromatography in the large scale synthesis of that compound (12). The resolution of urethane diastereomers by high-performance liquid chromatography has been recently used in the determination of the stereochemistry of alcohols produced in the reduction of ketones by an alcohol dehydro-

PRODUCTS

317

genase isolated from a thermophilic bacterium (13). The difficulty with the use of urethane derivatives is that for lipoxygenase products only the dehydroabietylamine-derived isocyanate was successful, and this requires a synthesis of the compound involving the preparation of the hazardous substance, phosgene. The stereochemistry of lipoxygenase-derived products has also been determined by nuclear magnetic resonance spectroscopy (NMR) of the (+)-cu-methoxy-ar-trifluoromethylphenylacetic acid (MTPA)3 esters of the appropriate alcohols using a lanthanide shift reagent ( 14). We report here a method for resolving the enantiomeric products that could be formed in the lipoxygenase reaction that employs a readily available reagent for chemical derivatization and requires only a normal-phase isocratic HPLC separation. Examples are presented that illustrate the utility of the procedure for determining the enantiomeric composition of individual hydroperoxides and its application to the determination of stereoselectivity in lipoxygenase-catalyzed reactions. MATERIALS

AND METHODS

Lipoxygenases were obtained as previously described from two soybean cultivars, Provar and Vickery, using a chromatofocusing procedure (15). The isoenzymes are referred to by number corresponding to the reverse order of elution from the column. The latest eluting isoenzyme was already known as type- 1. The polyunsaturated fatty acids were all obtained from Sigma Chemical Company. The (+)MTPA was from Aldrich Chemical Company. Derivatization of the methyl esters of the fatty alcohols corresponding to the products of lipoxygenase-catalyzed oxygenations were carried out as described by Dale et al. ( 16) and Hamberg (9). Briefly, the fatty acid derivatives were treated with the chiral acid chloride from 3 Abbreviation used: MTPA. (+)-a-methoxy-oc-trifluoromethylphenylacetic acid.

318

ANDRE

(+)-MTPA in carbon tetrachloride and pyridine. The reactions were terminated by addition of water followed by extraction with diethyl ether. The ether extracts were evaporated in wcuo and taken up in mobile phase for HPLC analysis. Individual enantiomers of products were prepared by the action of a lipoxygenase under previously established conditions 9 S (7), 13 S, and 15 S (17). The hydroperoxides were reduced with sodium borohydride in either borate buffer at pH 9 or in 2-propanol to give the alcohols. The methyl esters were prepared by treating the acids with a fivefold excess of diazomethane in diethyl ether for 1 h at 4°C. The ether and excess reagent were removed by evaporation in vacua. Racemic mixtures of the alcohols were prepared by chemical oxidation and reduction. An example of the procedure is provided for one of the compounds. In 45 ml of dry dimethylformamide, pyridinium dichromate (4.0 g, 10.6 mmol) and (IOE, 12Z)-94hydroxy10,12-octadecadienoic acid ( 135 mg, 0.46 mmol) were combined and stirred at 4°C under an inert atmosphere. After 1.5 h, the reaction mixture was poured into 450 ml of water and was extracted with diethyl ether (4X, 100 ml). The extracts were dried over sodium sulfate and evaporated in vacua. The product was purified by chromatography on silica gel using hexane/ethyl acetate/acetic acid (75/24/l) providing 79.6 mg (59%) of ( 1OE, 12Z)-9-0x0- 10,12-octadecadienoic acid. The ketone (26.5 mg, 0.09 mmol) was combined with sodium borohydride (100 mg, 2.65 mmol) in 75 ml of sodium borate buffer (0.05 M, pH 9.0) at room temperature under an inert atmosphere. After stirring for 2.5 h, the reaction mixture was acidified to pH 3 by addition of aqueous hydrochloric acid (6 M). The suspension was extracted with diethyl ether (3X, 70 ml). The extracts were dried over sodium sulfate and evaporated in vacua. The product was esterified by treatment with diazomethane and purified by chromatography on silica gel using hexane/ethyl acetate (83/17) to yield 17.3 mg (62%) of racemic methyl (lOE, 12Z)9-hydroxy- 10,12-octadecadienoate.

