The resolution of racemic hydroperoxides: a chromatography-based

The Bridged Mode (α-Tether). Scott E. Denmark, Vito Guagnano, Julie A. Dixon, and Andreas Stolle. The Journal of Organic Chemistry 1997 62 (14), 4610...
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Chem. Res. Toxicol. 1990, 3, 236-243

236

The Resolution of Racemic Hydroperoxides: A Chromatography-Based Separation of Perketals Derived from Arachidonic, Linoleic, and Oleic Acid Hydroperoxides Ned A. Porter,* Patrick Dussault,t Robert A. Breyer, Jere Kaplan, and Joseph Morelli Department of Chemistry, Duke University, Durham, North Carolina 27706 Received January 15, 1990

In spite of the importance of optically pure unsaturated hydroperoxides in biology, chemistry, and medicine, no general method for synthesizing these labile compounds has been reported. We have developed a chiral vinyl ether that reacts with racemic hydroperoxides t o give diastereomeric perketals in high yield. These perketals can be separated by liquid chromatography and the chiral group removed t o provide highly enriched hydroperoxide enantiomers (>99% enantiomeric excess). T h e chiral reagent t h a t has been most successful in our hands is the 2-propenyl ether derived from trans-2-phenylcyclohexanol.By use of this vinyl ether, perketals are readily formed from hydroperoxides, they are stable t o normal- and reverse-phase chromatography, and the hydroperoxide is regenerated from the perketal without racemization in high yield with mild acid. Several chiral hydroperoxides have been resolved by this procedure: a-phenethyl hydroperoxide, 2-octyl hydroperoxide, and a number of hydroperoxides derived from oleic, linoleic, and arachidonic acids.

Fatty acid hydroperoxides me formed by a lipoxygenase enzyme catalyzed conversion of polyunsaturated fatty acids ( 1 ) . These hydroperoxides serve as important intermediates in the formation of diverse compounds of biological importance. For example, fatty acid hydroperoxides are precursors to the leukotrienes (2)and the lipoxins (3),and recently a new biochemical pathway involving the conversion of fatty acid hydroperoxides to allene oxides has been reported in flax, corn, and coral ( 4 ) . A new stereocenter is generated in the lipoxygenase reaction, and fatty acid hydroperoxides isolated from natural sources are essentially one enantiomer if formed enzymatically (1,5).For example, the 13(S)-hydroperoxide is formed when soybean lipoxygenase acts on linoleic acid, while this enzyme gives the 15(S)-hydroperoxide, 15(S)HPETE,' when arachidonic acid is the substrate. On the other hand, lipoxygenase enzyme in coral converts arachidonic acid into 8(R)-HPETE (4). Nonenzymatic autoxidation of fatty acid substrates also gives fatty acid hydroperoxides ( 5 ) ,and racemic mixtures are formed in this process ( 6 ) . H 1 1 C 5 ~ ( C H 2 ) $ O O H OOH linoleate 1J(S)-hydroperoxide

OOH

H11C5-

1S(S)-HPETE

-

,OOH

/

8

-

(CH,),COOH

8(R)-HPETE

Despite the importance of unsaturated hydroperoxides in chemistry, biology, and medicine, no general method 'Present address: Department of Chemistry, University of Nebraska, Lincoln, NE 68588.

0893-228~/90/2703-0236$02.50/0

Scheme I

+

ROOH

H+

--c

H 1

ROOxo*

separate

diastereOmen

-H+

ROOH (Optically pure)

H

2

for the preparation of these labile compounds has been reported. Chemical syntheses of optically pure hydroperoxides have been attempted (7-9), but no general chemical approach has been found to prepare allylic and dienylic hydroperoxides like those formed from lipoxygenase enzymes. For example, nucleophilic displacement of mesylates or tosylates by hydrogen peroxide has been reported in the synthesis of 2-octyl hydroperoxide, but allylic and dienylic mesylates are unstable and reaction of these compounds (even at -100 "C) apparently gives racemic hydroperoxide products. Thus, the natural products are available only from enzymatic sources, while the unnatural enantiomers have not been reported. We report here what appears to be the first general solution to this problem: the resolution of unsaturated hydroperoxide enantiomers by liquid chromatography of diastereomeric derivatives. This method allows for the nonenzymatic preparation of optically pure allylic or dienylic hydroperoxide natural products through conversion Abbreviations: BHT, butylated hydroxytoluene;HETE, hydroxyeicosatetraenoate; HPETE, hydroperoxyeicosatetraenoate; HPLC,highpressure liquid chromatography; IR, infrared; NMR, nuclear magnetic resonance;PPTS, pyridinium p-toluenesulfonate; THF, tetrahydrofuran; TLC, thin-layer chromatography; TsOH, toluenesulfonic acid.

