[40]
SYNTHESIS OF EPOXYEICOSATRIENOIC ACIDS
357
conditions where substrate was near saturation and in haft-life experiments carried out at subsaturating levels of substrate (Table II). This is presumably due to the longer oxoacyl group esterifying the sn-2 position, in agreement with our findings with the valeroyl analog of PAF. Additional experiments, including antibody cross-reactivity, have allowed us to establish that in plasma, PAF and oxidized phospholipids are hydrolyzed by the same phospholipase, i.e., the PAF acetylhydrolase.
[40] S y n t h e s i s of E p o x y e i c o s a t r i e n o i c Acids a n d H e t e r o a t o m Analogs
By J. R. FALCK, PENDm YADAGIRI, and JORGE CAPDEVILA Various studies have established an alternative route for the enzymatic generation of biologically active, oxygenated eicosanoids. 1 This pathway is known as the "epoxygenase" branch of the arachidonate cascade and is mediated by cytochrome P-450. Several epoxygenase metabolites have been identified as endogenous constituents of mammalian systems,2-6 but their physiological significance is presently unclear. A comprehensive review of the current status of the epoxygenase pathway with a critical discussion of its implications has appeared. 7 The most characteristic and extensively studied epoxygenase metabolites are the four regioisomeric cis-epoxyeicosatrienoic acids (EETs) (Fig. 1). They are relatively stable to hydrolysis'at physiological pH [e.g., leukotriene A4 (LTA4) and hepoxilin A3].8 The exception is 5,6-EET I j. Capdevila, G. Snyder, and J. R. Falck, in "Microsomes and Drug Oxidations" (A. R. Boobis, J. Caldwell, F. DeMatteis, and C. R. Elcombie, eds.), p. 84. Taylor and Francis, Ltd., London and New York, 1985. 2 j. Capdevila, B. Pramanik, J. L. Napoli, S. Manna, and J. R. Falck, Arch. Biochem. Biophys. 231, 511 (1984). 3 R. Toto, A. Siddhanta, S. Manna, B. Pramanik, J. R. Falck, and J. Capdevila, Biochim. Biophys. Acta 919, 132 (1987). 4 j. R. Falck, V. J. Schueler, H. R. Jacobson, A. K. Siddhanta, B. Pramanik, and J. Capdevila, J. Lipid Res. 28, 840 (1987), R. C. Murphy, J. R, Falck, S. Lumin, P. Yadagiri, J. A. Zirrolli, M. Balazy, J. L. Masferrer, N. G. Abraham, and M. L. Schwartzman, J. Biol. Chem. 263, 17197 (1988). 6 p. M. Woollard, Biochem. Biophys. Res. Commun. 136, 169 (1986). 7 F. A. Fitzpatrick and R. C. Murphy, Pharmacol. Rev. 40, 229 (1989). 8 N. Chacos, J. Capdevila, J. R. Falck, S. Manna, C. Martin-Wixtrom, S. S. Gill, B. D. Hammock, and R. W. Estabrook, Arch. Biochem. Biophys. 223, 639 (1983).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
358
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS O
~
[40]
O O CO2H
CO2H
5,6-EET
8,9-EET
o
O 11,12-EET
14,15-EET
C02H
N H 14,15-EET AZIRIDINE
_ _
~
CO2H
S
14,15-EET THIIRANE
FIG. 1. Structure of epoxyeicosatrienoic acids and heteroatom analogs.
which, in its free-acid form, readily decomposes to 5,6-dihydroxyeicosatrienoic acid and/or the corresponding 8-1actone. 9 The absolute configuration of the EETs has been determined using material obtained from in vitro incubation of arachidonic acid with the major phenobarbital-inducible form of rat liver microsomal cytochrome P-450. lORecent results suggest, however, that the enantiomeric composition is highly dependent on the identity of the cytochrome P-450 isozyme. 1~This may be significant since in some instances biological activity is sensitive to EET stereochemistry.12 Chiral syntheses of all the EET enantiomers have been achieved (Table I). In general, these syntheses are impractical for laboratories not equipped for multistep organic synthesis. Most investigators have instead 9 D. Schlondorff, E. Petty, J. A. Oates, M. Jacoby, and S. D. Levine, Am. J. Physiol. 253, F464 (1987). ,o j. R. Falck, S. Manna, H. R. Jacobson, R. W. Estabrook, N. Chacos, and J. Capdevila, J. Am. Chem. Soc. 106, 3334 (1984). u j. Capdevila, unpublished results, 1989. 12 F. Fitzpatrick, M. Ennis, M. Baze, M. Wynalda, J. McGee, and W. Liggett, J. Biol. Chem. 261, 15334 (1986).
