Investigation of the chemical conversion of hydroperoxyeicosatetraenoate to leukotriene epoxide using stereospecifically labeled arachidonic acid. Comparison with the enzymatic reaction.

A series of stereospecifically labeled polyunsaturated fatty acids were prepared by biosynthesis from [8-DR-3H]- and [8-LS-3H]stearic acids. The labeled stearic acids were synthesized by a novel scheme employing readily available alkyne and aldehyde starting materials. The stereochemical purity of the prochiral tritium labels was judged to be greater than 99%, as determined by analysis of the octadec-1-yn-8(R)- and 8(S)-ol intermediates in the synthesis. Previously, the labeled arachidonic acids were used to investigate the stereoselectivity of hydrogen abstraction in the biosynthesis of leukotriene epoxides. We have now investigated the selectivity of hydrogen abstraction in a chemical synthesis of 14,15-leukotriene (LT) A4 from mixtures of [3-14C]- and either [10-DR-3H]- or [10-LS-3H]15(S)-HPETE methyl esters. Reaction with either chirally labeled precursor led to 70-95% retention of 3H relative to 14C in the 14,15-LTA4 and 10-Z-14,15-LTA4 products after purification by high performance liquid chromatography. The 15-dienone obtained from this reaction was consistently enriched in 3H relative to 14C after isolation and purification. Evidence was obtained to indicate that the majority of the 3H in the products was retained in its original location and configuration. These results indicate that the biomimetic chemical reaction is stereo-random with respect to hydrogen loss from carbon 10 and that, in contrast to the reaction as it occurs in leukocytes and platelets, in the chemical model the reaction begins by decomposition of the hydroperoxide group, with hydrogen loss from carbon 10 occurring as a late or final step.

A series of stereospecifically labeled polyunsaturated fatty acids were prepared by biosynthesis from [S-DR-3H]and [S-~S-~Hlstearic acids. The labeled stearic acids were synthesized by a novel scheme employing readily available alkyne and aldehyde starting materials. The stereochemical purity of the prochiral tritium labels was judged to be >99%, as determined by analysis of the octadec-l-yn-S(R)-and S(S)-ol intermediates in the synthesis.
Previously, the labeled arachidonic acids were used to investigate the stereoselectivity of hydrogen abstraction in the biosynthesis of leukotriene epoxides. We have now investigated the selectivity of hydrogen abstraction in a chemical synthesis of 14,15-leuko-

D~-~H ]or [~O-L~-"H]~B(S)-HPETE methyl esters.
Reaction with either chirally labeled precursor led to 70-95% retention of 'H relative to 14C in the 14,lB-LTA4 and 10-2-14,lB-LTA4 products after purification by high performance liquid chromatography. The 15dienone obtained from this reaction was consistently enriched in 3H relative to 14C after isolation and purification. Evidence was obtained to indicate that the majority of the 'H in the products was retained in its original location and configuration. These results indicate that the biomimetic chemical reaction is stereorandom with respect to hydrogen loss from carbon 10 and that, in contrast to the reaction as it occurs in leukocytes and platelets, in the chemical model the reaction begins by decomposition of the hydroperoxide group, with hydrogen loss from carbon 10 occurring as a late or final step.
Studies on leukotriene biosynthesis in leukocytes and platelets have revealed that, during the course of leukotriene A, and 14,15-leukotriene A4 formation from their respective hy-* This work was supported by Grant GM 15431 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$Supported by the Vivian Allen Fund of Vanderbilt Medical School. Present address: Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115. ** To whom correspondence should be addressed. droperoxide precursors 5(S)-HPETE' and 15(S)-HPETE, stereoselective hydrogen removal occurs from the prochiral center' at carbon 10 (1)(2)(3). While the hydrogen removed from carbon 10 in these two cases has the opposite absolute configurations, being pro-R in the case of LTA4 and pro-S in the case of 14,15-LTA4, it is of interest that in each case the hydrogen which is removed occupies the same relative configuration with respect to the parent hydroperoxide group. It has also been shown for LTA, biosynthesis that a large primary isotope effect is associated with substitution of 3H into the pro-R position at carbon 10 of 5(S)-HPETE (1, 2). These findings are reminiscent of results obtained in studies on the soybean, platelet, and corn lipoxygenase enzymes (4)(5)(6). Stereospecific hydrogen abstraction has also been demonstrated for the reactions catalyzed by the cyclooxygenase and RBL cell 5-lipoxygenase (7, 8). In each case, stereoselective hydrogen removal precedes oxygen insertion and, when 3H is substituted for hydrogen, an isotope effect can be detected. The similarity between the findings obtained for the platelet 12-lipoxygenase and the formation of 14,15-LTA4 recently led us to propose that biosynthesis of 14,15-LT& from 15(S)-HPETE is catalyzed by an enzyme with many mechanistic features in common with the 12-lipoxygenase (3).