0 1990 American Chemical Society

Resolution of Racemic Hydroperoxides

of the racemic hydroperoxide to diastereomeric perketals by reaction with the vinyl ether 1, separation of the perketal diastereomers 2 by liquid chromatography, and conversion of the separated perketals to the optically pure hydroperoxides (Scheme I). Experimental Sectlon Reactions were run under an atmosphere of argon except where indicated. T H F was distilled from sodium/benzophenone. CH2C12,CHCl,, and CDCl, were passed through activity I neutral alumina before use. Reagents were used as commercially supplied. Pyridinium p-toluenesdfonate (PPTS)was prepared by dissolving toluenesulfonic acid (TsOH) into excess pyridine, followed by removal of solvent in vacuo. Phenethyl hydroperoxide was isolated by extraction of >5 year old commercial ethylbenzene with 15% NaOH, followed by neutralization and extraction with CH2C12. Flash chromatography was performed on 230-400 mesh silica. Normal-phase HPLC separations were performed on Rainin Dynamax columns, 21 mm X 25 cm, 8 pm (preparative), or Rainin Microsorb columns, 4.6 mm X 25 cm, 5 pm (analytical). Reverse-phase separations were performed on Rainin Dynamax (2-18 columns, 21 mm X 25 cm, 8 pm (preparative), or Altex-Beckmann ODS columns, 4.6 mm, 2 X 25 cm, 5 pm (analytical). A Waters Associates Model 6000 pump and Model 480 variable-wavelength UV detector was used for most separations; alternatively, a Waters Model 401 refractive index detector was employed. Hydroperoxides were always concentrated and stored in the presence of 0.1-0.2% butylated hydroxytoluene (BHT), and neither the hydroperoxides nor the perketals were ever exposed to temperatures above 30 OC. Optical rotations were measured in a 1-dm cell by using a PE 241 polarimeter. Proton NMR ('H NMR) were recorded in CDC1, on a Varian XL-300 spectrometer a t 300 MHz. Proton spectral data are reported as follows: chemical shift (multiplicity, integrated intensity, coupling constants in Hz, assignment when possible). Shifts are reported in ppm values relative to tetramethylsilane (6 = 0) as an internal standard. Carbon NMR (13C NMR) spectra were recorded in CDC13 on a Varian XL-300 spectrometer at 75 MHz. TLC plates could be visualized for hydroperoxides and perketals by using a spray of 1.5 g of N,N'-dimethyl-p-phenylenediaminedihydrochloride in 1 mL of acetic acid/25 mL of water/125 mL of methanol; hydroperoxides gave a pink spot without heating, while perketals gave a green or red spot after charring. A solution of 1%ceric sulfate/2.5% ammonium molybdate in 10% sulfuric acid was used for general TLC staining. Elemental analyses were performed by Atlantic Microlab, Atlanta, GA. The synthesis of vinyl ether 1 was patterned on literature precedent (IO, 11) and is reported here in detail. Ethynyl trans-2-Phenylcyclohexyl Ether (3). A suspension of 1.2 g (10.1 mmol, 2 equiv) of KH, nominally 35% in oil, was washed with hexane (2 X 2 mL). T H F was added (25 mL), followed by two crystals of imidazole. In approximately 150200-mg portions, (-)-trans-2-phenylcyclohexanol(855 mg, 5.02 mmol) was added over a period of 30 min. The solution was then cooled in an ice bath, and trichloroethylene (450 pL, 5.02 mmol) was slowly added, resulting in an immediate brown color. The cooling bath was removed, and the solution gave way to a deep brown suspension. A small amount of T H F (5 mL) was added to aid stirring. After 30 min, the reaction was cooled to -78 "C and 5.2 mL of nominally 2.5 M nBuLi/hexane was added. The reaction was warmed to -40 "C and quenched with MeOH. The suspension was diluted with water and allowed to warm to room temperature. Extraction with petroleum ether (2 X 50 mL) was followed by washing the aqueous extracts (saturated NaCl). Drying (Na2S04)and concentration produced a brown oil which was chromatographed on 25 g of flash silica that had been previously equilibrated with 2.5% v/v EbN. Elution with 5% ethyl acetate/l% Et3N in petroleum ether afforded 965 mg (96%) of a light yellow oil which solidified in the freezer: R, = 0.55 (5% ethyl acetate/petroleum ether); deforms at 39-42 "C; mp 44-46 "C; [ a ]=~-83.4 (c = 1.7, CHCl,); IR (neat fh) 3325 (5, acetylenic H),3030, 2970 (s), 2850, 2150 (vs, alkyne), 1490, 1450, 1100, 780, 700 (s) cm-l; 'H NMR 6 7.34-7.19 (5 H, aromatic), 4.165 (dt, 1 H, J = 4.4,9 Hz, HCO), 2.757 (dt, 1 H, J = 3, 9 Hz, PhCH), 2.42

Chem. Res. Toxicol., Vol. 3, No. 3, 1990 237 (m, 1H), 1.95-1.90 (2 H), 1.77-1.28 (6 H); 13C NMR 6 141.88 (Ar), 127.98 (Ar), 127.02 (Ar), 126.24 (Ar),89.10 (CO), 88.62 (acetylenic CO), 48.395 (acetylene), 33.36 (PhC), 30.28, 27.38, 24.99, 24.19. Anal. Calcd for C14H1,O: C, 83.96; H, 8.05. Found: C, 83.79; H, 8.06. trans-2-Phenylcyclohexyl2-Propen-2-yl Ether (1). To a suspension of CuBr (1.54 g, 11.0 mmol, 1.5 equiv) in 14 mL of dry THF at -40 "C (CH3CN/C02)in an oven-dried 3 N roundbottom flask under argon was added MeMgBr, nominally 1.5 M in 3:l toluene/THF. The reaction progressed from intense blue to green and then finally to yellow green. After 10 min, the alkynyl ether 3 was added in a minimum of THF and the resulting suspension was stirred for 30 min. The reaction was allowed to warm to -20 "C and then brought to 0 "C for 1 h. The reaction was quenched with 10 mL of 1:l saturated NH4Cl/2 N NH,OH and stirred for 30 min. After dilution with petroleum ether (50 d) the ,phases were separated and the organic phase was washed (3 X 40 mL) with the ammonia buffer. The aqueous washes were back-extracted with 40 mL of petroleum ether. The combined organic phases were dried over Na2S04and concentrated to a light green oil. Bulb-to-bulb distillation (0.5 mmHg, 120-130 "C) afforded 1.335 g (86%) of the enol ether as a colorless oil with density -0.95: R, =. 0.50 (5% ethyl acetate/petroleum ether, alumina plates, partial decomposition); [a]D = -3.30 (c = 1.4, CHCI,); IR (neat film) 3040, 2940 (s), 1650 (s), 1600, 1440, 1390, 1280 (s), 1080 (s), 990, 790, 755, 700 (s) cm-'; 'H NMR 6 7.29-7.14 (5 H, aromatic), 4.04 (m, 1H, CHO), 3.78,3.75, (s, 2 H, CH2=), 2.6 (m, 1H, HCPh), 2.35 (m, 1 H), 2.0-1.7 (4 H), 1.5 (s, 3 H, Me), 1.5-1.25 (4 H); 13C NMR 6 158.51 (=CO), 144.1 (Ar), 127.99 (Ar), 127.40 (Ar), 125.93 (Ar), 81.06 (CH2=), 78.31 (CO), 50.19, 34.16, 31.11, 26.10, 24.78, 21.18. Anal. Calcd for C15Hm0: C, 83.29; H, 9.32. Found: C, 83.16; H, 9.36. General Method for Hydroperoxide Resolution. The resolution of hydroperoxides is illustrated here for the 13hydroperoxide derived from linoleic acid. The resolution was carried out on the hydroperoxide methyl ester derived from autoxidation of methyl linoleate. The racemic 9(Z),ll(E) 13hydroperoxide was purified by HPLC, as described below. (A) 13-Hydroperoxy-9(Z),11(E)-octadecadienoic Acid Methyl Ester (Racemic). Methyl linoleate (2 g, Sigma 99% grade) was stirred in air for 1week and loaded directly onto 40 g of flash silica. Elution with 15% ethyl acetate-petroleum ether afforded 467 mg of mixted hydroperoxides. Chromatography on normal-phase HPLC (7752251 hexane/ether/acetic acid) afforded 75 mg of pure 13-hydroperoxy-9(Z),l1(E)-octadecadienoic acid methyl ester (first eluting peak). (B) Perketal Formation. To a solution of 75 mg (0.23 mmol) of hydroperoxide methyl ester (derived from the autoxidation of linoleic acid) in 1 mL of CH2C12was added 1-2 mg (2.5 mol %) of PPTS and phenylcyclohexyl 2-propen-2-yl ether (1) (58 pL, -1.1 equiv). After 5 min, additional enol ether (10 pL) was added. The reaction was diluted with CC14,and the CH2C12was removed with an argon stream. The resulting solution was directly subjected to flash chromatography on 10 g of silica gel with 5% ethyl acetate/petroleum ether to afford 125.8 mg (quantitative) of a colorless oil. The mixture of diastereomers was resolved in neat CH3CN on a Dynamax C-18 column, at 9 mL/min and 20 mg/injection (58.72 and 64.45 min, k l = 6.34, k 2 = 7.06, a = 1.11). Mixed fractions (12 mg) were reinjected, and the leading and tailing fractions were individually repurified to afford, initially, 57.5 mg of the leading diastereomer which was shown to be at least 98% pure: Rf = 0.64 (10% ethyl acetate/petroleum ether); [a]D = +67.2 (c = 0.8, CHCl,); MS (CI, NH,) 560.45 (MH+ + NH,, 1.15); IR (neat film) 2940 (s), 2850,1740 (s), 1450,1380,1200,1060,700 cm-'; 'H NMR 6 7.21 (5 H), 6.4 [dd, 1 H, J = 15.3, 11.0 Hz, CH=C(H)OO], 5.9 [t, 1 H, J = 11.3 Hz, CH=CHC=CC(OO)],5.5 [dd, 1H, J = 15.1, 7.6 Hz, =CHC(OO)], 5.41 (m, 1 H, CH,CH=), 4.3 (apparent q, 1 H, J = 6.5 Hz, CHOO), 3.6, (s, 3 H, Me ester), 3.5 (dt, 1 H, J = 4.4, 9.7 Hz, CHO), 2.53 (m, 1 H), 2.42 (m, 1 H), 2.3 (t, 2 H, J = 7.3 Hz, CH2C=), 2.1 (apparent q, 1 H, J = 7 Hz), 1.95-1.55 (11 H), 1.5-1.2 (25-30 H), 1.2 (s, 3 H, Me), 0.9 (t, 3 H, J = 6.5 Hz, terminal Me), 0.5 (s, 3 H, Me); 13C NMR 6 174.19 (carbonyl), 144.80, 133.03, 132.36, 128.51, 128.08, 127.62, 127.41,125.91, 104.77 (perketal), 84.37 (CHOO), 75.80 (CHO), 51.39, 50.75, 34.92, 34.05, 33.07, 32.24, 31.80, 29.52, 29.11, 29.07, 29.01, 27.70, 26.01, 25.40,