[40]
359
SYNTHESIS OF EPOXYE1COSATRIENOIC ACIDS TABLE I ASYMMETRICSYNTHESESOF EPOXYEICOSATRIENOICACIDS AND ANALOGS
EET isomer
Overall yield (%)
Chirality source
Source"
5(S),6(R) 5(R),6(S) 5(S),6(R)-20-OH 8(S),9(R) 8(R),9(S) 11(S), 12(R) 11(S), 12(R) 1I(R), 12(S) 14(S),15(R) 14(S), 15(R) 14(R),15(S) 14(R),15(S) 14(R), 15(S) trans-14(S),15(S) trans-14(R), 15(R) trans-14(R),15(R) 14(R), 15(S)-20-OH
5 7 11 15-20 15-20 15-20 1-3 15-20 2-6 15 3 13 15 b b 33 11
2-Deoxy-D-glucose 2-Deoxy-D-glucose D~methyl L-malate Dimethyl L-malate Dimethyl D-malate Dimethyl D-malate Methyl 15(S)-HETE Dimethyl L-malate Methyl 15(S)-HETE Sharpless epoxidation Methyl 15(S)-HETE 2-Deoxy-D-glucose Sharpless epoxidation Sharpless epoxidation Sharpless epoxidation Methyl 15(S)-HETE Dimethyl L-malate
1 I 2 3 3 3 4 3 4 5 4 1 5 5 5 4 2
a Key to references: (1) C. A. Moustakis, J. Viala, J. Capdevila, and J. R. Falck, J. Am. Chem. Soc. 1tl7, 5283 (1985); (2) S. Lumin, P. Yadagiri, J. R. Falck, J. Capdevila, P. Mosset, and R. Gree, J. Chem. Soc. Chem. Commun. p. 389 (1987); (3) P. Mosset, P. Yadagiri, S. Lumin, J. Capdevila, and J. R. Falck, Tetrahedron Lett. 27, 6035 (1986); (4) J. R. Falck, S. Manna, and J. Capdevila, Tetrahedron Lett. 25, 2443 (1984); (5) M. D. Ennis and M. E. Baze, Tetrahedron Lett. 27, 6031 (1986). b Incomplete data.
relied on racemic EETs prepared by Corey's site-specific oxidation methodology 13'~4 o r , more conveniently, by nonselective peracid epoxidation of arachidonic acid. t5 This chapter describes procedures based on the latter reaction that have proved reliable in the authors' laboratories for the production and purification of nanomole-millimole amounts of EETs and their methyl esters. In light of the increasing interest in EET heteroatom analogs, protocols 16for converting EETs to cis-thiiranes and cis-aziridines (Fig. 1) are also included. 13 E. J. Corey, H. Niwa, and J. R. Falck, J. Am. Chem. Soc. 101, 1586 (1979). 14 E. J. Corey, A. Marfat, J. R. Falck, andJ. O. Albright, J. Am. Chem. Soc. 102, 1433 (1980). is S.-K. Chung and A. I. Scott, Tetrahedron Lett. p. 3023 (1974). t6 j. R. Falck, S. Manna, J. Viala, A. K. Siddhanta, C. A. Moustakis, and J. Capdevila, Tetrahedron Lett. 26, 2287 (1985).
360
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[40]
Experimental Section
General Procedures. Dichloromethane is distilled from calcium hydride. Tetrahydrofuran and ether are distilled from sodium benzophenone ketyl. All other solvents and solutions are peroxide free and sparged with argon to remove dissolved oxygen immediately prior to use. All reactions and distillations are conducted under an inert atmosphere of nitrogen or argon. Methyl arachidonate (>99%) and arachidonic acid (>99%) are obtained from Nu-Chek-Prep (Elysian, MN). Radiolabeled arachidonate (14C or 3H) is purchased from DuPont-New England Nuclear (Boston, MA) or Amersham (Arlington Heights, IL) and mixed with unlabeled arachidonate to give the desired specific activity prior to epoxidation. 3-Chloroperoxybenzoic acid (80-85%, technical grade) and all other chemicals which are reagent grade or better are obtained from Aldrich (Milwaukee, WI). Woelm neutral alumina W-200 (activity grade I) is obtained from ICN Pharmaceuticals (Cleveland, OH). Eicosanoids are stored at - 7 8 ° under argon in glass containers with fluorocarbon-faced caps. Extracts are dried over anhydrous sodium sulfate. Solvent removal refers to evaporation under reduced pressure on a rotary evaporator. Thin-layer chromatography (TLC) is performed on silica gel 60 F-254 plates (20 x 20 cm, 0.25 mm thickness) from E. Merck (Darmstadt, FRG) in glass tanks preequilibrated with solvent. Products are visualized by transillumination of the plate with a bright, white light, by exposure to iodine vapor, or by spraying with 5% ethanolic phosphomolybdic acid and heating briefly at 170°. Silica gel is removed from the TLC plate with a razor blade and extracted with methanol/dichloromethane (CH2C12) (1:10). Radioactivity of HPLC eluants is measured on-line with a Ramona-LS (Raytest Gmbh). Epoxidation of Arachidonic Acid 3-Chloroperoxybenzoic acid (19 mg, 0.11 mmol) is added portionwise to a stirring, 0° solution of arachidonic acid (31 mg, 0.10 mmol) in CH2C12 (8 ml). After 12 hr at 0°, methyl sulfide (Me2S) (50/~1) is added and the mixture maintained at ambient temperature for 20 min to quench any remaining peracid. The reaction is diluted with CH2C12 (15 ml), transferred to a separatory funnel, washed with water (3 x 15 ml), then with saturated NaCI solution (15 ml), and dried. Solvent removal gives a viscous oil which is purified immediately. Normal-phase HPLC purification (Fig. 2) yields unreacted arachidonic acid (8 mg), 14,15-EET (7 mg), ll,12-EET (5 mg), 8,9-EET (3 mg), and 5,6-EET (1.5 mg). Comparable results are obtained using TLC and methanol/CH2Cl2 (5:95, 5 elutions) as eluant: arachidonic acid ( g f ~-"
[40]
SYNTHESIS OF EPOXYEICOSATRIENOIC ACIDS
361
100.$U)
75 w
50 rr
E ~ 25 0
A I
5
A I
1'0
15
,
20
2=5
Minutes
FIG. 2. Normal-phase HPLC analysis of the reaction products from epoxidation of arachidonicacid. The crude productis resolvedon a Beckman(Fullerton,CA) UltrasphereSi (5/zm, 25 x 0.46 cm) using0.4%2-propanol/0.1% acetic acid/99.5% hexaneat a flowrate of 2 rnl/min:arachidonicacid (Rt ~ 8.14 min), 14,15-EET(Rt ~ 11.13rain), 11,12-EET(Rt -~ I 1.47 rain), 8,9-EET (Rt ~ 15.40), and 5,6-EET (Rt ~ 23.44 rain). Radioactivityis monitored on-line with a Ramona-LS.