AND

FUNK

Substrate solutions for the incubations with various isoenzymes were prepared by combining 0.13 ml of 10% linoleic acid solution (ethanol) with 0.05 ml of 10% Tween 20 solution (ethanol). After removing the solvent in a stream of nitrogen gas, the substrate was first dissolved in 2.0 ml of phosphate buffer (0.05 M, pH 9.0) and then combined with a further 3.0 ml of phosphate buffer (0.2 M, pH 6.8). The enzyme (0.27 nmol) was added and the reaction was allowed to proceed at room temperature for 20 min under an oxygen atmosphere. The reactions were terminated by addition of 9.0 ml of phosphate buffer (0.2 ml, pH 2.1). The reaction mixture was passed through a solid-phase extractor (J. T. Baker 7020-l). The extractor was rinsed with water (2X, 1 ml) and the products were eluted in methanol (2X, 1 ml). The solvent was removed, and the products were reduced, esterified, and purified by chromatography on silica gel as previously described ( 18). Chromatographic determinations were carried out at room temperature on a system consisting of a pump (Altex 1 IOA), injector (Rheodyne 7 120) 4.6 X 250-mm silica column (Fisher Resolvex SIL 6-650-2) and detector (Gilson Holochrome). The effluent was monitored for absorbance at 236 nm. The mobile phase employed for the resolutions was hexane/ethyl acetate (98/2). The ratios of components in the chromatograms were determined from peak areas. RESULTS

AND

DISCUSSION

Samples of the products of lipoxygenase catalysis were derivatized for stereochemical analysis using the method developed by Dale et al. for alcohols in general. The enzymatic reactions were terminated by treatment with sodium borohydride. The alcohols were extracted following acidification and were subsequently esterified with diazomethane. Derivatives were formed by treatment with MTP chloride. The procedure is illustrated in Eq. PI.

STEREOCHEMISTRY

OF LIPOXYGENASE

Racemic mixtures of the alcohols were prepared by the oxidation of the enzymatically prepared, reduced, and esterified materials to the corresponding ketones using pyridinium dichromate, followed by reduction back to the alcohols using sodium borohydride. The mixtures of diastereomeric derivatives were then prepared as for individual enantiomers. Chromatography was carried out on a simple isocratic HPLC system using a silica column packed with spherical 10 pm particles. The resolution for the column was modest (N = 6000), meaning that the separations were far from being optimized in terms of currently available technology. It was found that ethyl

FIG. 1. Chromatographic profiles (silica) for MTPA derivatives of methyl-(9Z, 11E)- 13-hydroxy-9, I I-octadecadienoate. (A) 13 S derivative from soybean lipoxygenase1. (B) 13 (R, 5’) synthetic racemic mixture. Mobile phase, hexane/ethyl acetate (98/2).

PRODUCTS

319

FIG. 2. Chromatographic profiles (silica) for MTPA derivatives of methyl-( lOE, I2Z)-9-hydroxy- IO, I2-octadecadienoate. (A) 9 S derivative from tomato lipoxygenase. (B) 9 (R. S) synthetic racemic mixture. Mobile phase, hexane/ethyl acetate (98/2).

acetate provided better selectivity as a polar modifier of the mobile phase in the resolution of diastereomeric derivatives than 2-propanol. Because the proportions of the ethyl acetate were quite low in the mobile phase, this choice of solvent did not interfere with detection based on ultraviolet absorption even at 236 nm. Examples of the application of the HPLC technique to the resolution of stereoisomers of lipoxygenase products are presented in Fig. 1 and 2. Soybean type- 1 lipoxygenase is known to produce nearly exclusively the 13-hydroperoxide derivative of linoleic acid when the incubation is carried out in borate buffer at pH 9. The absolute configuration of this compound, S, has been previously established. As shown in Fig. 1, one isomer of (9Z, 1 lE)-I 3hydroperoxy-9,11 -octadecadienoic acid determined as the MTPA derivative of the methyl ester was formed predominately by lipoxygenase-1 at pH 9. The diastereomeric mixture of derivatives formed from this material by the chemical oxidation-reduction procedure was clearly resolved by this chromatographic system. In a similar fashion, samples of the derivatives of the lipoxygenase products resulting from oxygenation at position 9 of linoleic acid were analyzed (Fig. 2).