Porter et al.

238 Chem. Res. Toxicol., Vol. 3, No. 3, 1990 25.29,25.02, 24.90, 22.49, 21.51, 14.00. Anal. Calcd for (&HMO5: C, 75.23; H, 10.03. Found: C, 74.51; H, 10.07. From the mixed diastereomers, 58.7 mg of the trailing diastereomer of 99% purity was recovered: R, = 0.64 (10% ethyl acetate/petroleum ether); [ a ] D = +81.2 (c = 0.8, CHCl,); MS (CI, NH3) 560.45 (MHt + NH,, 3); IR (neat film)2940 (s), 2850,1740 (s), 1450, 1380, 1200, 1160, 1060, 700 cm-'; 'H NMR 6 7.26-7.19 (5 H), 6.3 [dd, 1 H, J = 15.4, 11.2 Hz, CH=C(H)OO], 5.9 [t, 1 H, J = 10.9 Hz, CH=CH-C=CC(OO)], 5.5 [dd, 1 H, J = 15.2, 8.1 Hz, -CHC(OO)], 5.404 (dt, 1 H, J = 10.7, 7.8 Hz, CHZCH=), 4.3 (dd, 1 H, J = 7.6, 6.2 Hz, CHOO), 3.666, (s, 3 H, Me ester), 3.5 (m, 1 H, CHO), 2.538 (m, 1 H), 2.37 (m, 1 H), 2.29 (t, 2 H, J = 7.4 Hz, CH,C=), 2.137 (apparent q, 1 H, J = 6.5-7 Hz), 2.0-1.55 (12 H), 1.6-1.35 (25-30 H), 1.16 (s, 3 H, Me), 0.9 (t, 3 H, J = 6.5 Hz, terminal Me), 0.5 (s, 3 H, Me); 13CNMR (75 MHz, CDCl,) 6 174.22 (carbonyl), 144.752, 132.85, 132.56,128.50, 127.96, 127.62, 125.91, 104.66 (perketal), 83.91 (CHOO), 75.79 (CHO), 51.407, 50.74, 34.99, 34.05, 32.96, 32.22, 31.73, 29.51, 29.09, 29.05, 29.00, 27.69, 26.00, 25.55, 25.39, 25.01, 24.90, 22.54, 21.39, 14.01. Anal. Calcd for C34H6406:C, 75.23; H, 10.03. Found: C, 75.34; H, 10.09. (C) 13(R)-Hydroperoxy-9(2),1l(E)-octadecadienoic Acid Methyl Ester (13EZ-00H). The leading 13-E,Z perketal(30.9 mg, 57 mmol) was dissolved in 1.5 mL of 4:2:1 THF/HOAc/H20 in the presence of 1 mol % BHT. The reaction was stirred overnight and then concentrated to dryness: Rf = 0.37 (10% ethyl acetate/petroleum ether); [ a ] D = -8.7 (c = 0.84, CHCl,), +5.0 (c = 0.8, MeOH); 'H NMR 6 7.8 (s, 0.6 H, OOH), 6.575 [dd, 1 H, J = 15.2, 11.1 Hz, CH=CHC(OOH)]; 6.0 (t, 1 H, J = 11.0 Hz, CH,CH=CH), 5.6 [dd, 1 H, J = 15.3, 8.3 Hz,=CHCH(OOH)], 5.5 (dt, 1 H, J = 10.8, 7.6 Hz, CH,CH=), 4.4 (apparent q, 1 H, J = 6.3-8.1 Hz, CHOO), 3.7 (s, 3 H, Me ester), 2.3 (t, 2 H, J = 7.3 Hz, CH,COO), 2.2 (4, 2 H, J = 6.9-7.2 Hz, CH,CH=), 1.8-1.6 (4 H), 1.5-1.2 (15 H), 0.9 (t, 3 H, terminal Me). (D) 13(R)-Hydroxy-9(Z),11(E)-octadecadienoic Acid Methyl Ester (13EZ-OH). The 13(R)-hydroperoxide (14 mg) was dissolved in 500 pL of ether along with 15 mg of Ph3P and concentrated to dryness. The crude product was directly loaded onto 5 g of silica and eluted with 30% diethyl ether/petroleum ether to afford 11.9 mg of alcohol: R, = 0.23 (10% ethyl acetate/petroleum ether); [ a ] D = -6.7 (c = 1.2, CHCl,), -4.2 (c = 0.6, hexane), -5.4 (c = 1.2, hexane; implies concentration dependence). Both points fall on a line with literature values [lit. (11) [aID= -7.5 (c = 2.3, hexane), (12) ["ID = +6.0 (e = 1.6, hexane)] when plotted as rotation vs concentration. 'H NMR 6 6.5 (dd, 1 H, J = 15.2 Hz, ll.l),6.0 (t, 1 H, J = 10.9 Hz), 5.7 (dd, 1 H, J = 15.3, 6.8 Hz), 5.4 (dt, 1 H, J = 10.7, 7.7 Hz), 4.2 (m, 1 H), 3.7 (s, 3 H), 2.304 (t, 2 H, J = 7.6 Hz), 2.2 (apparent q, 1 H, J = 6.6-7.3 Hz), 1.7-1.5 (6 H), 1.4-1.2 (12 H), 0.890 (t, 3 H, J = 6.8 Hz). (E) 13(S)-Hydroperoxy-9(Z),ll(E)-octadecadienoicAcid Methyl Ester (13EZ-00H). The trailing 13-E,Zperketal(31.0 mg) was dissolved in 1.5 mL of 4:2:1 THF/HOAc/H,O in the presence of 1 mmol of BHT and stirred for 22 h. The reaction was concentrated to dryness and directly subjected to flash chromatography on 10 g of silica gel with 20% ethyl acetate/ petroleum ether to afford 16.2 mg (91%) of the 13(S)-hydroperoxide: R, = 0.26 in 20% ethyl acetate/petroleum ether; ["ID = +9.1 (c = 0.75, CHCl,), -5.2 (c = 0.75, MeOH); 'H NMR 6 7.8 (s, 0.6 H, OOH), 6.5 [dd, 1H, J = 15.2 Hz, 11.1CH=CHC(OOH)]; 6.0 (t, 1 H, J = 11.0 Hz, CH,CH=CH), 5.5 [dd, 1 H, J = 15.3, 8.3 Hz, =CHCH(OOH)], 5.5 (dt, 1 H, J = 10.8,7.6 Hz, CH,CH=), 4.4 (apparent q, 1H, J = 6.3-8.1 Hz, CHOO), 3.7 (s, 3 H, Me ester), 2.3 (t, 2 H, J = 7.3 Hz, CHZCOO), 2.2 (q, 2 H, J = 6.9-7.2 Hz, CH,CH=), 1.8-1.6 (4 H), 1.5-1.2 (15 H), 0.9 (t,3 H, terminal Me). (F) Saponification [ 13(S)-Hydroperoxy-9(Z),ll(E)-octadecadienoic Acid]. To a solution of 128 mg (0.236 mmol) of 13(S)-peroxyketal in 1.5 mL of THF was added 250 pL of 4 M aqueous LiOH. The reaction was stirred overnight, furnishing a soapy solution. No starting material was visible by TLC. Acetic acid (1 mL), THF (2 mL), and BHT (2 pmol) were added, and the reaction was stirred for 1 h. The reaction mixture was then pipetted into 30 mL of saturated NH,Cl and extracted with ethyl acetate (2 x 40 mL). After concentration, the crude product was subjected t o flash chromatography on 15 g of Silicar CC-4 (Mallinckrodt) in a gradient of 10-20% EtOAc/PE, affording 71 mg of hydroperoxy acid (quantitative). Purification by nor-