0.76), 14,15-EET (Rf ~ 0.70), 11,12-EET (Rf ~ 0.68), 8,9-EET (gf ~ 0.60), 5,6-EET (Rf ~ 0.54), and by-products (Rf ~ 0.47-0.40).
Epoxidation of Methyl Arachidonate 3-Chloroperoxybenzoic acid (25 mg, 0.14 mmol) is added portionwise to a stirring, 0° mixture of methyl arachidonate (43 rag, 0.135 mmol) and anhydrous NaECO3 (17 mg, 0.16 mmol) in CHECI2 (4 ml). After stirring for 15 hr at 0°, Me2S (50/xl) is added and the mixture maintained at ambient temperature for 20 min to quench any remaining peracid. The reaction is diluted with CHEC12 (15 ml), transferred to a separatory funnel, washed with water (3 x 10 ml), then with saturated NaC1 solution (10 ml), and dried. Solvent removal gives a viscous oil (43 mg). Reversed-phase HPLC purification (Fig. 3) affords unreacted methyl arachidonate (12 rag), a coeluting mixture of three methyl EETs (i.e., methyl 5,6-, 8,9-, and ll,12-EET), methyl 14,15-EET (6 rag), and polar by-products. Resolution of the mixed methyl EET fraction by normalphase HPLC (Fig. 4) furnishes methyl 5,6-EET (3 mg), methyl 8,9-EET (5 mg), and methyl II,12-EET (5 rag). TLC purification of the crude reaction product using ethyl ether (EtEO)/CHECl2/hexane (1 : 1 : 8, 5 elutions) gives incomplete resolution of the regioisomeric methyl EETs: methyl arachidonate (Rf ~- 0.88), methyl 14,15-, and ll,12-EET (Rf
362
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[40]
tu
,b
tu 0~
oJ 100
o O~
75
> n"
E
Q. O
50
25 0
0
30
40
Minutes FIG. 3. Reversed-phase HPLC analysis of the reaction products from epoxidation of methyl arachidonate. The crude product is resolved partially on a Rainin Microsorb C~8 (5/~m, 25 × 0.46 cm) using a linear gradient of 50% solvent A/50% solvent B to 100% solvent B over 40 min at a flow rate of 1 ml/min: methyl arachidonate (R t = 39.2 min), methyl 5,6-, 8,9-, and 11,12-EET coeluted (Rt ~ 34.1 min), methyl 14,15-EET (Rt ~ 32.7 rain). Solvent A is 0.1% acetic acid/99.9% H20; solvent B is 0.1% acetic acid/99.9% CH3CN. Radioactivity is monitored on-line with a Ramona-LS.
uJ
dJ
100 O
o~ _>
75 tlJ
rr
& Q6
50
E e~ o 25
A; t6
d
I
0
5
,0
ts
20
Minutes FIG. 4. Normal-phase HPLC analysis of methyl 5,6-, 8,9- and II,12-EET. The mixed methyl EET fraction from Fig. 3 is resolved on a Waters (Milford, MA)/zPorasil (5/xm, 25 × 0.46 cm) using a linear gradient from 0.15% 2-propanol/99.85% hexane to 0.4% 2-propanol/ 99.6% hexane over 30 min at a flow rate of 2 ml/min: methyl 5,6-EET ( R t ~ , 18.0 min), methyl 8,9-EET (Rt ~ 11.5 rain), and methyl 11,12-EET (Rt ~ 9.6 min). Radioactivity is monitored on-line with a Ramona-LS.
[40]
SYNTHESIS OF EPOXYEICOSATRIENOIC ACIDS
363
0.55), methyl 8,9-EET (Rf ~ 0.51), methyl 5,6-EET (Rf ~ 0.42), and polar by-products (Rf = 0.16).