While the results on leukotriene biosynthesis are clearly consistent with an enzymatic mechanism of formation, it is possible that similar findings might obtain in the nonenzymatic formation of leukotriene epoxides from HPETE precursors. The possibility exists that the observed stereospecificity of hydrogen removal and the accompanying isotope TLC, thin layer chromatography; THF, tetrahydrofuran.
* The 10-carbon is prochiral in the sense that replacement of either of the two hydrogen atoms attached to it with a new point ligand of different priority results in a chiral assembly. The pro-R (or ~g ) hydrogen is that which, when arbitrarily accorded a priority higher than that of the other enantiotopic hydrogen (e.g. replacement with tritium) but not higher than that of the other remaining ligands, results in an assignment of R configuration according to the R/S system. Similarly, the pro-S (or LS) hydrogen is that which yields an S configuration by application of the same rule. By convention, the p r o 3 and pro-S nomenclature refers to the configuration in the parent arachidonic acid. This is because introduction of the 15(S)hydroperoxy group in the formation of 15(S)-HPETE from arachidonic acid results in a reversal of configuration according to strict application of the priority system. 4217 effect might actually be general features of the leukotriene epoxide-forming reaction mechanism and not necessarily reflections of enzymatic control of the reaction. This notion is plausible because the pre-existing chirality of the precursor 5(S)or 15(S)-hydroperoxide group could conceivably direct the chiral loss of hydrogen from carbon 10, even in a nonenzyme-catalyzed process. Two different nonenzymatic reactions are known to lead to leukotriene epoxide formation from HPETEs. The first of these is the chemical biomimetic reaction, in which a HPETE as the methyl ester is derivatized as the triflate or mesylate in the presence of a highly hindered non-nucleophilic base, with formation of leukotriene epoxide as the major product (9)(10)(11)(12). Hydroxy-epoxy derivatives have also been used as precursors for this type of reaction (13). In addition, leukotriene epoxides can also be formed from their corresponding hydroperoxides by autoxidative processes (14,15).
In order to test the idea that the stereoselectivity and isotope effects observed in the cellular formation of LTA, and 14,15-LT& from 5(S)-and 15(S)-HPETEs really do result from enzymatic control of the reaction, [~O-LS-~HI-and [lo-~~-~H]arachidonic acids were prepared and used to investigate the mechanism involved in the two nonenzymatic routes of leukotriene epoxide formation. The present report discusses the stereochemical fidelity with respect to hydrogen loss at carbon 10 in the chemical biomimetic formation of 14,15-LTA4 from 15(S)-HPETE. An accompanying report describes the findings for the autoxidative formation of HPETEs and leukotriene epoxides (15). In neither case is the same pattern of hydrogen loss and isotope effect observed as is found with incubation of these stereolabeled materials with leukocytes and platelets. From these results, we conclude that the key steps in leukotriene epoxide formation by leukocytes and platelets are in fact enzyme catalyzed.

RESULTS
Stereochemical Purity of Starting Materials-In the resolution step of the chemical synthesis, Fig. 1, octadec-6-yn-8(R,S)-ol was converted to the carbamate of dehydroabietylamine, and the R and S enantiomers were separated by HPLC (see Miniprint). The final preparations of R and S enantiomers were 99.3 and 100% pure, respectively (see Miniprint, Fig. S4). Acetylenic migration to give the terminal alkynols did not change the stereochemical purity. Tosylation  (15-dienone methyl ester). Small amounts of conjugated pentaene, presumably formed as an elimination product, were observed in some reactions. This side product showed Amax (hexane) at 376,355, 337, and 319 nm and eluted just after the solvent front on SP-HPLC. In experiments employing radioactive substrates, no other radioactive reaction products could be detected, and no 15(S)-HPETE methyl ester or 15(S)-HETE methyl ester were recovered. Following extractive isolation, the crude yield of the three main products assayed from Amax at 280 nm totaled 4040%.