mal-phase HPLC removed traces of the 2-phenylcyclohexanol auxiliary, providing 45.3 mg (65%) of pure product which was identical by TLC and NMR with the product derived from the action of lipoxygenase on linoleic acid, except that the acidic protons were now visible as broad peaks at 8.23-7.5 ppm in the NMR spectrum: ["ID = -4.5 (c = 0.91, MeOH). The hydroperoxy acid produced by saponification/hydrolysis was reconverted to the methyl ester perketal by subsequent treatment with diazomethane and chiral auxiliary 1. HPLC analysis showed >99% purity of the 13(S) perketal, indicating that no epimerization occurred during the saponification or acidic hydrolysis. Autoxidation of the Methyl Ester of Arachidonic Acid. The methyl ester of arachidonic acid (2.0 g, 6.3 mmol) was placed in a dry 500-mL round-bottomed flask and stirred under a slow flow of dry air for 48 h. The reaction mixture was then loaded directly into a flash chromatography column (silica 50:l) with hexane/diethyl ether, 4:1, as the eluent. The unreacted ester was present in the first eluting fraction and could be recycled. The next fractions contained the peroxides. The peroxide-containing fractions were combined, and a small amount of BHT was added to prevent degradation. The solvent was removed to leave a viscous yellow oil (140 mg, 6.4% yield). The oil was then loaded onto a normal-phase HPLC (silica, hexane/2-propanol, 99:l) and separated into the different diastereomeric peroxides; the order of elution was 15-HPETE, 12-HPETE, 11-HPETE, 8-HPETE, g-HPETE, and 5-HPETE (Figure 2). See ref 18 and references cited therein for assignment of HPETE positional isomers. (A) Formation of Diastereomeric Perketal of 5-HPETE Methyl Ester. To a solution of 5-HPETE methyl ester (18.5 mg, 0.053 mmol in 1 mL of dry dichloromethane) was added 13 pL (0.057 mmol) of the vinyl ether and a catalytic amount of PPTS. The resulting solution was stirred at room temperature under argon for 20 min, and then a second 13 mL of the auxiliary was added. Stirring was continued for an additional 20 min, and then sodium bicarbonate was added to quench the reaction. Carbon tetrachloride (1mL) was added and the dichloromethane was removed under a flow of argon. Polar impurities were removed by flushing the sample through a short plug of silica with petroleum ether/ethyl acetate, 4:1, as the eluent. The petroleum ether and ethyl acetate were removed under reduced pressure, and the resulting residue was loaded directly onto a reverse-phase HPLC C-18 column with acetonitrile as the eluent; a trace amount of triethylamine was added to the eluent to prevent degradation of the perketal. The first eluting perketal was determined to be derived from 5(R)-HPETE (9.7 mg) and the second from 5(S)-HPETE (9.7 mg) (total yield 57.2%). 'H NMR (perketal derived from 5(S)-HPETE methyl ester) 6 7.2-7.1 (m), 6.40 [dd, 1 H, J = 10.88, 15.28 Hz, CH=C(OO)], 5.9 [t, 1 H, J = 10.4 Hz, CH=CHC=CC(OO)], 5.5 [dd, 1 H, J = 8.06, 15.27 Hz, CHC(OO)], 5.35 (m, 5 H), 4.3 (m, 1 H, CHOO), 3.6 (s, 3 H, OCHJ, 3.5 (m, 1 H, CHO), 2.9 (m, 2 H, CH,), 2.8 (m, 2 H, CHJ, 2.5-0.8 (32 H). (B) Regeneration of the Resolved 5-HPETE's. The perketal (9.7 mg, 0.16 mmol) was dissolved in 1 mL of THF/acetic acid/water, 4:2:1, and a small quantity of BHT, a radical inhibitor, was added. The solution was stirred overnight and then added to 30 mL of water and extracted with ether (3 X 30 mL). The organic phases were combinaed, washed with water, and dried over MgSO,, and the ether was removed under reduced pressure to leave to thick oil [4.8 mg (84%)]. (C) Reduction of 5-HPETE To Provide 5-HETE. To a methanolic solution of the 5-HPETE methyl ester (4.8 mg, 0.014 mmol in 1mL) at 0 "C was added 20 mg of sodium borohydride. The solution was stirred at 0 OC under an argon atmosphere for 20 min and then allowed to warm to room temperature. The reaction mixture was acidified with dilute acetic acid and extracted with diethyl ether (3 X 5 mL). The combined organic layers were dried over MgS04, and the solvent was removed under reduced pressure to leave to crude alcohol that was loaded onto a reverse-phase HPLC (C-18,acetonitrile as eluent) to afford 2.1 mg (46%) of the pure 5-HETE methyl ester. The optical rotation was measured for both the 5(S)-HETE and the 5(R)-HETE methyl esters. The specific rotation of 5(S)-HETE was [ a ] D = +10.9 (c = 0.1, benzene) (lit. [a]D = +14); this indicates a purity of 78%. For 5(R)-HETE, [ a ]=~-14.1 (C = 0.49, benzene), which indicates a purity of >99%.