Saponification of Methyl 8,9-, 11,12-, or 14,15-EET: General Procedure To a stirring, 0° solution of methyl EET (16.7 mg, 0.05 mmot) in MeOH (4.5 ml) is slowly added 0.33 N aqueous NaOH (1.5 ml). The homogeneous reaction is stirred at ambient temperature and monitored by TLC analysis until complete (=12 hr). After cooling to 0°, the reaction is diluted with water (5 ml) and carefully acidified with 0.25 M aqueous oxalic acid (1.0 ml) to pH 4-4.5. The solution is transferred to a separatory funnel and extracted with Et20 (3 x 25 ml). The combined ethereal extracts are washed with water until the aqueous layer is neutral, then washed with saturated NaCI solution (30 ml), and dried. Solvent removal affords EETfree acid in essentially quantitative yield. The products can be purified by HPLC as described in Fig. 2. TLC analysis using methanol/CH2Cl2 (5:95) as eluant shows: 8,9-EET (Rf 0.17), ll,12-EET (Rf = 0.18), and 14,15-EET (Rf -~ 0.18).
Saponification of Methyl 5,6-EET To a stirring, 0° solution of methyl 5,6-EET (16.7 mg, 0.05 mmol) in tetrahydrofuran (4.5 ml) is slowly added 0.41 N aqueous NaOH (1.5 ml). The homogeneous mixture is stirred at ambient temperature until the hydrolysis is judged complete (~12 hr) by TLC analysis (ethyl acetate/ hexane, 1 : 2, ester Rf ~ 0.49). The reaction is diluted with ice chips (15 g) and transferred to a separatory funnel containing ice-cold water (30 ml), Et20 (30 ml), 0.25 M aqueous oxalic acid (1.1 ml), and glacial acetic acid (100/xl). After vigorous mixing, the upper organic layer is separated. The aqueous layer is extracted again with fresh EtzO (2 x 20 ml) and the combined organic extracts are washed with water, until the washings are neutral, then with saturated NaC1 solution (25 ml), and dried. Solvent removal affords 5,6-EET-free acid accompanied by a variable amount of the corresponding 5,6-diol. The products are somewhat labile and should be used without delay following purification by HPLC (Fig. 2) or by TLC using methanol/CH2C12 (1 : I0) as eluant: 5,6-EET (Rf ~ 0.38), 5,6-diol (Rf = 0.17).
Thiirane (Episulfide ) Heteroatom Analog Preparation of the cis-14,15-thiirane (episulfide) heteroatom analog ~6 from methyl 14,15-EET is representative. A mixture of methyl 14,15-EET (25 mg, 0.075 mmol) and anhydrous KSCN (145 mg, 1.5 mmol) in dry methanol (10 ml) is heated to reflux for
364
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[40]
72 hr. After cooling to 0°, the reaction is diluted with water (30 ml), carefully acidified to pH 4.5 with dilute hydrochloric acid, and extracted in a separatory funnel with Et20/hexanes (1 : I, 3 × 20 ml). The combined organic extracts are washed with water (3 x 20 ml), then with saturated NaCI solution (30 ml), and dried. Solvent removal gives an oily residue which is purified by TLC eluted with Et20/hexanes (1 : 2) to furnish methyl 14,15-thiirane (20.5 mg, Rf ~ 0.55) and 14,15-thiirane-free acid (2 mg, Rf -~ 0.24). The methyl ester is hydrolyzed as described for methyl 14,15-EET. Aziridine Heteroatom Analog
Preparation of the cis-14,15-aziridine heteroatom analog16 from methyl 14,15-EET is representative. A solution of sodium azide (45 nag, 0.69 mmol) in water (0.6 ml) is added with stirring to benzene (1 ml) followed by concentrated H2SO4 (17/zl). Caution: hydrazoic acid is toxic and should be handled in a hood while wearing gloves and eye protection. After 10 min, the upper organic layer is removed by decantation and dried. According to the method of Posner and Rogers, I7 the resultant benzene solution of hydrazoic acid is added to a well-stirred suspension of Woelm neutral alumina W-200 (600 rag) in dry Et20 (3 ml). A solution of methyl 14,15-EET (25 mg, 0.075 mmol) in dry Et20 (1 ml) is added after 5 min and the stirring continued until the reaction is judged complete (= 10 min) by TLC analysis. The reaction is diluted with methanol (20 ml), stirred vigorously for 15 min, filtered through a Celite (diatomaceous earth) pad, and the filter cake washed thoroughly with methanol. Solvent removal provides the regioisomeric methyl 14,15azidohydrins (23 mg) as an oil which are used directly in the next step. TLC analysis utilizing Et20/hexanes (1 : 2) as eluent gives the following: methyl 14,15-EET (gf ~ 0.40), methyl 14,15-azidohydrins (Rf ~- 0.26 and 0.28). After azeotropic drying with benzene, the above crude product is dissolved in dry Et20 (4 ml). To this is added triphenylphosphine (Ph3P) (27 mg, 0.10 mmol) and the mixture is stirred at ambient temperature for 18 hr, then at reflux for 12 hr. Solvent removal and rapid chromatography of the residue on a column of silica gel (2 g) using CH3CN as eluant gives methyl 14,15-aziridine (17 mg). TLC analysis using CH3CN:Rf ~- 0.14. The ester is hydrolyzed as described for methyl 14,15-EET. Acknowledgments Work in the authors' laboratories was supported by grants from the Robert A. Welch Foundation,JuvenileDiabetesFoundation,Kroc Foundation,NATO,and the USPHSNIH. 17G. H. Posner and D. Z. Rogers,J. Am. Chem. Soc. 99, 8208(1977).
