Resolution of enantiomers of hydroxyeicosatetraenoate derivatives by chiral phase high-pressure liquid chromatography

ANALYTICAL

BIOCHEMISTRY

Resolution

173,456-462

(1988)

of Enantiomers of Hydroxyeicosatetraenoate Derivatives Chiral Phase High-Pressure Liquid Chromatography

DAN J. HAWKINS,

HARTMUT

K~~HN, ERIC H. PETTY,

AND ALAN

by

R. BRASH

Division of Clinical Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Received March 11, I988 A new method was developed for analyzing the steric configuration ofhydroxyeicosatetraenoates (HETEs) and other hydroxy fatty acids. Racemic HETE methyl esters were reacted with either benzoyl or naphthoyl chloride in pyridine and the resulting aromatic ester derivatives purified by reversed phase HPLC and subsequently chromatographed on a chiral stationary phar; HPLC column [(R)-(-)-N-3.5-dinitrobenzoyl-a-phenylglycine)]. In contrast to the enantiomers of the underivatized HETE methyl esters which were only partially resolved, the enantiomers of their aromatic ester derivatives were completely separable on this chiral phase. Chiral HETEs can be retrieved from the aromatic derivatives by alkaline hydrolysis. Thus, this method has both analytical and preparative applications. o 1988 Academic press, I~C.

In the last few years, enantiomers of a variety of molecules have been shown to be separable by high-pressure liquid chromatography upon chiral stationary phases (l-6). Recently, we demonstrated that chiral phase HPLC on (R)-(-)-N-(3,5-dinitrobenzoyl)-cr-phenylglycine (DNBPG)’ columns is a convenient means of analyzing the steric configuration of hydroxyeicosatetraenoates (HETEs) (78). However, with the existing methodology, chiral phase HPLC has been shown to effect only partial separation of HETE enantiomers. Thus, its use as an analytical and preparative tool has been limited to some degree. The separation of enantiomers by chiral phase HPLC is generally believed to require a three point stereochemical interaction between the solute enantiomers and the chiral stationary phase (l-6). The stereospecific interaction(s) may involve spatial factors, bonding, and/or repulsive forces (l-6). Structure-activity relationships for model com-

pounds and chiral phase columns indicate that K--K donor-acceptor interactions between aromatic moieties of solute enantiomers and “electron-deficient” dinitrobenzoyl residues of the chiral stationary phase play a crucial role in the chiral molecular recognition process and differential retention of enantiomers ( l-6). The chiral center and conjugated diene system of underivatized HETE methyl esters has a relatively weak steric interaction with the chiral DNBPG stationary phase, resulting in only partial separation of their enantiomers (7,8). Therefore, the introduction of an electron-rich aromatic residue into the molecule, connected to the chiral center via an ester linkage, might be expected to increase the separation ofthe enantiomeric derivatives on the DNBPG column. This idea prompted us to examine the separation of HETE enantiomers after conversion of the free alcohol moiety to benzoyl and naphthoyl esters. In this report, we describe the synthesis and purification of these derivatives and the chromatographic conditions for separation of the enantiomers by chiral phase HPLC. MATERIALS

’ Abbreviations used: HETEs, hydroxyeicosatetraenoic acids; DNBPG, (R)-(-)-N-(3,5dinitrobenzoyl)-olphenylglycine. 0003-2697/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