SP-HPLC analysis demonstrated the presence of two main peaks absorbing in the UV at 280 nm, as shown for the case of an experiment involving the 15(S)-HPETE methyl ester derived from the pro-S-labeled arachidonic acid, Fig. 2. The first eluting peak was 14,15-LTA4 methyl ester, which after SP-HPLC was nearly, but not completely, pure. The second peak consisted of 102-14,15-LT& methyl ester and the 15dienone methyl ester, which was marginally resolved from the epoxide, chromatographing in the back half of the second UV absorbing peak. In later experiments, using a newer chromatographic column, the 15-dienone and 102-14,15-LT& could be resolved to about a 10% trough. In either case, the three reaction products were purified to homogeneity, Fig. 3, by recycling them over SP-HPLC or, as described below, by RP-HPLC.
Resolution of these products by SP-HPLC revealed that 14,15-LT& methyl ester was the major product in all experiments. When the 102-14,15-LTA4 methyl ester and the 15dienone methyl ester in the second UV absorbing peak were resolved, the molar distribution of the three products of the reaction could be calculated as follows: 49 f 8% 14,15-LT& methyl ester, 25 f 11% 102-14,15-LT& methyl ester, and 26 f 6% 15-dienone methyl ester (mean f S.D., n = 6), or a ratio of about 2:l:l. As reported by Corey and Barton (12), we observed that recovery of 14,15-LT& methyl ester from SP-HPLC was highly dependent on the length of time spent on the chromatographic column. Using a solvent system composed of 1% triethylamine in hexane and varying the column flow rate from 2.0 to 1.0 to 0.5 ml/min resulted in ratios of the first, 14,15-LTA4 peak to the second peak, comprised of 102-14,15-LT& and the stable 15-dienone, of 2.6, 2.2, and 1.6, respectively. A flow rate of 1.0 ml/min was selected as offering the best compromise between resolution and yield. The final isolated yield of analytically pure 14,15-LT& methyl ester in these experiments after SP-HPLC purification ranged from 8 to 18%, with corresponding recoveries of the other two products.
Epoxide Reactions Employing lS(S)-HPETE Methyl Esters Prepared from [3-"C,lo-~~-~H]and [3-'4C,10-~R-3HlArachidonic Acids-The biomimetic epoxide reaction was conducted using 15(S)-HPETE methyl ester (3.6 mg, 150,000 dpm 3H, 40,000 dpm I4C) derived from pr~-S-[~H]arachidonic acid and the resulting products separated by SP-HPLC. The eluate was monitored with UV detection at 280 nm, and aliquots of individual fractions were counted by liquid scintillation counting. The three main products appeared as two main radioactive and UV absorbing peaks, with the 15-dienone present as a shoulder on the backside of the peak corre-

FIG. 1. Synthetic scheme used to prepare stereospecifically labeled [S-D~-'H]-and [8-~~-'H]stearic acids and [lo-D~-'Hl-and [lO-Ls-'HJarachidonic acid and l6(S)-
HPETEs. The tosylation-LiA13H~ reduction sequence is associated with inversion of configuration at the asymmetric center. Absolute stereochemistry and enantomeric purity were assigned from the propargyl intermediate and the terminal alkynol (intermediates 2 and 3 in the scheme). For experimental details and pertinent spectroscopic data see Miniprint Supplement. The 15(S) HPETEs used in the experiment were formed from the arachidonic acids by reaction with the soybean lipoxygenase followed by extraction and esterification in CH2N2. sponding to 10Z-14,15-LTA4, Fig. 2.
In a second experiment involving the pro-S-labeled material, the 102-14,15-LT& methyl ester isolated by SP-HPLC was heated at 55 "C in benzene under argon for 12 h. This resulted in quantitative conversion to the more stable non- The results of liquid scintillation counting to high accuracy of the chromatographically pure products obtained from the leukotriene epoxide reaction using 15(S)-HPETE methyl ester derived from [lO-~s-~H]arachidonic acid are shown in Table I. The retention of 3H relative to I 4 C in the 14,15-LT& and 102-14,15-LT& products ranged from 77 to 85%, while the 15-dienone methyl ester underwent an enrichment in 3H relative to 14C, with 109 to 129% 3H/14C relative to starting 15(S)-HPETE methyl ester. These results indicate a modest apparent loss of 3H in the formation of the epoxide reaction products. On a molar basis, the 3H enrichment observed in the 15-dienone can account only for about 30% of the 3H loss in the epoxide products. It is likely that the remainder of the residual 3H is accounted for by that found in the aqueous phase after extraction of the reaction products with organic solvent.
The workup and analysis of experiments conducted using 15(S)-HPETE methyl ester obtained from pro-R-[3H]arachidonic acid were similar to that described for the pro-S experiments. 14,15-LT& methyl ester was purified by SP-HPLC. These experiments led to the result that the 14,15-LT& methyl ester retained from 89 to 95% of the 3H to 14C ratio of specific activities found in the starting material.