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Chem. Res. Toxicol., Vol. 3, No. 3, 1990 239

Resolution of Racemic Hydroperoxides

(D) Enzymatic Formation of 15(S)-HPETE. An ethanolic solution of arachidonic acid (100 mg, 0.329 mmol in 4 mL) was added in one aliquot to 407 mL of pH 9 boric acid buffer that had been previously saturated with oxygen. To this slightly cloudy solution was added 1 mg of soybean lipoxygenase enzyme in 1 mL of deionized water. Oxygen was slowly bubbled into the stirred solution for 10 min, by which time the solution had cleared. The reaction mixture was acidified to pH 4 with 0.1 N HC1, and the resulting cloudy suspension was extracted with diethyl ether (3 x 100 mL). The combined organic phases were dried over MgSO,, and the solvent was removed under reduced pressure to leave 84.5 mg (76%) of the crude peroxide. Diazomethane was added to the peroxide to afford the ester, which was purified and perketalized as described before. The perketal derived from 15(S)-HPETE methyl ester gave the following: 'H NMR b 7.5-7.2 (m), 6.52 [dd, 1 H, J = 10.91, 15.46 Hz, CH=C(OO)], 6.1 [t, 1 H, J = 10.8 Hz, CH=CHC=CC(OO)], 5.6 [dd, 1H, J = 8.06,15.2 Hz, CHC(OO)], 5.5 (m, 5 H), 4.39 (4, 1 H, J = 7.4 Hz, CHOO), 3.7 (s, 3 H, OCH,), 3.6 (m, 1 H, CHO), 3.04 (m, 2 H, CH,), 2.79 (m, 2 H, CH2),2.5-0.8 (m, 32 H). Anal. Calcd for C36H5405:C, 76.28; H, 9.60. Found: C, 76.02; H, 9.63. (E) Data for the Perketal Derivatives of a-Phenethyl Hydroperoxide. The diastereomers were resolved by RP-HPLC in 950:50:0.1 CH3CN/H20/Et3N at 9 mL/min and 35 mg/injection, with detection at 254 nm (21.23 and 23.23 min, k1 = 1.73, k 2 = 1.99, CY = 1.15). After reinjection of 30 mg of the mixed fractions, the leading and tailing diastereomers were individually "shaved" to afford 319.2 mg of the leading diastereomer. Analytical RP-HPLC (same solvent) showed the material to be 100% diastereomerically pure: R, = 0.5 (7 % ethyl acetate/petroleum acetate); ["ID = +179.6 ( c = 0.91, CHCl,); MS (CI, NHJ 372.2 (MH+ + NH,, 0.7); IR (neat film) 2980,2930 (s), 2850,1450,1370, 1200, 1160, 1060 (s), 875, 860, 840, 760, 700 (9) cm-'; 'H NMR 6 7.33-7.27 (bs, 5 H, Ar), 7.25-7.12 (m, 5 H, Ar), 4.9 [q, 1 H, J = 6.6 Hz, ArCH(Me)OO],3.5 (dt, 1 H, J = 4.4,9 Hz, OCH), 2.5 (dt, 1 H, J = 3.5, 10 Hz), 2.4 (m, 1 H), 1.92-1.6 (4 H, cyclohexyl), 1.4 (d, 3 H, J = 6.6 Hz,ArCHCH,OO), 1.36-1.25 (3 H, cyclohexyl), 1.2 (s, 3 H, Me), 0.442 (s, 3 H, Me); 13C NMR 6 144.72, 142.57, 128.47, 128.12, 127.61, 127.46, 126.29, 125.90, 104.80, 81.62, 75.85, 50.67, 34.85, 32.17, 25.96, 25.25, 25.11, 21.34, 20.62. Anal. Calcd for C23H3003: C, 77.93; H, 8.53. Found: C, 77.96; H, 8.55. Then 317.8 mg of the trailing diastereomer was recovered, which was shown by RP-HPLC to be 298% pure: R, = 0.5 (7% ethyl acetate/petroleum acetate); ["ID = +16.7 (c = 0.7, CHC1,); MS (CI, NH3) 372.2 (MH' + NH3,2.7), 217 (MH' - ROOH, 100);IR (neat film) 2980, 2930 (s), 2850, 1450, 1370, 1200, 1160, 1060 (s), 875, 860, 840, 760, 700 (s) cm-'; 'H NMR 6 7.29 (bs, 5 H, Ar), 7.28-7.15 (m, 5 H, Ar), 4.9 [q, 1 H, J = 6.4 Hz, ArCH(Me)OO], 3.5 (dt, 1 H, J = 4.4,9.5 Hz, OCH), 2.5 (m, 1 H, PhCH), 2.40 (m, 1 H), 1.9-1.6 (4 H, cyclohexyl), 1.43 (d, 3 H, J = 6.6 Hz, ArCHCH,OO), 1.4-1.25 (4 H, cyclohexyl), 1.081 (s, 3 H, Me), 0.43 (s, 3 H, Me); 13C NMR 6 144.68, 142.11, 128.47, 128.04, 127.61, 127.55,126.62,125.91,104.65,80.93,75.79,50.70,34.85,32.17,25.96, 25.38, 25.29, 21.31, 20.35. Anal. Calcd for C23H3003: C, 77.93; H, 8.53. Found: C, 77.82; H, 8.55. (F) Data for the Perketal Derivatives of 2-Octyl Hydroperoxide. (1) (S)-(+)-Perketa1Derivative of 7: 'H NMR (300 MHz, CDClJ 6 0.50 (s, 3 H), 0.88 (t, 3 H, J = 7 Hz), 1.14 (d, 3 H, J = 6 Hz), 1.20 (s, 3 H), 1.25-1.45 (m, 14 H), 1.50-1.95 (m, 3 H), 2.40 (m, 1 H), 2.55 (d, t, 1 H, J = 11 Hz, J = 3.5 Hz), 3.55 (d, t, 1 H, J = 11 Hz, J = 4.7 Hz), 3.95 (sx, 1 H, J = 6 Hz), 7.2 (m, 5 H); 13C NMR (300 MHz, CDCl,) 6 14.09, 18.74, 21.50, 22.60, 25.27, 25.40, 25.52, 26.02, 29.40, 31.78, 32.22, 34.50, 34.86, 50.75, 75.80, 79.40, 104.49, 125.93, 127.54, 128.64,144.83; IR (KBr plate) 2950 (s), 1600 (w),1450 (s), 1375 (s), 1200 (s), 1160 (s), 1060 (s) cm-'. Anal. Calcd for C23H3803:C, 76.20; H, 10.56. Found: C, 76.27; H, 1055. (2) @)-(-)-Perketa1 Derivative of 7: 'H NMR (300 MHz, CDCl,) 6 0.52 (s, 3 H), 0.90 (t, 3 H, J = 7 Hz), 1.12 (d, 3 H, J = 6 Hz), 1.19 (s, 3 H), 1.25-1.45 (m, 14 H), 1.50-1.95 (m, 3 H), 2.40 (m, 1 H), 2.55 (d, t, 1 H, J = 11 Hz, J = 3.5 Hz), 3.55 (d, t, 1 H, J = 11 Hz, J = 4.7 Hz), 3.95 (sx, 1 H, J = 6 Hz), 7.2 (m, 5 H); NMR (300 MHz, CDC13) 6 14.12, 18.59, 21.40, 22.66, 25.42, 25.52, 26.02, 29.37, 31.60,31.83, 32.20, 34.49, 34.96,50.76, 75.85, 79.08, 104.46, 125.93, 127.64, 128.54, 144.80; IR (KBr plate) 2950 (s), 1600 (w), 1450 (SI, 1375 (s), 1200 (s), 1160 (s), 1060 (s). Anal.