AND

METHODS

Chemicals. Arachidonic acid (purity, >99%) was obtained from NuChek Prep 456

CHIRAL

PHASE

CHROMATOGRAPHY

(Elysian, MN). Benzoyl chloride (>99% purity) and 1-naphthoyl chloride (98% purity) were obtained from Aldrich Chemical (Milwaukee, WI). Anhydrous pyridine was maintained over calcium hydride. Bis(trimethylsi1yl)trifluoroacetamide was obtained from Supelco (Bellefonte, PA). Organic solvents for HPLC were distilled-in-glass grade obtained from Burdick and Jackson (Muskegon, MI). Triphenylphosphine was obtained from Eastman Chemicals. Preparation of racemic and chiraE HETE standards. Racemic HETE standards were prepared by controlled autoxidation of methyl arachidonate (9). The resulting hydroperoxy fatty acid methyl esters were reduced with triphenylphosphine to the corresponding hydroxy methyl esters and separated by silica HPLC on an Alltech 5-pm column in 100:0.5 hexane:2-propanol (v/v) (8). Mass spectra of HETEs were obtained after conversion ofthe methyl esters to trimethylsilyl ether derivatives as described previously (8,lO). Chiral HETE standards were prepared biosynthetically from arachidonic acid using enzymes of known stereospecificity. 15(S)HETE was prepared using soybean lipoxygenase (Sigma, St. Louis, MO) (11). 12(S)HETE was prepared using the 100,OOOg supernatant fraction of disrupted porcine white blood cells as a source of 12(S)-lipoxygenase ( 12). 11 (R)-HETE was isolated from the eggs of the sea urchin, Strongylocentrotus purpuratus. as described previously (8). 8(R)-HETE was prepared using an acetone powder of the sea whip coral, Plexaura homomalla, as a source of 8(R)-lipoxygenase (11). S(S)-HETE was prepared as described (13). Synthesis and purijication of HETE derivatives. HETE methyl esters were prepared by treatment of the hydroxy fatty acid with an excess of ethereal diazomethane for 5 min at room temperature. HETE methyl esters were then further derivatized by reaction of 25 pg of the methyl ester with 3 ~1 of either benzoyl chloride or 1-naphthoyl chloride in 30 ~1 of anhydrous pyridine; reaction mixtures were

OF

HYDROXYEICOSANOIDS

457

routinely incubated for 40 min at 60°C but it was later established that incubation for 15 min at room temperature was sufficient. The reaction mixtures were taken to dryness under a stream of nitrogen and the aromatic ester derivatives extracted into hexane. The hexane-soluble products were dried under a stream of nitrogen and purified by reversed phase HPLC (Altex Ultrasphere ODS 5-ym column, 250 X 4.6 mm with a 4-cm guard column) in 96:4 methanol: water (v/v). Products were eluted isocratically at a flow rate of l-l .5 ml per minute; uv absorbance was monitored at 235 nm. The HETE methyl esters eluted in 4.3-5 ml, the benzoyl esters in 7.1-7.6 ml, and the naphthoyl esters in 10. l11.9 ml. On the basis of uv absorbance at 235 nm, the yield of the aromatic ester derivatives was 75% after reaction for 15 min at room temperature. Steric analyses ofHETE derivatives by chiral phase HPLC. HETE methyl esters and the benzoylated and naphthoylated HETE methyl esters were chromatographed on a DNBPG chiral stationary phase HPLC column (Pirkle Type 1-A, ionically linked; 5 pm, modified Spherisorb; 25 cm X 4.6 mm i.d.) obtained from Regis Chemical Co. (Morton Grove, IL). HETE derivatives were eluted with a mobile phase solvent system of n-hexane containing 0.08-0.5% 2-propanol (v/v); products were eluted isocratically at a flow rate of 0.5- 1.2 ml per minute. The uv absorbance of the effluent was monitored at 235 nm and uv spectra of products recorded using a Hewlett-Packard 1040A diode array detector. Resolution values were calculated as described ( 14). Preparation of chiral HETEs from derivatized enantiomers separated by chiral phase HPLC. The enantiomers of naphthoylated HETE methyl ester were isolated by chiral phase HPLC as described above. The derivatives were then treated with a 4: 1 mixture of methanol:5 N KOH (v/v) for 30 min at 60°C under argon to hydrolyze the ester linkages. The time course of the reaction was followed by injecting aliquots on reverse phase HPLC.

458

HAWKINS F’ c-o 00 03 I-naphthoyl

F; +

chloride

ET AL.

~wH3 S(FI.S)-HETE

\py,id,ns methyl

ester \

naphthoylaled

SCHEME

I. Formation

S(R,S)-HETE

methyl

ester

of naphthoyl and benzoyl ester derivatives of a HETE methyl ester

After complete hydrolysis, the reaction mixture was acidified with glacial acetic acid and the HETEs isolated by direct injection on reversed phase HPLC. Circular dichroism. A Jasco J-500A recording spectropolarimeter was used to obtain circular dichroism measurements. HETE enantiomers (free acids) were dissolved in ethanol (10 pg/ml) and scanned from 300 to 205 nm in a l-ml sample holder with a l-cm pathlength. The spectra were recorded four times and the signals averaged. RESULTS

Chiral phase HPLC analyses of HETE benzoyl and naphthoyl esters. HETE methyl esters were reacted with either benzoyl or naphthoyl chloride in pyridine and the resulting aromatic ester derivatives were purified by reversed phase HPLC (Scheme I and Fig. 1). The chiral phase HPLC separation of the enantiomers of 8-HETE methyl ester and its corresponding benzoyl and naphthoyl ester derivatives are shown in Fig. 2, and their uv spectra in Fig. 3. Derivatization improved the separation of the enantiomers of all HETE positional isomers (i.e., 5-,8-, 9-, 1 l-, 12-, and 15-HETE) on the chiral column. Resolution of the naphthoyl ester derivatives was as good or better than for the benzoyl esters (Table 1).