SYNTHESIS OF EPOXYEICOSATRIENOIC ACIDS
357
conditions where substrate was near saturation and in haft-life experiments carried out at subsaturating levels of substrate (Table II). This is presumably due to the longer oxoacyl group esterifying the sn-2 position, in agreement with our findings with the valeroyl analog of PAF. Additional experiments, including antibody cross-reactivity, have allowed us to establish that in plasma, PAF and oxidized phospholipids are hydrolyzed by the same phospholipase, i.e., the PAF acetylhydrolase.
[40] S y n t h e s i s of E p o x y e i c o s a t r i e n o i c Acids a n d H e t e r o a t o m Analogs
By J. R. FALCK, PENDm YADAGIRI, and JORGE CAPDEVILA Various studies have established an alternative route for the enzymatic generation of biologically active, oxygenated eicosanoids. 1 This pathway is known as the "epoxygenase" branch of the arachidonate cascade and is mediated by cytochrome P-450. Several epoxygenase metabolites have been identified as endogenous constituents of mammalian systems,2-6 but their physiological significance is presently unclear. A comprehensive review of the current status of the epoxygenase pathway with a critical discussion of its implications has appeared. 7 The most characteristic and extensively studied epoxygenase metabolites are the four regioisomeric cis-epoxyeicosatrienoic acids (EETs) (Fig. 1). They are relatively stable to hydrolysis'at physiological pH [e.g., leukotriene A4 (LTA4) and hepoxilin A3].8 The exception is 5,6-EET I j. Capdevila, G. Snyder, and J. R. Falck, in "Microsomes and Drug Oxidations" (A. R. Boobis, J. Caldwell, F. DeMatteis, and C. R. Elcombie, eds.), p. 84. Taylor and Francis, Ltd., London and New York, 1985. 2 j. Capdevila, B. Pramanik, J. L. Napoli, S. Manna, and J. R. Falck, Arch. Biochem. Biophys. 231, 511 (1984). 3 R. Toto, A. Siddhanta, S. Manna, B. Pramanik, J. R. Falck, and J. Capdevila, Biochim. Biophys. Acta 919, 132 (1987). 4 j. R. Falck, V. J. Schueler, H. R. Jacobson, A. K. Siddhanta, B. Pramanik, and J. Capdevila, J. Lipid Res. 28, 840 (1987), R. C. Murphy, J. R, Falck, S. Lumin, P. Yadagiri, J. A. Zirrolli, M. Balazy, J. L. Masferrer, N. G. Abraham, and M. L. Schwartzman, J. Biol. Chem. 263, 17197 (1988). 6 p. M. Woollard, Biochem. Biophys. Res. Commun. 136, 169 (1986). 7 F. A. Fitzpatrick and R. C. Murphy, Pharmacol. Rev. 40, 229 (1989). 8 N. Chacos, J. Capdevila, J. R. Falck, S. Manna, C. Martin-Wixtrom, S. S. Gill, B. D. Hammock, and R. W. Estabrook, Arch. Biochem. Biophys. 223, 639 (1983).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
358
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS O
~
[40]
O O CO2H
CO2H
5,6-EET
8,9-EET
o
O 11,12-EET
14,15-EET
C02H
N H 14,15-EET AZIRIDINE
_ _
~
CO2H
S
14,15-EET THIIRANE
FIG. 1. Structure of epoxyeicosatrienoic acids and heteroatom analogs.
which, in its free-acid form, readily decomposes to 5,6-dihydroxyeicosatrienoic acid and/or the corresponding 8-1actone. 9 The absolute configuration of the EETs has been determined using material obtained from in vitro incubation of arachidonic acid with the major phenobarbital-inducible form of rat liver microsomal cytochrome P-450. lORecent results suggest, however, that the enantiomeric composition is highly dependent on the identity of the cytochrome P-450 isozyme. 1~This may be significant since in some instances biological activity is sensitive to EET stereochemistry.12 Chiral syntheses of all the EET enantiomers have been achieved (Table I). In general, these syntheses are impractical for laboratories not equipped for multistep organic synthesis. Most investigators have instead 9 D. Schlondorff, E. Petty, J. A. Oates, M. Jacoby, and S. D. Levine, Am. J. Physiol. 253, F464 (1987). ,o j. R. Falck, S. Manna, H. R. Jacobson, R. W. Estabrook, N. Chacos, and J. Capdevila, J. Am. Chem. Soc. 106, 3334 (1984). u j. Capdevila, unpublished results, 1989. 12 F. Fitzpatrick, M. Ennis, M. Baze, M. Wynalda, J. McGee, and W. Liggett, J. Biol. Chem. 261, 15334 (1986).