320

ANDRE AND FUNK

In this case, the tomato 9-lipoxygenase was used to produce a single enantiomer of known absolute configuration S. Again, the diastereomeric mixture of derivatives was well resolved on chromatography. In fact, all four of the products commonly obtained from the oxidation of linoleic acid by lipoxygenases (two diastereomers each at positions 9 and 13) could be obtained simultaneously, since an adequate resolution of the components was obtained. The structures of the derivatives were verified by C-l 3 as well as H-l NMR spectroscopy, and the assignment of stereochemistry for individual enantiomers was confirmed by H-l NMR spectroscopy in the presence of a lanthanide shift reagent. Control experiments in which the polyunsaturated fatty acid was incubated without lipoxygenase revealed that autoxidation resulted in the formation of less than 0.2% of the hydroperoxide product obtained from the slowest of the enzymatic oxygenations (V2 with linoleic acid). Therefore, under the conditions of the experiments, the yield from the reaction that was not enzyme catalyzed was negligible and would not contribute in a significant way to the distribution of stereoisomers in the products. The efficient resolution of lipoxygenase isoenzymes from two soybean cultivars was recently achieved in this laboratory (15). The newly developed chromatographic method

was used to compare the product distributions for selected isoenzymes. The results of the determination of stereochemistry for lipoxygenase products derived from various sources and under different conditions of incubation are collected in Table 1. The observations for the type- 1 enzyme from soybeans and the tomato enzyme were comparable to those from previous reports (9,10,14). The other soybean isoenzymes (V2, P4) gave a much more even distribution of products than the type- 1 or tomato enzymes (8). Two electrophoretically distinct soybean enzymes (V2, P4) gave similar results upon stereochemical determination of the product distribution. There was a small difference in the distribution of the geometrical isomers in the products obtained from these isoenzymes. The application of the HPLC of MTPA derivatives to the determination of stereochemistry of arachidonate lipoxygenase products is illustrated in Fig. 3. The oxidation of arachidonic acid by soybean lipoxygenase-1 at pH 9 in borate buffer is known to occur exclusively at position 15 with the production of S absolute configuration. As would be expected for this experiment, one isomer was obtained nearly exclusively in the HPLC determination of the MTPA derivative of the product. An adequate separation of the appropriate mixture of diastereomers was also obtained. An example of the result of the HPLC determi-

TABLE I STEREOCHEMICAL AND

ANALYSES

ARACHID~NIC

ACID

OF THE PRODUCTS BY LIPOXYGENASES

OF OXYGENATIONS UNDER

VARIOUS

OF LINOLEIC

ACID

CONDITIONS

Lipoxygenase

PH

c,t:t,t

9:13

9R:9S

13R:13S

Vl Vl v2 P4 Tomato VI (20:4) Chemical (20:4) Chemical Chemical

9 7 7 7 5.5 9 -

97:3 92:8 72128 63:37 9O:lO -

0:lOO 22:78 48:52 50:50 95:5 -

20:80 67~33 64136 3x97

14:86 20:80 54~46 57:43 13:87” 50:50" 50:50 -

’ Oxygenated at C- 15.