Because the combination of initial SP-HPLC purification, followed by conversion to conjugated tetraene and RP-HPLC purification, was considered a particularly powerful purification scheme, the 102-14,15-LT& methyl ester in both cases was subjected to this sequence. The rigorously purified conjugated tetraene epoxide methyl ester obtained in this manner had from 70 to 74% of the starting 15(S)-HPETE methyl ester ratio of 3H-to 14C-specific activities. The 15-dienone isolated from reaction with 15(S)-HPETE methyl ester prepared from pro-R-labeled arachidonic acid was also purified by sequential SP-HPLC and RP-HPLC. In addition to HPLC, the 15dienone was found homogeneous by UV spectroscopy. Liquid scintillation counting showed 111 to 113% of the 'H/i% ratio of specific activities as found for the starting 15(S)-HPETE methyl ester. These results are summarized in Table I.
Purity of Products-The purity of all products was assayed by SP-HPLC with UV detection and by UV spectroscopy (Figs. 3 and 5). Liquid scintillation of individual SP-and RP-HPLC fractions was also performed. In some instances, epoxide samples were subject to 'H Fourier transform NMR at 90 MHz. In other cases, an aliquot of the SP-HPLC-purified epoxides was treated with dimethoxyethane-pH 3 water to effect conversion to hydrolysis products, and the purity of these was assessed by RP-HPLC with UV detection. Following initial SP-HPLC separation of the reaction products (Fig. 2)  The absolute ratio of counts/min and percentage relative to the 15(S)-HPETE starting material, arbitrarily defined as lOO%, are given. The counting error varied, but was nominally less than -~5% (2 S.D.). All values given are for chromatographically purified and spectroscopically homogeneous samples. A ratio for 14,15-LTA1 in pro-S experiment II (+) is not given because although pure by HPLC and UV, the sample contained impurities apparent by 'H NMR and when counted gave a clearly spurious result. PrO tion-The present results clearly demonstrate that the cellular and chemical formation of leukotriene epoxides from their HPETE precursors are mechanistically distinct. The cellular biosynthesis of LT& or 14,15-LTA4 from 5 or 15HPETE is accompanied by stereoselective hydrogen loss from carbon 10 and also by a substantial primary isotope effect, detected by progressive tritium enrichment in unused substrate as the reaction proceeds. In addition, a smaller secondary isotope effect can also be detected when experiments are performed using substrate containing 3H label geminal to the site of stereoselective hydrogen abstraction. In contrast, the data presented in Table I indicate that hydrogen loss from carbon In the absence of isotope effects, it would be predicted that each leukotriene epoxide would retain 50% of its original 3H relative to 14C if a strictly stereorandom process were operative. In the chemical reactions described here, 3H retentions on the order of 70-95% relative to 14C were observed for the two isomeric epoxides formed from pro-R-and pro-S-stereolabeled material. The best explanation for this finding is that hydrogen loss from carbon 10 does not occur as the initial step in the chemical reaction mechanism as it does in leukotriene biosynthesis. In leukotriene biosynthesis, hydrogen abstraction from carbon 10 precedes or coincides with the attainment of the activation energy required for the reaction (1-3). In all likelihood, hydrogen removal is in fact the activation energy-requiring process. Hence, the 3H-labeled substrate reaches the transition state at a slower rate than the I4C standard, thus leaving the unconverted substrate pool progressively 3H. enriched as the reaction progresses. In contrast, in the chemical reaction, the data presented in Table I would be consistent with hydrogen loss from carbon 10 occurring after the required activation energy along the reaction coordinate has been attained. This would be the case if the chemical reaction were initiated by elimination of triflic anion accompanied by concomitant formation of an allylic carbocation at C-13, followed by loss of a proton from carbon 10 as a late or ultimate step. Such a scheme, involving formation of a carbonium ion intermediate via electrophilic attack of the peroxy triflate, with final loss of a proton from C-10, is summarized in Fig. 6. In this case, once having surpassed the transition state in the rate-determining step, the carbonium ion intermediate must either undergo obligate loss of a proton from C-10 to yield the leukotriene epoxides or, alternatively, decompose to a different product. In the event of stereospecific substitution with tritium at C-10, the geminal protium to C-10 bond is broken more rapidly than the tritium to C-10 bond, resulting in net 3H retention in the epoxide products, in excess of 50%. This thus accounts for 3H retentions on the order of 70-95% in the leukotriene epoxides in the present experiments. It should be noted that such a finding is not entirely surprising. Previously, Corey et al. (16) reported an analogous reaction involving the triterpene 11a,12a-oxidotaraxerol in which it was postulated that oxirane formation from a hydroperoxide precursor might occur with electrophilic attack of the hydroperoxide group on an allylic double bond to yield a carbocation intermediate, followed by a subsequent rearrangement and proton loss. Thus, the biomimetic chemical formation of leukotriene epoxides is not truly "biomimetic," because although both the cellular enzymatic and chemical processes result in conversion of a hydroperoxide to a leukotriene epoxide, they proceed by entirely different mechanisms.