[40]
359
SYNTHESIS OF EPOXYE1COSATRIENOIC ACIDS TABLE I ASYMMETRICSYNTHESESOF EPOXYEICOSATRIENOICACIDS AND ANALOGS
EET isomer
Overall yield (%)
Chirality source
Source"
5(S),6(R) 5(R),6(S) 5(S),6(R)-20-OH 8(S),9(R) 8(R),9(S) 11(S), 12(R) 11(S), 12(R) 1I(R), 12(S) 14(S),15(R) 14(S), 15(R) 14(R),15(S) 14(R),15(S) 14(R), 15(S) trans-14(S),15(S) trans-14(R), 15(R) trans-14(R),15(R) 14(R), 15(S)-20-OH
5 7 11 15-20 15-20 15-20 1-3 15-20 2-6 15 3 13 15 b b 33 11
2-Deoxy-D-glucose 2-Deoxy-D-glucose D~methyl L-malate Dimethyl L-malate Dimethyl D-malate Dimethyl D-malate Methyl 15(S)-HETE Dimethyl L-malate Methyl 15(S)-HETE Sharpless epoxidation Methyl 15(S)-HETE 2-Deoxy-D-glucose Sharpless epoxidation Sharpless epoxidation Sharpless epoxidation Methyl 15(S)-HETE Dimethyl L-malate
1 I 2 3 3 3 4 3 4 5 4 1 5 5 5 4 2
a Key to references: (1) C. A. Moustakis, J. Viala, J. Capdevila, and J. R. Falck, J. Am. Chem. Soc. 1tl7, 5283 (1985); (2) S. Lumin, P. Yadagiri, J. R. Falck, J. Capdevila, P. Mosset, and R. Gree, J. Chem. Soc. Chem. Commun. p. 389 (1987); (3) P. Mosset, P. Yadagiri, S. Lumin, J. Capdevila, and J. R. Falck, Tetrahedron Lett. 27, 6035 (1986); (4) J. R. Falck, S. Manna, and J. Capdevila, Tetrahedron Lett. 25, 2443 (1984); (5) M. D. Ennis and M. E. Baze, Tetrahedron Lett. 27, 6031 (1986). b Incomplete data.
relied on racemic EETs prepared by Corey's site-specific oxidation methodology 13'~4 o r , more conveniently, by nonselective peracid epoxidation of arachidonic acid. t5 This chapter describes procedures based on the latter reaction that have proved reliable in the authors' laboratories for the production and purification of nanomole-millimole amounts of EETs and their methyl esters. In light of the increasing interest in EET heteroatom analogs, protocols 16for converting EETs to cis-thiiranes and cis-aziridines (Fig. 1) are also included. 13 E. J. Corey, H. Niwa, and J. R. Falck, J. Am. Chem. Soc. 101, 1586 (1979). 14 E. J. Corey, A. Marfat, J. R. Falck, andJ. O. Albright, J. Am. Chem. Soc. 102, 1433 (1980). is S.-K. Chung and A. I. Scott, Tetrahedron Lett. p. 3023 (1974). t6 j. R. Falck, S. Manna, J. Viala, A. K. Siddhanta, C. A. Moustakis, and J. Capdevila, Tetrahedron Lett. 26, 2287 (1985).
360
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[40]
Experimental Section
General Procedures. Dichloromethane is distilled from calcium hydride. Tetrahydrofuran and ether are distilled from sodium benzophenone ketyl. All other solvents and solutions are peroxide free and sparged with argon to remove dissolved oxygen immediately prior to use. All reactions and distillations are conducted under an inert atmosphere of nitrogen or argon. Methyl arachidonate (>99%) and arachidonic acid (>99%) are obtained from Nu-Chek-Prep (Elysian, MN). Radiolabeled arachidonate (14C or 3H) is purchased from DuPont-New England Nuclear (Boston, MA) or Amersham (Arlington Heights, IL) and mixed with unlabeled arachidonate to give the desired specific activity prior to epoxidation. 3-Chloroperoxybenzoic acid (80-85%, technical grade) and all other chemicals which are reagent grade or better are obtained from Aldrich (Milwaukee, WI). Woelm neutral alumina W-200 (activity grade I) is obtained from ICN Pharmaceuticals (Cleveland, OH). Eicosanoids are stored at - 7 8 ° under argon in glass containers with fluorocarbon-faced caps. Extracts are dried over anhydrous sodium sulfate. Solvent removal refers to evaporation under reduced pressure on a rotary evaporator. Thin-layer chromatography (TLC) is performed on silica gel 60 F-254 plates (20 x 20 cm, 0.25 mm thickness) from E. Merck (Darmstadt, FRG) in glass tanks preequilibrated with solvent. Products are visualized by transillumination of the plate with a bright, white light, by exposure to iodine vapor, or by spraying with 5% ethanolic phosphomolybdic acid and heating briefly at 170°. Silica gel is removed from the TLC plate with a razor blade and extracted with methanol/dichloromethane (CH2C12) (1:10). Radioactivity of HPLC eluants is measured on-line with a Ramona-LS (Raytest Gmbh). Epoxidation of Arachidonic Acid 3-Chloroperoxybenzoic acid (19 mg, 0.11 mmol) is added portionwise to a stirring, 0° solution of arachidonic acid (31 mg, 0.10 mmol) in CH2C12 (8 ml). After 12 hr at 0°, methyl sulfide (Me2S) (50/~1) is added and the mixture maintained at ambient temperature for 20 min to quench any remaining peracid. The reaction is diluted with CH2C12 (15 ml), transferred to a separatory funnel, washed with water (3 x 15 ml), then with saturated NaCI solution (15 ml), and dried. Solvent removal gives a viscous oil which is purified immediately. Normal-phase HPLC purification (Fig. 2) yields unreacted arachidonic acid (8 mg), 14,15-EET (7 mg), ll,12-EET (5 mg), 8,9-EET (3 mg), and 5,6-EET (1.5 mg). Comparable results are obtained using TLC and methanol/CH2Cl2 (5:95, 5 elutions) as eluant: arachidonic acid ( g f ~-"
[40]
SYNTHESIS OF EPOXYEICOSATRIENOIC ACIDS
361
100.$U)
75 w
50 rr
E ~ 25 0
A I
5
A I
1'0
15
,
20
2=5
Minutes
FIG. 2. Normal-phase HPLC analysis of the reaction products from epoxidation of arachidonicacid. The crude productis resolvedon a Beckman(Fullerton,CA) UltrasphereSi (5/zm, 25 x 0.46 cm) using0.4%2-propanol/0.1% acetic acid/99.5% hexaneat a flowrate of 2 rnl/min:arachidonicacid (Rt ~ 8.14 min), 14,15-EET(Rt ~ 11.13rain), 11,12-EET(Rt -~ I 1.47 rain), 8,9-EET (Rt ~ 15.40), and 5,6-EET (Rt ~ 23.44 rain). Radioactivityis monitored on-line with a Ramona-LS.
