Absolute configurations of K-region epoxide enantiomers of 3

Nov 15, 1987 - Shen K. Yang , Mohammad Mushtaq , Ziping Bao , Henri B. Weems , Magang Shou , Xiang-Lin Lu. Journal of Chromatography A 1989 461, ...
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Anal. Chem. 1987, 59, 2679-2688

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Absolute Configurations of K-Region Epoxide Enantiomers of 3-Methylcholanthrene, Benz[ a ]anthracene, and Benzo[ a Ipyrene Henri B. Weems, Mohammad Mushtaq, and Shen K. Yang* Department of Pharmacology, F. Edward H6bert School of Medicine, Uniformed Services, University of the Health Sciences, Bethesda, Maryland 20814-4799

The absolute conflguratlons of K-region epoxide enantlomers of B-methyichoianthrene, benz[a]anthracene, and benzo[a ]pyrene have been determined via their monomethyl ether derlvatives. Methoxylatlon of each racemic or enantiomeric epoxlde by sodium methoxlde resulted In a pair of monomethyl ether derlvatlves, whlch were separated by normalphase high-performance liquid chromatography (HPLC). The position of the methoxy group was determined by products formed by acid-catalyzed dehydration and/or demethanoiization of each monomethyl ether derivative. Enantiomers of each epoxide and its methoxyiated derivatlves were resolved by at least two of the four Pirkle chlrai stationary phase HPLC columns utilized In this study. The absolute stereochemistries of enantiomeric monomethyl ether derivatives were established by comparlng their clrcular dlchrolsm spectra wlth those of enantlomerk monomethyl ether derlvatives derlved from trans dihydrodlol enantlomers of known absolute configurations. The absolute configuration of each epoxide enantlomer was deduced from the location of the methoxy group and the absolute configuration of enantlomerlc monomethyl ether derlvatlves. Results Indicate that the method described Is useful in general for the determlnation of absolute conflguratlons of K-region epoxkle enantlomers of polycyclic aromatic hydrocarbons.

Epoxides (arene oxides) are major initial products formed in the metabolism of polycyclic aromatic hydrocarbons (PAHs) by mammalian cytochrome P-450 isozymes (1, 2). While some PAH epoxides possess mutagenic and/or cytotoxic activities ( I , 2), others are further metabolized either to nontoxic or to mutagenic and carcinogenic products (3, 4). The stereoselective natures of microsomal cytochrome P-450 isozymes and epoxide hydrolase account for the formation of optically active epoxides, dihydrodiols, and dihydrodiol epoxides, some of which are proximate and ultimate carcinogenic metabolites of some PAHs (3-6). Enantiomeric compositions of some metabolically formed K-region epoxides have been found to be highly dependent on the type of cytochrome P-450 isozymes present in the microsomal enzyme preparations (7-11). Thus, the stereoheterotopic enzyme-substrate interaction determines the absolute configuration and enantiomeric purity of epoxides formed. Hence, the enantiomeric composition of a metabolically formed epoxide reflects the asymmetric binding interaction between the K-region double bond of a PAH and the catalytic site of a cytochrome P-450 isoenzyme. K-region as well as non-K-region epoxides formed in the metabolism of some PAHs can be stabilized and isolated by normal-phase HPLC (7-11). Enantiomers of many of these epoxides can be resolved directly by chiral stationary phase (CSP) HPLC (8,9). Thus, the enantiomeric composition of many K-region epoxides formed in the metabolism of PAHs

by various drug-metabolizing enzyme systems can be determined either directly by CSP HPLC or by CD spectral analysis when the circular dichroism (CD) spectral data of enantiomerically pure epoxides are known (7-11). However, the exact asymmetric enzyme-substrate interaction requires the knowledge of the absolute configurations of epoxide enantiomers. In this report we describe the procedure for determining the absolute configuration of the K-region epoxide enantiomers of 3-methylcholanthrene (MC), benz[a]anthracene (BA), and benzo[a]pyrene (BP). Results indicate that the method is useful in general in the determination of absolute configuration of K-region epoxide enantiomers of PAHs.