The separation of enantiomers of each positional isomer of the HETE methyl esters and their corresponding benzoyl and naphthoyl ester derivatives is shown in the partial uv chromatograms of Fig. 4. For HETEs with a free alcohol group, the “S” enantiomers elute before the “R” enantiomer. This has been established for 5-, 8-, 1 1-, 12-, and 15HETE methyl esters (Refs (7) and (8) and this study, not shown). The R and S assignments of the naphthoyl derivatives were established as shown in Fig. 5. The order of elution is R before S, i.e., opposite to the underivatized HETE methyl esters. (The R and S assignment of the chiral center is not changed by derivatization.) With the benzoyl derivatives, only 12- and 15-HETEs have been tested: again, “R” eluted before “S.” Modification of a chiral center has also been shown to effect a reversal of elution order of enantiomers for other types of molecules chromatographed on a phthalide-based chiral stationary phase column (5). In additional experiments (data not shown), the enantiomers of the naphthoyl derivatives of 9- and 13-hydroxyoctadecadienoic acid were separated. The chromatogmphic resolution of these compounds was comparable to that of 15HETE and 5-HETE, respectively. Preparation of chiral HETEs. In order to evaluate the suitability of this method for the

CHIRAL

PHASE CHROMATOGRAPHY

459

OF HYDROXYEICOSANOIDS

Reagent Blank

*-HETE methyl esters with underivatized alcohol moiety derivatives

b

lb

SR

50

40

i0

Time hwlutes)

FIG. 2. Separation of enantiomers of an HETE methyl ester and its aromatic ester derivatives by chiral phase HPLC. A mixture of 8(&S)-HETE methyl ester and its corresponding benzoyl and naphthoyl ester derivatives were chromatographed on a DNBPG chiral phase HPLC column (Pirkle Type l-A, Regis; 250 X 4.6 mm i.d.). Compounds were eluted isocratically at a flow rate of I .2 ml/min with 100:0.5 hexane:isopropanol (v/v). 15-HETE Benzoyl Ester

hydroxy-92,ll E-octadecadienoic acid also exhibit negative and positive Cotton effects, respectively (7).

J

Benzoylated 8-HEX

!

\

;,

k

ELUTION

lb

1’5

VOLUME

;o

Naphthoylated

(mls)

FIG. 1. Purification of an HETE aromatic ester derivative by reversed phase HPLC. 1S(R,S)-HETE methyl ester was reacted with benzoyl chloride as described under Materials and Methods and the aromatic ester derivative was extracted and eluted isocratically at a flow rate of 1 ml/min with 96:4 methanol:water (v/v) from a reversed phase HPLC column (Altex Ultrasphere ODS 5-pm column, 250 X 4.6 mm plus 4-cm guard column).

WAVELENGTH

preparation of chiral HETEs, 1 mg of 5(&S)HETE methyl ester was converted to its naphthoyl derivative and the enantiomers resolved on the chiral column. The HETEs recovered after alkaline hydrolysis were indistinguishable by HPLC, uv, and GC-MS, but they exhibited opposite Cotton effects in the CD. The 5(R)- and 5(S)-HETE exhibited a negative and positive Cotton effect, respectively. The “R” and “S” enantiomers of 13-

(nm)

FIG. 3. Ultraviolet spectra of enantiomers of HETE methyl esters and aromatic ester derivatives. The uv spectra of the enantiomers of 8-HETE methyl ester and its corresponding benzoyl and naphthoyl ester derivatives shown in Fig. 2 were recorded using a HewlettPackard 1040A diode array detector. Ultraviolet spectra for the enantiomers of each derivative were indistinguishable. Naphthoyl ester derivatives had a new strong absorbance maximum at 22 1 nm and a weaker band at around 300 nm with significant absorbance remaining at 235 nm. The extinction coefficients were not determined.

460

HAWKINS

ET AL.