0.76), 14,15-EET (Rf ~ 0.70), 11,12-EET (Rf ~ 0.68), 8,9-EET (gf ~ 0.60), 5,6-EET (Rf ~ 0.54), and by-products (Rf ~ 0.47-0.40).
Epoxidation of Methyl Arachidonate 3-Chloroperoxybenzoic acid (25 mg, 0.14 mmol) is added portionwise to a stirring, 0° mixture of methyl arachidonate (43 rag, 0.135 mmol) and anhydrous NaECO3 (17 mg, 0.16 mmol) in CHECI2 (4 ml). After stirring for 15 hr at 0°, Me2S (50/xl) is added and the mixture maintained at ambient temperature for 20 min to quench any remaining peracid. The reaction is diluted with CHEC12 (15 ml), transferred to a separatory funnel, washed with water (3 x 10 ml), then with saturated NaC1 solution (10 ml), and dried. Solvent removal gives a viscous oil (43 mg). Reversed-phase HPLC purification (Fig. 3) affords unreacted methyl arachidonate (12 rag), a coeluting mixture of three methyl EETs (i.e., methyl 5,6-, 8,9-, and ll,12-EET), methyl 14,15-EET (6 rag), and polar by-products. Resolution of the mixed methyl EET fraction by normalphase HPLC (Fig. 4) furnishes methyl 5,6-EET (3 mg), methyl 8,9-EET (5 mg), and methyl II,12-EET (5 rag). TLC purification of the crude reaction product using ethyl ether (EtEO)/CHECl2/hexane (1 : 1 : 8, 5 elutions) gives incomplete resolution of the regioisomeric methyl EETs: methyl arachidonate (Rf ~- 0.88), methyl 14,15-, and ll,12-EET (Rf
362
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[40]
tu
,b
tu 0~
oJ 100
o O~
75
> n"
E
Q. O
50
25 0
0
30
40
Minutes FIG. 3. Reversed-phase HPLC analysis of the reaction products from epoxidation of methyl arachidonate. The crude product is resolved partially on a Rainin Microsorb C~8 (5/~m, 25 × 0.46 cm) using a linear gradient of 50% solvent A/50% solvent B to 100% solvent B over 40 min at a flow rate of 1 ml/min: methyl arachidonate (R t = 39.2 min), methyl 5,6-, 8,9-, and 11,12-EET coeluted (Rt ~ 34.1 min), methyl 14,15-EET (Rt ~ 32.7 rain). Solvent A is 0.1% acetic acid/99.9% H20; solvent B is 0.1% acetic acid/99.9% CH3CN. Radioactivity is monitored on-line with a Ramona-LS.
uJ
dJ
100 O
o~ _>
75 tlJ
rr
& Q6
50
E e~ o 25
A; t6
d
I
0
5
,0
ts
20
Minutes FIG. 4. Normal-phase HPLC analysis of methyl 5,6-, 8,9- and II,12-EET. The mixed methyl EET fraction from Fig. 3 is resolved on a Waters (Milford, MA)/zPorasil (5/xm, 25 × 0.46 cm) using a linear gradient from 0.15% 2-propanol/99.85% hexane to 0.4% 2-propanol/ 99.6% hexane over 30 min at a flow rate of 2 ml/min: methyl 5,6-EET ( R t ~ , 18.0 min), methyl 8,9-EET (Rt ~ 11.5 rain), and methyl 11,12-EET (Rt ~ 9.6 min). Radioactivity is monitored on-line with a Ramona-LS.
[40]
SYNTHESIS OF EPOXYEICOSATRIENOIC ACIDS
363
0.55), methyl 8,9-EET (Rf ~ 0.51), methyl 5,6-EET (Rf ~ 0.42), and polar by-products (Rf = 0.16).