EXPERIMENTAL SECTION Chemicals. Racemic K-region epoxides and cis dihydrodiols of MC, BA, and BP, 11-hydroxy-MC, 5-hydroxy-BA, 6hydroxy-BA, 4-hydroxy-BP, and 5-hydroxy-BP were obtained from the Chemical Repository of the National Cancer Institute. 12-Hydroxy-MCwas not available for this study. Acid-catalyzed dehydration with HC1 of MC truns-11,12-dihydrodiol yielded only 11-hydroxy-MC. The phenolic derivatives of MC, BA, and BP were each methoxylated or ethoxylated by reaction with an excess amount of CHJ or C2HJ in acetone containing 10% (v/v) of 0.5 N NaOH. K-region trans dihydrodiols of MC, BA, and BP enriched in R,R enantiomer were obtained by incubation of the respective racemic epoxide with rat liver microsomes in the absence of NADPH or by incubation of the respective parent hydrocarbon with rat liver microsomes and an NADPH-regenerating system (7). NaOCH3 (sodium methylate) and NaH were purchased from Fisher Scientific Co. (Silver Spring, MD). Diethyl ether and HPLC solvents were purchased from Mallinckrodt (Paris, KY). BF3etherate, CH31,and C2H&were purchased from Aldrich Chemical Co. (Milwaukee, WI). High-Performance Liquid Chromatography. HPLC was performed with a Waters Associates (Milford, MA) liquid chromatograph consisting of a Model 6000A solvent delivery system, a Model M45 solvent delivery system, a Model 660 solvent programmer, and a Model 440 absorbance detector (254 nm). Samples were injected via a Valco (Houston, TX) Model N60 loop injector. Retention times and ratios of enantiomers, determined as areas under the chromatographic peaks, were recorded with a Hewlett-Packard (Palo Alto, CA) Model 3390A integrator. Reversed-Phase HPLC. This was performed with either a Du Pont (Du Pont Co., Wilmington, DE) Zorbax ODS column (4.6 mm i.d. X 25 cm) or a Du Pont Golden Series ODS column (6.2 mm i.d. X 8 cm). Products were eluted with a 15-min linear gradient from methanol/water (3/1, v/v) to methanol at a flow rate of 1.5 mL/min. Under the chromatographic conditions described, the retention times of the compounds are as follows: MC monomethyl ether 1, 4.8 min; MC monomethyl ether 2, 5.3 min; ethoxylated derivative of MC monomethyl ether l, 10.4 min; ethoxylated derivative of MC monometthyl ether 2, 11.2 min; ll-hydroxy-MC, 12.6 min; 11-methoxy-MC,17.1 min; 11-ethoxy-MC,18.8min; BA monomethyl ethers 1 and 2, 3.6 min; 5-hydroxy-BA, 6.7 min; 6-hydroxy-BA,6.6 min; 5-methoxy-BA,12.0 min; 6-methoxy-BA, 12.2 min; BP monomethyl ethers 1and 2,4.3 min; 4-hydroxy-BP,

This article not subject to US. Copyright. Published 1987 by the American Chemlcal Society

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ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987