Scheme I1

M e 0 OMe R'XH H+

Calcd for CBHB03: C, 76.20; H, 10.56. Found: C, 76.34; H, 1059.

Results Preliminary screens of several hydroperoxide derivatives

of the structure ROOCR'XOR", were carried out to achieve three specific goals: (1)to easily convert the hydroperoxide to the derivative, (2) to easily remove the protecting group without racemization or destruction of the unsaturated hydroperoxide substructure, and (3) t o determine acceptable chromatographic characteristics of t h e derivative including resolution of diastereomers and stability to the conditions of chromatography. T h e strategy chosen was based upon known conversions of hydroperoxides to peracetals as reported [e.g., Rieche (13)or Rigaudy (14),a n d as shown in Scheme 111. Four hydroperoxide derivatives based on ROOCR'XOR'' were considered as candidates: peracetals (with R' = alkyl or H, X = H), perorthoesters (R' = H, aryl, or alkyl a n d X = OR"), which our perliminary experiments show are unstable to chromatography, peraminals (R' = H, aryl, or alkyl a n d X = NR2/1), also unstable to chromatography, a n d perketals (with R' = X = alkyl), which, like peracetals, are readily prepared from percursor hydroperoxides a n d are stable to most chromatographic conditions. While several hydroperoxide derivatives were examined in preliminary experiments, more detailed studies were carried out with t h e peracetal4 a n d t h e perketals 5 a n d 2. These detailed studies involved assessment of chro-

n 4

5

2

matographic characteristics of the derivatives from assorted hydroperoxides, determination of conditions for deprotection, a n d evaluation of hydroperoxide stability under deprotection conditions. Peracetal derivatives like 4 were easy to prepare (1.9, were stable to chromatography, a n d generally provided separation of diastereomers when t h e stereocenter was located a t the a position of the hydroperoxide. The biggest problem associated with these derivatives was the vigorous conditions required for deprotection. Generally, sulfonic acids were necessary a s catalysts for t h e conversion of peracetal t o starting hydroperoxide, a n d while some hydroperoxides would tolerate these conditions of deprotection, sensitive hydroperoxides such as those derived from unsaturated fatty acids gave decomposition under t h e conditions of deprotection. Perketals 2 (16) a n d 5 were readily formed a n d were stable t o chromatography under appropriate conditions, a n d t h e perketal protecting group was removed without t h e need t o use strong acid catalysts. Although perketals

240 Chem. Res. Toxicol., Vol. 3, No. 3, 1990

Porter et al. 15

6

,

OOH

Arachidonate Hydroperoxides

-C 51H 1

-

-

OOH

-

/

5

(CH2)3COOH

8, 5-HPETE OOH (CH2)3COOH

9, 8-HPETE

HIIC5A

HOO

-

-

9

\

(CH2)3COOH

-

0

10, 9-HPETE C -5lHl

-

OOH 0

1

-

-

1

11,

(CH&COOH

11-HPETE

10

20

30

40

Volume (ml) Figure 2. HPLC for separation of methyl arachidonate hydroperoxides, 8-13, as the methyl ester. Normal-phase separation on silica with hexane/2-propanol, 99:l. Numbers indicate the position of OOH substitution. Detection with 234-nm light.