One additional experimental finding that must be incorporated into the proposed mechanism for the chemical biomimetic reaction relates to the observed 'H/'*C enrichment in the 15-dienone reaction product. While this product clearly can and undoubtedly does in part originate from 15(S)- The retention of 3H in the reaction products following reactionemployingeither [~O-DR-~HIor [~O-LS-~H]~~(S)-HPETE methyl ester as substrate indicates that loss of hydrogen from carbon 10 is a late step in the reaction mechanism. In the scheme shown, this is postulated as occurring after initial decomposition of the trifluoromethane sulfono peroxide to give a carbonium ion. The formation of the 15-dienone from the carbonium ion intermediate is proposed in order to account for the consistent enrichment in 3H observed in the 15-dienone relative to the 15(S)-HPETE methyl ester starting material.
On the Chemical Conversion of HPETE to Leukotriene Epoxide HPETE by simple, 1,2-elimination, the finding of 3H/14C enrichment in the dienone indicates that the situation in the present reaction is probably more complex. This is because there is no required loss of hydrogen from carbon 10 in conversion of 15(S)-HPETE methyl ester to 15-dienone methyl ester. Thus, in the case of simple, 1,2-elimination, there should be no change in 3H/14C ratio. Tritium enrichment in the 15-dienone likely indicates that this reaction by-product can also be formed from intermediateb) generated after collapse of the hydroperoxy triflate but prior to the expulsion of hydrogen from C-10 that occurs as a late if not ultimate step in the reaction process. The fact that some 3H is apparently lost from the epoxide reaction products is consistent with either actual loss of 3H from the carbocation intermediate or, alternatively, with decomposition to some other reaction product which would be tritium containing and possibly, tritium-enriched.
An alternate explanation for the above findings which was excluded related to the unlikely possibility that tritium migration to other sites within the product molecules during the course of the reaction was responsible for the observed retention of 3H in excess of 5096, following reaction with stereolabeled substrate. This possibility was dismissed by the findings that 1) reaction conducted using [5,6,8,9,1,12,14,15-2Hs] 15(S)-HPETE methyl ester led to complete retention of all eight deuteriums in their original positions in the epoxidederived hydrolysis products when the latter were analyzed by gas chromatography-mass spectrometry, and 2) reductive ozonolysis of the 8,15-dihydroxy acids derived from 14,15-LTA3 as their methyl ester acetates and also analysis of the 15-dienone by sodium borohydride reduction, saponification, and reaction with soybean lipoxygenase, which established that the 3H in these products was in its original position and configuration (Miniprint).
The present study indicates that the chemical conversion of 15(S)-HPETE to 14,15-LT&, and presumably also the analogous conversion of 5(S)-HPETE to LT&, occurs by a mechanism which is different from that involved in the biosynthesis of these compounds. The chemical conversion of hydroperoxide to leukotriene epoxide would thus not appear to be a useful model of the enzyme-catalyzed reaction. Studies described in the companion paper concerning the autoxidative formation of 14,15-LT& from 15(S)-HPETE reveal that process to be stereorandom with respect to hydrogen loss from carbon 10 and to proceed with hydrogen loss as an early or initial step. The chemical, autoxidative, and cellular formation of leukotriene epoxides can all three be clearly distinguished by this type of analysis, which thus establishes the premise that the latter process, as it occurs in platelets and leukocytes, is an enzymatic process.  91 (t, 2Hl. 2.26 11. 2Hi, ll.P2Im  hepfanal Cpd 11. There fragment. were treated with either lAc12-0 or BSiFA. and pyridine wifh analysis ?-hydroxy methyl dadecenoate, obcained by oxidative ozonolysis shorn. The resulting diastereomers of occsdec-6-yn-8(S)-ol and 8(R)-ol were resolved on GC (3% QF-1) as described (24) with the 8(S) diastereomer having the shorter retention time.