8.4 min; 5-hydroxy-BP, 8.5 min; 4-methoxy-BP, 13.6 min; 5methoxy-BP, 13.8 min. Normal-Phase HPLC. Monomethyl ethers derived from K-region epoxides were separated by use of a Du Pont Golden Series SIL column (6.2 mm i.d. X 8 cm). Two monomethyl ethers derived from MC 11,12-epoxidewere separated by using methanol/tetrahydrofuran (THF)/hexane (1.5/5/93.5, vol ratio) at 2 mL/min. Two monomethyl ethers derived from BA 5,6-epoxide and BP 4,5-epoxide respectively were separated using 0.2/0.2/ 5.0/94.6 (volume ratio) and 0.25/0.25/5.0/94.5 (volume ratio) of THF/methanol/ethyl acetate/ hexane, respectively, at 2 mL/min. Monomethyl ethers derived by methylation of K-region cis dihydrodiols of MC, BA, and BP, respectively, have different retention times on normal-phase HPLC from those derived from the corresponding trans dihydrodiols. CSP HPLC. The enantiomers of epoxides and their monomethyl ether derivatives were resolved by using a CSP column (4.6 mm i.d. X 25 cm; Regis Chemical Co., Morton Grove, IL) packed with spherical particles of 5-pm diameter of y-aminopropyl silanized silica to which either (R)-N-(3,5-dinitrobenzoyl)phenylglycine (R-DNBPG-I or R-DNBPG-C) or (S)-N-(3,5-dinitrobenzoy1)leucine(S-DNBL-I or S-DNBL-C) was either ionically (I) or covalently (C) bonded. The elution solvent was 1-10% (v/v) of ethanol/acetonitrile (2:1, v/v) in hexane at 2 mL/min. Methylation of K-RegionEpoxides. Racemic or enantiomeric epoxide was dissolved in methanol saturated with NaOCH, and heated at 50 "C for 1 h and then stored overnight at room temperature. The resulting two isomeric monomethyl ethers were separated by normal-phase HPLC. Enantiomers of each monomethyl ether were resolved by CSP HPLC. Methylation of Trans Dihydrodiol. Trans and cis dihydrodiols were each methylated to a pair of isomeric monomethyl ethers by dissolving in NaH-treated THF (1 mL) with an 500-fold excess of CH31and a catalytic amount of NaH, added at 0,15,30, and 45 min at room temperature in the dark. Fifteen minutes after the last addition of CH,I, reaction was stopped by dropwise additions of methanol. The resulting isomeric monomethyl ethers were separated by normal-phase HPLC m described above. Due to the use of an excess amount of CHJ, some dimethyl ether was formed in addition to monomethyl ethers. Location of the Methoxy (or Hydroxyl) Group in Monomethyl Ethers. In order to establish the position of the methoxy and/or hydroxy group in each of the monomethyl ethers, one of the two following methods was used. Method 1. Each monomethyl ether (-50 pg) was dissolved in 0.5 mL of acetone and 5 drops (approximately 0.25 mL) of 6 N HC1 was added and heated at 50 "C for 2 h. Method 2. Each monomethyl ether (-50 fig) was dissolved in 1 mL of NaH-treated THF and 10 drops of CzHJ was added and heated at 45 "C for 1.5 h. Methanol (2 mL) was added to quench the reaction and the solvent was evaporated under a stream of nitrogen at 50 "C. The residue, containing a methoxy-ethoxy derivative, was dissolved in THF/methanol (l/l,v/v) and was analyzed by reversed-phase HPLC as described above. Each ethoxylated derivative was then dissolved in 1mL of diethyl ether which was pretreated with sodium as previously described (16), and 10 drops (-0.5 mL) of BF, etherate was added. The mixture was kept under nitrogen and refluxed at 45 "C for 1.5 h. Products formed by the methods described above were partitioned between ethyl acetate and water. The organic phase was washed three times with water and dried under a stream of nitrogen at 50 "C. The residue was dissolved in THF/methanol (1/1,v/v) and was analyzed by reversed-phase HPLC as described above. Purified products were each characterized by UV-vis absorption and mass spectral analyses, as well as by retention times on reversed-phase and normal-phase HPLC. Spectral Analysis. Mass spectral analysis was performed on a Finnigan Model 4000 gas chromatograph-mass spectrometerdata system by electron impact with a solid probe at 70 eV and 250 "C ionizer temperature. Ultraviolet-visible absorption spectra of samples in methanol were determined by using a 1-cm path length quartz cuvette with a Varian Model Cary 118C spectrophotometer. CD spectra of samples in methanol in a quartz cell of I-cm path length at room temperature were measured with a Jasco Model 500A spectropolarimeter equipped with a Model

-

DP500 data processor. The concentration of the sample is indicated by Ak2/mL (absorbance units at wavelength A2 per milliliter of solvent). CD spectra are expressed by ellipticity (@.X1/Ah2, in millidegrees) for solutions that have an absorbance of 4 2 unit per milliliter of solvent at wavelength Xz (usually the wavelength of maximal absorption). Under conditions of measurements indicated above, the molecular ellipticity ( [ o ] in deg.cm2dmo1-') and ellipticity (@Al/An,in millidegrees) are related to the extinction coefficient (ex2, in cm-'.M-') as follows:

,,,

[olkl

=

O.~d@kl/AhZ)