Saponification of Methyl 8,9-, 11,12-, or 14,15-EET: General Procedure To a stirring, 0° solution of methyl EET (16.7 mg, 0.05 mmot) in MeOH (4.5 ml) is slowly added 0.33 N aqueous NaOH (1.5 ml). The homogeneous reaction is stirred at ambient temperature and monitored by TLC analysis until complete (=12 hr). After cooling to 0°, the reaction is diluted with water (5 ml) and carefully acidified with 0.25 M aqueous oxalic acid (1.0 ml) to pH 4-4.5. The solution is transferred to a separatory funnel and extracted with Et20 (3 x 25 ml). The combined ethereal extracts are washed with water until the aqueous layer is neutral, then washed with saturated NaCI solution (30 ml), and dried. Solvent removal affords EETfree acid in essentially quantitative yield. The products can be purified by HPLC as described in Fig. 2. TLC analysis using methanol/CH2Cl2 (5:95) as eluant shows: 8,9-EET (Rf 0.17), ll,12-EET (Rf = 0.18), and 14,15-EET (Rf -~ 0.18).
Saponification of Methyl 5,6-EET To a stirring, 0° solution of methyl 5,6-EET (16.7 mg, 0.05 mmol) in tetrahydrofuran (4.5 ml) is slowly added 0.41 N aqueous NaOH (1.5 ml). The homogeneous mixture is stirred at ambient temperature until the hydrolysis is judged complete (~12 hr) by TLC analysis (ethyl acetate/ hexane, 1 : 2, ester Rf ~ 0.49). The reaction is diluted with ice chips (15 g) and transferred to a separatory funnel containing ice-cold water (30 ml), Et20 (30 ml), 0.25 M aqueous oxalic acid (1.1 ml), and glacial acetic acid (100/xl). After vigorous mixing, the upper organic layer is separated. The aqueous layer is extracted again with fresh EtzO (2 x 20 ml) and the combined organic extracts are washed with water, until the washings are neutral, then with saturated NaC1 solution (25 ml), and dried. Solvent removal affords 5,6-EET-free acid accompanied by a variable amount of the corresponding 5,6-diol. The products are somewhat labile and should be used without delay following purification by HPLC (Fig. 2) or by TLC using methanol/CH2C12 (1 : I0) as eluant: 5,6-EET (Rf ~ 0.38), 5,6-diol (Rf = 0.17).
Thiirane (Episulfide ) Heteroatom Analog Preparation of the cis-14,15-thiirane (episulfide) heteroatom analog ~6 from methyl 14,15-EET is representative. A mixture of methyl 14,15-EET (25 mg, 0.075 mmol) and anhydrous KSCN (145 mg, 1.5 mmol) in dry methanol (10 ml) is heated to reflux for
364
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[40]
72 hr. After cooling to 0°, the reaction is diluted with water (30 ml), carefully acidified to pH 4.5 with dilute hydrochloric acid, and extracted in a separatory funnel with Et20/hexanes (1 : I, 3 × 20 ml). The combined organic extracts are washed with water (3 x 20 ml), then with saturated NaCI solution (30 ml), and dried. Solvent removal gives an oily residue which is purified by TLC eluted with Et20/hexanes (1 : 2) to furnish methyl 14,15-thiirane (20.5 mg, Rf ~ 0.55) and 14,15-thiirane-free acid (2 mg, Rf -~ 0.24). The methyl ester is hydrolyzed as described for methyl 14,15-EET. Aziridine Heteroatom Analog
Preparation of the cis-14,15-aziridine heteroatom analog16 from methyl 14,15-EET is representative. A solution of sodium azide (45 nag, 0.69 mmol) in water (0.6 ml) is added with stirring to benzene (1 ml) followed by concentrated H2SO4 (17/zl). Caution: hydrazoic acid is toxic and should be handled in a hood while wearing gloves and eye protection. After 10 min, the upper organic layer is removed by decantation and dried. According to the method of Posner and Rogers, I7 the resultant benzene solution of hydrazoic acid is added to a well-stirred suspension of Woelm neutral alumina W-200 (600 rag) in dry Et20 (3 ml). A solution of methyl 14,15-EET (25 mg, 0.075 mmol) in dry Et20 (1 ml) is added after 5 min and the stirring continued until the reaction is judged complete (= 10 min) by TLC analysis. The reaction is diluted with methanol (20 ml), stirred vigorously for 15 min, filtered through a Celite (diatomaceous earth) pad, and the filter cake washed thoroughly with methanol. Solvent removal provides the regioisomeric methyl 14,15azidohydrins (23 mg) as an oil which are used directly in the next step. TLC analysis utilizing Et20/hexanes (1 : 2) as eluent gives the following: methyl 14,15-EET (gf ~ 0.40), methyl 14,15-azidohydrins (Rf ~- 0.26 and 0.28). After azeotropic drying with benzene, the above crude product is dissolved in dry Et20 (4 ml). To this is added triphenylphosphine (Ph3P) (27 mg, 0.10 mmol) and the mixture is stirred at ambient temperature for 18 hr, then at reflux for 12 hr. Solvent removal and rapid chromatography of the residue on a column of silica gel (2 g) using CH3CN as eluant gives methyl 14,15-aziridine (17 mg). TLC analysis using CH3CN:Rf ~- 0.14. The ester is hydrolyzed as described for methyl 14,15-EET. Acknowledgments Work in the authors' laboratories was supported by grants from the Robert A. Welch Foundation,JuvenileDiabetesFoundation,Kroc Foundation,NATO,and the USPHSNIH. 17G. H. Posner and D. Z. Rogers,J. Am. Chem. Soc. 99, 8208(1977).