HOO (CH&COOH

12,

12-HPETE (CHz)3COOH

13,

‘OH

15-HPETE

Oleate Hydroperoxides

OOH 16

Linoleate Hydroperoxides y ( C H z ) $ O O H OOH

/

H

I

I

C

(CHz)7COOH T OOH 9

S

20

Figure 1. Hydroperoxides resolved in this study.

are completely stable to normal-phase chromatography, some decomposition occurs in reverse-phase solvents, such as acetonitrile/water or methanol/water, unless 0.01% Et,N is added. Diastereomers could be isolated with isomeric purities of 99% or better from perketals 2 or 5 derived from a-phenethyl hydroperoxide. The chromatographic resolution of the diastereomers of a phenethyl hydroperoxide was more efficient as the perketal2 than with 5. The phenylcyclohexanol derivative was therefore chosen for a broader screen of hydroperoxide resolution. It should be pointed out that our exploration of perketal structure as it relates to resolution has not been exhaustive, since we have been able to resolve every hydroperoxide we have examined as the perketal 2. The hydroperoxides investigated in this study are shown in Figure 1, and they include derivatives of oleic, linoleic, and arachidonic acid. These hydroperoxides were first separated as their methyl esters (as shown in Figure l), derivatized, and separated as the perketals and then reconverted to the optically pure hydroperoxide. In every case where configuration of the resolved hydroperoxide was proven, the R perketal was the first to elute on reversephase HPLC. Figure 3 shows the separation of the 5(R) and 5(S) perketal of HPETE methyl ester; Figure 4 shows the separation of the 15-HPETE diastereomeric perketals. For the perketals of 5-HPETE, the resolved hydroperoxide was reduced to the corresponding alcohol (HETE) and an optical rotation was taken to prove stereochemistry (I 7). For the 15-HPETE perketals, the 15(R) perketal elutes first on reverse-phase HPLC, as is shown upon coinjection of the authentic 15(S)diastereomer prepared from arachidonic acid and soybean lipoxygenase (18). The 5(R) perketal was the first to elute on reverse-phase HPLC. The configuration of the eluting diastereomeric perketals was proved for only the 5- and 15-substituted HPETE’s in the arachidonic acid series, 8 and 13 in Figure 1. The configurations of the perketals of the 8 , 9 , 11, and 12 isomers were inferred from the general observation that the R perketal elutes first on reverse-phase HPLC in all cases where the stereochemistry has been unambiguously determined. The configurations of perketals 6, 14, 16, 17,

Chem. Res. Toxicol., Vol. 3, No. 3, 1990 241

Resolution of Racemic Hydroperoxides I

6

Ib

1

20

1

40

30

Volume (mi)

Figure 3. HPLC for separation of methyl arachidonate 5hydroxyperoxide perketals. Reverse-phase separation on C-18 with acetonitrile solvent (0.1% Et3N added). Detection with 254-nm light.

Table I. Data for Hydroperoxide Resolution via Perketal 2 chromaracemic elution tography hydroperoxide order phase solvent 6 R/S" reverse acetonitrile/5% H,0/0.1% Et3N 7 SIR" normal hexane/l.O% diethyl ether 5-HPETE (8) R/S" reverse acetonitrile/O.l% Et3N SIR" normal hexane/l% ethyl acetate 5-HPETE (8) R/Sb reverse acetonitrile/O.l% Et3N 8-HPETE (9) SIRb normal hexane/l% ethyl acetate 8-HPETE (9) R/Sb reverse acetonitrile/O.l% Et3N 9-HPETE (10) R/Sb normal hexane/l% ethyl acetate 9-HPETE (10) R/S: reverse acetonitrile/O.l% Et3N 11-HPETE (11) SIR normal hexane/l% ethyl acetate 11-HPETE (11) R/Sb reverse acetonitrile/O.l% Et3N 12-HPETE (12) ? normal hexane/ 1% ethyl acetate 12-HPETE (12) R/S" reverse acetonitrile/O.l% Et3N 15-HPETE (13) R/S" normal hexane/l% ethyl acetate 15-HPETE (13) R/Sc reverse acetonitrile/O.l% Et3N 14 SIR' normal hexane/l% ethyl acetate 14 R/S' reverse acetonitrile/O.l% Et3N 16 R/Sc normal hexane/l% ethyl acetate 16 R/S" reverse acetonitrile/O.l% Et3N 17 R/Sc reverse acetonitrile/O.l% Et3N 20 a Stereochemical assignment based on comparison of rotation or with authentic material. bBased on R eluting first on reversephase HPLC (general observation, eight examples). Based on circular dichroism of derivative p-bromobenzoate ester.

.Ph

I!

a * n

12. Stereoisomers

N

I

I

0

60

I

I20

I80

I

240

Volume (ml) I

l

0

IO

l

20

310

Volume (ml)

Figure 4. HPLC for separation of methyl arachidonak 15hydroperoxide perketals. Reverse-phase separation on C-18 with acetonitrile solvent (0.1% Et3N added). Detection with 254-nm light. Top trace shows mixture with perketal derived from lipoxygenase added.

and 19 were also proven by conversion to compounds of known configuration or comparison with materials prepared by soybean lipoxygenase. In each case where stereochemistry of the perketal was proven, the R perketal was the first to elute on reverse-phase HPLC. In Table I is presented the elution order for the perketals studied on normal- and reverse-phase chromatography. A mixture of all of the arachidonate HPETE's was derivatized by the vinyl ether l . Figure 5 shows the normal-phase chromatogram of this mixture of perketals. While the separation of all 12 perketals (from the 6HPETE's) is not complete, we do see complete separation for the 11-and 5-HPETE diastereomers in this mixture of 12 stereoisomers and one each of the 9 and 8 diastereomers is obtained without contamination. We have not explored the separation of individual diastereomeric pairs on normal-phase chromatography, but the chromatogram

Figure 5. HPLC for separation of all perketals analogous to 2 derived from methyl arachidonate hydroperoxides. Normal-phase separation on silica with hexane/ethyl acetate, 99:l. Numbers indicate the position of OOH substitution and absolute configuration of the stereocenter. Detection with 254-nm light.

presented in Figure 5 of the mixture of all 12 isomers indicates that all pairs of diastereomers do separate on silica.

Discussion The hydroperoxide functional group, ROOH, is an important reactive group in chemistry and biology. The Sharpless epoxidation is a recent example of the utility of hydroperoxides in synthesis (19),and hydroperoxides are also formed in biological systems by lipoxygenase enzymes present in plants and animals (1-5). Even though hydroperoxides play an important role in chemistry and biology, general approaches for the resolution of chiral hydroperoxides have not been reported and chemical approaches to optically pure hydroperoxides by synthesis have not always been successful. In fact, only a few reports of successful syntheses of optically pure hydroperoxides by nucleophilic substitution methods have appeared in the literature.

242 Chem. Res. Toxicol., Vol. 3, No. 3, 1990

Porter et al.