RESULTS We previously reported that K-region epoxide enantiomers of a large number of PAHs can be directly resolved by HPLC using CSP columns (8,9). Each pair of enantiomeric epoxides has CD spectra that are mirror images of each other. The next obvious step is to ascertain the absolute configurations of resolved epoxide enantiomers. Experimental approaches in elucidating the absolute confiiations of the resolved epoxide enantiomers are summarized in Figure 1, using MC 11,12epoxide as an example. Similar procedures were used to elucidate the absolute configuration of the K-region epoxide enantiomers of BA and of BP. The experimental procedures summarized in below are not necessarily carried out in the order described. 1. A racemic epoxide in methanol is reacted with NaOCH,, resulting in two monomethyl ether derivatives. Methyl ethers are separated by normal-phase HPLC. Both monomethyl ethers are trans isomers. 2. The position of methoxy (or hydroxyl) group in each monomethyl ether derivative is determined. In some cases, the ethoxylation reaction may not be necessary. 3. The enantiomers of each monomethyl ether are separated by CSP HPLC. CD spectra of enantiomeric monomethyl ethers are determined. 4. Enantiomers of MC tmns-11,lZ-dihydrodiol are resolved by CSP HPLC. K-region trans dihydrodiols enriched in R,R enantiomer are obtained by incubation of BA and BP, respectively, with rat liver microsomes and an NADPH-regenerating system (7). The absolute configuration of either an enantiomerically pure trans dihydrodiol or a trans dihydrodiol highly enriched in one enantiomer can be established by the exciton chirality CD method (17). 5. Either an enantiomerically pure trans dihydrodiol or a trans dihydrodiol enriched (preferably with an enantiomeric excess 120%) in one enantiomer of known absolute configuration is methylated with CHJ to yield a pair of monomethyl ethers, which are separated by normal-phase HPLC under the same chromatographic conditions as used in step 1. CD spectra of these two monomethyl ethers are determined. 6. The enantiomers of a racemic epoxide are resolved by CSP HPLC (8, 9). '7. Either an enantiomerically pure epoxide enantiomer or an epoxide highly enriched (preferably with an enantiomeric excess 220%)in one enantiomer is reacted with NaOCH, in methanol to yield a pair of isomeric monomethyl ethers. The monomethyl ethers are separated by normal-phase HPLC and their CD spectra are determined. The absolute configurations of the epoxide enantiomers can be deduced from the results obtained as described above. It is important to point out, for the purpose of elucidating the absolute configuration of epoxide (or trans dihydrodiol) enantiomers, the procedures described do not require chemically synthesized racemic K-region epoxide (or trans dihydrodiol), which may not be readily available. Epoxides and trans dihydrodiol highly enriched in one of the two enantiomers can often be obtained biosynthetically by incubation of the parent hydrocarbon with rat liver microsomes and a NADPH-regenerating system with and without the presence of an epoxide

ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987

9

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c2H5'

csp

(f)

HpLcl @ HPLCl @ csp

11s,12s

- C H 3 0 H\

11R,12R

Figure 1. Summary of experimental approach in determiningthe absolute configurations of MC 11,12-epoxide enantiomers. The Same approach has been used in the determination of absolute configurations of K-region epoxide enantiomers of other PAHs. See text for description of steps 1 to 7 and experimental details.

hydrolase inhibitor such as 3,3,3-trichloropropylene1,2-oxide, respectively (7-12).Consequently, steps 3,4,and 6 may be eliminated. The use of an epoxide either enantiomerically pure or highly enriched in one enantiomer in the reaction with NaOCH3will produce two monomethyl ethers that are either enantiomerically pure or highly enriched in one enantiomer (in this case step 1is synonymous to steps 6 and 7 combined). Thus CD spectra of the two monomethyl ethers can be readily obtained, bypassing steps 3 and 6 (Figure 1). Availability of either an enantiomerically pure trans dihydrodiol or a trans dihydrodiol highly enriched in one enantiomer also allows bypassing step 4 (Figure 1). In this study, the monomethyl ethers derived from K-region epoxides of MC, BA, and BP were found to have identical retention times on normal-phase HPLC to the monomethyl ether derivatives derived from methylation of the corresponding trans dihydrodiol. Furthermore, CD Cotton effects of trans dihydrodiol and monomethyl ether enantiomers are characteristically different from those of cis dihydrodiol enantiomers derived from the same PAH (15,18). Earlier studies established that methoxylation of 7,12-dimethyl-BA 5,6-epoxide (19-229, 12-methyl-BA 5,6-epoxide (11), benzo[c]phenanthrene 5,6-epoxide (9,22), and chrysene, 5,6-epoxide (16,22) each resulted in a pair of trans monomethyl ethers. These were established by NMR spectral studies as well as by comparison of the retention times of the monomethyl ethers with those of monomethyl ethers derived from methylation of K-region trans and cis dihydrodiols ( I 1,16,20-22). Thus only trans addition products are known to be formed to date by methoxylation of PAH K-region epoxides. CSP HPLC Resolution of Enantiomeric Monomethyl Ethers. The retention times, absolute configurations, and resolution values in the separation of enantiomeric monomethyl ethers derived either by methoxylation of K-region