47:53

STEREOCHEMISTRY

4 4”

OF LIPOXYGENASE

1: d C

3. Chromatographic profiles (silica) for M_--. 1 I’A and. menthyloxycarbonyl derivatives of methyl-(5Z,8Z, 1lZ, 13E)- 1%hydroxy-5,8,11,13-eicosatetraenoate. (A) 15 S MTPA derivative from soybean Iipoxygenase- 1. (B) 15 (R, S) MTPA derivatives, synthetic racemic mixture. (C) 15 (R. S) menthyloxycarbonyl derivatives, synthetic racemic mixture. Mobile phase, hexane/ethyl acetate (98/2).

PRODUCTS

321

isocratic chromatographic system. Improvements in the procedure could be introduced in a number of ways as dictated by the conditions of the isolation of lipoxygenase products. The use of a high efficiency silica column would be expected to lead to substantially greater resolution of diastereomers which could be translated into shorter analysis times by adjustment of the mobile phase composition. In addition, the application of an HPLC technique opens the possibility of using radiometric or mass spectroscopic detection in a straightforward fashion for the further enhancement of sensitivity. REFERENCES

FIG.

1. Samuelsson, B. (1983) Science 220, 568-575. 2. Hamberg, M., and Samuelsson, B. (1974) Proc. Nutl. Acud. Sci. US.4 71,3400-3404.

3. Borgeat, P., Hamberg, M., and Samuelsson, B. (1977) J. Biol.

Chem.

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242,5329-5335.

5. Galliard, T., and Philips. D. R. (197 I) Biochem.

J.

124,431-438.

nation of the diastereomeric menthyloxycarbony1 derivatives obtained from the 1%hydroperoxide of arachidonic acid is also presented in the figure. It was found that the mixture was not resolved under conditions that were successful in the separation of the MTPA derivatives. The separation of the menthyloxycarbonyl derivatives was not affected by other polar modifiers of the mobile phase, i.e., 2-propanol. This represents a demonstration of the superiority of the MTPA derivatives for the HPLC determination of stereochemistry of lipoxygenase products derived from arachidonic acid. The method introduced here involves the determination of the stereochemistry of lipoxygenase-derived products of polyunsaturated fatty acid metabolism using HPLC of the MTPA derivatives. The derivatization takes place in a single step and does not involve the generation of ozone or the preparation of phosgene as is required for the preparation of other derivatives. The analysis of samples is sensitive and can be carried out on a simple

6. Gardner, H. W., and Weisleder, D. (1970) Lipids 5, 678-683. 7. Matthew, H., Chan, H. W.-S, and Galliard, T. (1977) Lipids

12, 324-326.

8. Van OS, C. P. A., Rijke-Schilder, G. P. M., and Vliegenthart, J. F. G. (1979) Biochim. Biophys. Acta 575,479-484.

Hamberg, M. (197 1) Anal. Biochem. 43, 5 15-526. 10. Van Os, C. P. A., Rijke-Schilder, G. P. M., Kamerling, J. P., Gerwig. G. J., and Vliegenthart, J. F. G. (1980) Biochim. Biophys. Acta 620, 326-33 1. 11. Brash. A. R.. Porter, A. T., and Mass, R. L. (1985) J. Biol. Chem. 260,42 1O-42 16. 12. Corey, E. J., and Hashimoto. S. (1981) Tetrahedron 9.

Lett. 22, 299-302.

13. Keinan. E.. Hafeli, E. K., Seth, K. K., and Lamed, R. (1986) J. Amer. Chem. Sot. 108, 162-169. 14. Van OS, C. P. A., Vente, M., and Vliegenthart, J. F. G. (1979) Biochim. Biophys. Acta 574, 103111. 15. Funk, M. 0.. Whitney, M. A., Hausknecht, E. C., and O’Brien, E. M. (1985) Anal. Biochem. 146, 246251.

16. Dale, J. A., Dull, D. L., Mosher, H. S. (1969) J. Org. Chem.

34,2543-2549.

17. Funk, M. O., Levison, B., and Keller, M. B. (1980) Lipids

15, 1051-1054.

18. Porter. N. A., Logan, J.. and Kontoyiannidou, (1979) J. Org. Chem. 44, 3177-3181.

V.