TABLE 1 RESOLUTION VALUES FOR ENANTIOMERS OF HETE METHYL ESTERSAND THEIR BENZOYL AND NAPHTHOYL ESTER DERIVATIVES SEPARATED BY CHIRAL PHASE HPLC Methyl esters hexane: isopropanol 100:0.5 (v/v) Resolution value 5-HETE 8-HETE 9-HETE 11-HETE 12-HETE 1.5-HETE

0.58 0.61 0.89 0.67 0.67 0.82

Retention times (1.2 ml per min) 65.6;61 39.0;40.4 38.3;39.6 30.7;31.5 22.8;23.4 27.2;28.1

Benzoyl esters hexane: isopropanol 100:0.25 (v/v) Resolution value

Naphthoyl esters hexane: isopropanol 100:0.25 (v/v)

Retention times (1.2 ml/min)

Resolution value

0.95

16.0; 16.8

1.0 1.1 1.0

13.4; 13.8 15.7; 16.5 12.7; 13.0

2.6 2.7

1.4 1.3

13.7; 14.4 12.9: 13.6

1.2 2.1 1.3 1.5

Retention times (1.2 ml per min) 31.5;38.5 25.k28.7 32.2i33.9 23.3;25.5 26.7;28.3 24.6i26.2

Note. Racemic HETE methyl esters were chromatographed on a (R)-(-)-N-(3,5-dinitrobenzoyl)-rY-phenylglycine chiral stationary phase HPLC column (Pirkle Type l-A, Regis: 25 cm X 4.6 mm i.d.). Enantiomers were eluted isocratically at a flow rate of 1.2 ml/min; uv absorbance was monitored at 235 nm. HETE methyl esters were eluted with 100:0.5 hexane:isopropanol (v/v); benzoyl and naphthoyl derivatives of HETE methyl esters were eluted with 100:0.25 hexane:isopropanol (v/v).

The overall yield for this procedure, calculated on the basis of the uv absorbance at 235 nm, was about 70%. To test for racemization in this preparative procedure, an aliquot of each enantiomer was rederivatized and analyzed on the chiral phase HPLC column; each afforded a single peak with an undetectable level of epimerization (~2%). DISCUSSION

chirality of HETEs. The conversion of HETE methyl esters to their benzoyl and naphthoyl ester derivatives greatly improved the separation of HETE enantiomers on a (R)-(-)-N(3,5-dinitrobenzoyl)-cr-phenylglycine chiral stationary phase. In principle, the approach will also work the other way around; namely,

NAPHTHOVL ESTERS

SHETE

EHETE

I,-HETE

12.HETE

IMETE

Chiral phase HPLC represents the most straightforward method for determining the

“cm” CHlRAl

FIG. 4. Partial uv chromatograms of enantiomers of HETE methyl esters and their aromatic ester derivatives separated by chiral phase HPLC. Racemic HETE methyl esters and their benzoyl and naphthoyl ester derivatives were chromatographed on the chiral phase HPLC column as described in Table 1.

-‘“-

FIG. 5. Partial uv chromatogram of chirally pure and racemic naphthoyl ester derivatives of HETE methyl esters analyzed by chiral phase HPLC. Naphthoyl ester derivatives of racemic HETE methyl esters and enzymatitally prepared chiral standards (see Materials and Methods) of 5(S)-, 8(R)-, 1l(R)-, 12(S)-, and 15(S)-HETE methyl esters were isocratically eluted from the chiral phase HPLC column with 100~0.25 hexane:isopropanol (v/v) at a flow rate of 1.2 ml/min. Co-chromatographing the chiral standards with the racemic naphthoyl ester derivatives established that R enantiomers elute first.