Over 30 years ago, Mosher reported that 2-octyl tosylate undergoes reaction with hydrogen peroxide to give 2-octyl hydroperoxide, presumably with inversion of configuration (7). Johnson reported a synthesis of the same hydroperoxide from optically pure 2-bromooctane using potassium superoxide. The sequence 2-octanol - 2-bromooctane 2-octyl hydroperoxide 2-octanol results in the formation of octanol with 96% retention of configuration by two inversion steps. The reagent Ag+/H202has also been a useful reagent for converting alkyl bromides to hydroperoxides or dialkyl peroxides with inversion of configuration occurring during the reaction. For example, Davies prepared 2-octyl peroxide by this method (20), and Porter and co-workers (21) successfully employed this procedure for the preparation of prostaglandin endoperoxides. While nucleophilic displacement utilizing superoxide or hydrogen peroxide is a useful approach for preparing saturated hydroperoxides, this approach is unsuccessful for preparing optically pure allylic and dienylic hydroperoxides from lipoxygenase enzymes. Thus, while Corey and co-workers reported that the mesylate of 5-HETE methyl ester could be converted to the 5-HPETE methyl ester (22) by the use of hydrogen peroxide at -100 “C, Zamboni and Rokach (23) showed that this reaction proceeds with loss of optical purity with a perfectly racemic product mixture being formed. Our attempts directed toward the preparation of optically pure allylic or dienylic hydroperoxides based upon nucleophilic displacement reactions also failed (24), and we therefore sought an alternate approach for the preparation of these important compounds.

-

-

H11C5d

-

OMS /

5

Scheme I11

-

(CH2)3COOMe

W z

(optically pure)

racemic 5-HPETE methyl ester

HPLC techniques have gained widespread use in the separation of enantiomers and diastereomers. For example, chiral HPLC columns have been used to separate enantiomers, and this method has proved to be useful for the separation of hydroxy fatty acids, such as the HETE’s and the hydroxy linoleates (25). Our initial screens of hydroperoxide resolution using chiral HPLC columns proved unsuccessful; therefore, we sought an alternate approach to hydroperoxide resolution. The approach we settled upon was a classical resolution scheme in which the chiral hydroperoxide was converted to a diastereomeric derivative using an optically pure resolving agent. The derivative of the hydroperoxide to be made was chosen with ease of formation and reconversion to the hydroperoxide in mind, and the strategy also was developed with the notion that the diastereomeric hydroperoxide derivatives would be separated by the use of HPLC. This approach has been used effectively in the resolution of carboxylic acids and amines (26). We quickly settled on perketal derivatives such as 2 as resolving reagents, and we have yet to fail in an attempted hydroperoxide resolution by the use of this strategy. The most difficult of the separations was 2-octyl hydroperoxide (7), for which the perketal reverse-phase separation was unsuccessful. However, normal-phase chromatography of the 7 perketal could achieve base-line separation on an analytical or a preparative scale. Each of the other hydroperoxides (Figure 1)we investigated showed perketal separation on reverse-phase chromatography, and although we have not studied all of them by normal-phase chromatography, each one that we examined by this method did successfully separate.

It should be noted that the perketal derivatives are reasonably robust compounds. For example, the perketal functional group survives the conditions required for hydrolysis of a methyl ester as shown in the chemistry of the hydroperoxide 17. Thus, the methyl ester perketal of 17 can be hydrolyzed with LiOH and the perketal functional group survives the hydrolysis (Scheme 111). Upon acidification of the solution, the free acid is formed and the perketal is hydrolyzed. The ability of the perketal to withstand these hydrolysis conditions suggests that simple synthetic conversions on functional groups elsewhere in a perketal-containing molecule might be possible. While many approaches have been taken for the preparation of optically pure hydroperoxides, the strategy presented in this paper would appear, at the present time, to be the most convenient and general of the methods described in the literature. Nucleophilic displacement of alkyl halides or mesylates with hydrogen peroxide anion or superoxide is apparently limited to the preparation of simple alkyl hydroperoxides, these approaches failing for halides or sulfonate esters that lead to stabilized carbocations. Resolution of hydroperoxides by the use of chiral HPLC columns is not yet a reality, and classical methods of resolution therefore remain as the only reasonable alternative. Classical resolution via HPLC separation of hydroperoxide derivative diastereomers is, in fact, a powerful method for preparation of optically pure hydroperoxides, and separation of hundreds of milligrams of diastereomeric perketals is possible. The structure of the auxiliary can also be altered if necessary, although the perketal functional group has the proper reactivity and stability required for this strategy and 2-phenylcyclohexanol was uniformly successful for resolution of the hydroperoxides reported in this study. Acknowledgment. This work was supported by USPHS Grant HL-17921. P.D. was supported by a NCI Postdoctoral Fellowship (CA 08282-Ol), 1987-1988. J.K. was supported by a NIEHS Toxicology Grant (ES 07031), 1988-1990. Registry No. 1,116102-43-3;3,116102-44-4;(*)-6,116181-13-6; (R)-6,78833-98-4; (S)-6,116102-42-2;(R)-6 perketal, 116102-45-5; (S)-6 perketal, 116181-21-6; (&)-7,126873-43-6; (R)-7,68570-62-7; (S)-7, 78856-79-8; (S)-7 perketal, 126788-61-2; (R)-7 perketal, 126873-59-4;(&)-8,73307-51-4;(R)-8,86562-49-4;(S)-8,71774-08-8; (&)-8 methyl ester, 80629-41-0; (R)-8 perketal methyl ester, 126788-60-1; (S)-8 perketal methyl ester, 126873-58-3; (&)-9, 120143-52-4; (R)-9, 100896-35-3; (S)-9, 114128-54-0; (*)-lo, 120048-07-9; (R)-10, 126873-46-9; (S)-10, 126873-47-0; (&)-11, 126873-44-7; (R)-11, 73347-42-9; (S)-11, 126873-48-1; (*)-12, 126873-45-8; (R)-12, 126873-49-2; (S)-12, 71774-10-2; (*)-13, 73804-66-7;(R)-13,126873-50-5;(S)-13,70981-96-3;( 8 - 1 3 perketal methyl ester, 12678862-3; (&)-14,126821-23-6;(R)-14,126873-51-6; (S)-14, 126873-52-7; (&)-15, 126788-58-7; (R)-15, 126873-53-8; (S)-15, 126873-54-9; (*)-16, 126788-59-8; (R)-16, 126873-55-0; (S)-16,126873-56-1;(&)-17,97672-38-3;(R)-17,73036-16-5;(S)-17, 33964-75-9; (&)-17methyl ester, 116181-14-7;(R)-17methyl ester,

Resolution of Racemic Hydroperoxides 116181-17-0;(S)-17 methyl ester, 96192-73-3; (*)-18,123410-62-8; (R)-18, 126873-57-2; (S)-18, 104832-62-4; (f)-19,123410-63-9; (R)-19,104759-96-8; (S)-19,122046-44-0; (*)-20, 100018-30-2; (R)-20,67597-24-4; (S)-20,29774-12-7; 13(R)-hydroxy-9(2),11(E)-octadecadienoic acid methyl ester, 10219-70-2; trans-2phenylcyclohexanol, 2362-61-0; trichloroethylene, 79-01-6.

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