CHIRAL

PHASE CHROMATOGRAPHY

OF HYDROXYEICOSANOIDS

461

derivatization of the HETE methyl esters (Fig. 2). Using this derivative, hydroxy fatty with the 3$dinitrobenzoyl ester should en- acids which lack the conjugated diene of the hance the separation of enantiomers on a HETEs can be detected on HPLC. In fact, we naphthoyl-based chiral stationary phase. found that the naphthoyl esters of hydrogeDerivatization allows complete resolution nated 8- and 1 I-HETE methyl esters are of all HETE enantiomers on the DNBPG chi- readily amenable to uv detection and separaral phase column. In addition to permitting tion on the DNBPG chiral column (data not the precise determination of enantiomeric shown). composition on an analytical scale, the Derivatization of the hydroxyl group remethod can be used for the preparative isola- verses the order of elution of the R and S ention of chiral HETEs. Other HPLC methods antiomers of HETEs on the chiral column. for the separation of racemic HETEs involve The free alcohol of a HETE probably interreaction with a chiral resolving agent, giving acts with the DNBPG chiral phase by hydrodiastereoisomers which are amenable to sep- gen bonding, while the conjugated diene sysaration on conventional columns. These tem functions as the r-donor. After derivatimethods include the use of chiral menthzation, the naphthoyl or benzoyl residues oxycarbonyl, dehydroabietylisocyanate, or constitute r-donors which can interact with a-methoxy-a-trifluoromethyl phenylacetate the electron-deficient dinitrobenzoyl moiety derivatives ( 13,15- 17). These derivatives give of the chiral stationary phase. Thus, the basis excellent separation of 5-HETE and an ade- for the chiral recognition process is reversed. quate resolution ofthe other HETEs, with the This type of reciprocity has also been noted exceptions that 9RS-, 12R4, and 15RS- with other secondary alcohols (5). HETEs are not resolved as the menthoxycarbony1 derivatives (15) and long retention ACKNOWLEDGMENTS times are required to resolve the 12RS-HETE We thank Dr. Fu Ming Chen for his help with the CD dehydroabietylamine urethanes ( 16). The procedure we describe offers three sig- measurements and we gratefully acknowledge Dr. W. H. Pirkle for his valuable advice and suggestions. This work nificant advantages over methods using chi- was supported by NIH Grants GM 15431 and DK ral “resolving agents”: (i) The benzoyl and 35275. naphthoyl esters are enantiomers, not diastereoisomers, and therefore there is no chance REFERENCES of chiral discrimination in the derivatization 1. Pirkle, W. H., House, D. W., and Finn, J. M. (1980) itself. This is a theoretical problem in any J. Chromatogr. 192, 143-158. synthesis of diastereoisomers. Recently, we 2. Pirkle, W. H., Finn, J. M., Schreiner, J. L., and Hamencountered this phenomenon in the derivaper, B. C. (198 I) J. Amer. Chem. Sot. 103,3964tization of racemic hydroxy derivatives of 3966. phytodienoic acid (18); under mild reaction 3. Pirkle, W. H., Mahler, G., and Hyun, M. H. (1986) conditions one enantiomer reacted preferenJ. Liquid Chromatogr. 9,443-453. 4. Pirkle, W. H., Mahler, G. S., Pochapsky, T. C., and tially with (-)-menthylchloroformate, and Hyun. M. H. (1987) J. Chromatogr. 388, 307heating was required to drive the reaction of 314. both enantiomers to completion. (ii) There is 5. Pirkle, W. H., and Sowin, T. J. (1987) J. Chromano possibility of changing the ratio of enanlogr.396,83-92. tiomers during the preliminary purification. 6. Pirkle. W. H., and Pochapsky, T. C. (1987) J. Amer. Chem. Sot. 109,5975-5982. (iii) Recovery of the free alcohols involves a 7. Kuhn, H., Wiesner, R., Lankin, V. Z., Nekrasov. A., simple alkaline hydrolysis. Alder, L., and Schewe, T. (1987) Anal. Biochem. The naphthoyl derivatives exhibited a uv 160,24-34. maximum absorbance at 221 nm and a 8. Hawkins, D. J., and Brash, A. R. (1987) J. Biol. weaker absorbance band at around 300 nm Chem. 262,7629-7634.

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HAWKINS

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13. Corey, E. J., and Hashimoto, S. (198 I ) Tetrahedron Lett. 22,299-302.

ET AL. 14. Johnson, E. L., and Stevenson, R. (1978) Basic Liquid Chromatography, pp. 15-22, Varian Assoc., Inc., Palo Alto, CA. 15. Brash, A. R., Porter, A. T., and Maas, R. L. (1985) J. Biol. Chem. 260,42 lo-42 16. 16. Falck, J. R., Manna, S., Jacobson, H. R., Estabrook, R. W., Chacos, N., and Capdevilla, J. (1984) J. Amer. Chem. Sot. 106,3334-3336. 17. Andre, J. C., and Funk, M. 0. (1986) Anal. Biochem. 158,316-321. 18. Baertschi, S. W., Ingram, C. D., Harris, T. M., and Brash. A. R. (1988) Biochemistry 27, 18-24.