epoxides or by methylation of trans dihydrodiols of MC, BA, and BP by ionically and covalently bonded R-DNBPG and S-DNBL columns are shown in Table I. The data on the enantiomeric resolutions of K-region epoxides and trans dihydrodiols of MC, BA, and BP were partially reported earlier (8, 14, 15) and are updated in Table I for comparison; new chromatographic data were obtained by using different elution solvent compositions and/or CSP columns which were not previously reported. Enantiomers can be considered to have base-line resolution if the chromatographic peaks of separated enantiomers are both perfectly symmetrical and have a resolution value 21.0. In practice, however, two compounds are said to have base-line separation when the resolution value is 1.5 or greater. Identities of resolved enantiomers were confirmed by UV-vis absorption, CD, and mass spectral analyses. Except that the enantiomers of MC monomethyl ether 1 have different elution orders on R-DNBPG-I and RDNBPG-C, other enantiomers, if resolved on a particular CSP, were found to have the same elution order regardless whether the CSP was ionically or covalently bonded (Table I). The llR,12R enantiomer of MC monomethyl ether 1 was more strongly retained on R-DNBPG-I, S-DNBL-I, and S-DNBL-C, but was less strongly retained on R-DNBPG-C. DMBA 5R,GR-dihydrodiol is also found to be less strongly retained on the covalently bonded R-DNBPG and more strongly retained on the ionically bonded R-DNBPG (18). The R,R enantiomers of monomethyl ether 1of both BA and BP were more strongly retained on both ionically and covalently bonded R-DNBPG and less strongly retained on both ionically and covalently bonded S-DNBL. The S,S enantiomers of monomethyl ether 2 derived from the K-region epoxides of MC, BA, and BP, if resolved, were all more strongly retained on R-DNBPG and S-DNBL.

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T a b l e I. C S P H P L C Resolution of Enantiomeric K-Region Derivatives of 3-Methylcholanthrene (MC), B e n z [ a ] a n t h r a c e n e (BA), and Benzo[a]pyrene ( B P )

Chemical"

CSPb

% Ac

MC 11,12-epoxide

R-DNBPG-I

5.0 2.5 2.5 1.0 1.0 0.5 1.0 0.5 10.0 5.0 10.0 10.0 10.0 5.0 5.0 2.5 10.0 5.0 5.0 2.5 5.0 10.0 5.0 10.0 5.0 10.0 5.0 10.0 5.0 2.5 1.0 0.5 1.0 0.5 1.0 0.25 1.0 0.25 5.0 5.0 5.0 5.0 5.0 2.5 2.5 2.5 2.5 1.0 5.0 2.5 2.5 2.5 2.5 1.0 2.3 1.0 2.5 1.0 0.5 0.5 10.0 10.0 5.0 10.0 5.0 10.0 5.0 5.0 2.5 5.0 2.5 5.0 5.0

R-DNBPG-C S-DNBL-I S-DNBL-C MC-trans-l1,12dihydrodiol (lla12a) MC monomethyl ether 1 (lla12a)

R-DNBPG-I R-DNBPG-C S-DNBL-I S-DNBL-C R-DNBPG-I R-DNBPG-C S-DNBL-I

MC monomethyl ether 2 (lla12a)

S-DNBL-C R-DNBPG-I R-DNBPG-C S-DNBL-I S-DNBL-C

BA 5,g-epoxide

R-DNBPG-I R-DNBPG-C S-DNBL-I S-DNBL-C

BA-trans-5,6dihydrodiol (5e6e) BA monomethyl ether 1 (5e6e)

R-DNBPG-I R-DNBPG-C S-DNBL-I S-DNBL-C R-DNBPG-I R-DNBPG-C S-DNBL-I S-DNBL-C

BA monomethyl ether 2 (5a6a)

R-DNBPG-I

B P 4,5-epoxide

R-DNBPG-I

R-DNBPG-C S-DNBL-I S-DNBL-C

R-DNBPG-C

BP-trans-4,5dihydrodiol (4e5e)

S-DNBL-I S-DNBL-C R-DNBPG-I R-DNBPG-C S-DNBL-I S-DNBL-C

B P monomethyl ether 1 (4a5a)

R-DNBPG-I R-DNBPG-C S-DNBL-I S-DNBL-C

retention timed enantiomer 1 enantiomer 2 9.2 (llR,12S) 14.4 (llR,12S) 14.6 (llR,12S) 24.2 (llR,12S) 9.3 (llS,12R) 14.1 (llS,12R) 10.8 (llR,12S) 16.6 (llR,12S) 20.4 (llR,12R) 50.5 (llR,12R) 16.1 (llR,12R) 23.2 (llR,12R) 12.1 26.9 32.8 ( l l S , l 2 S ) 58.2 (llS,12S) 11.3 (llR,12R) 27.6 (llR,12R) 15.2 (llS,lZS) 28.2 ( l l S , l 2 S ) 11.1 (llS,12S) 11.4 (llR,12R) 18.5 (llR,12R) 9.3 (llR,12R) 17.7 (llR,12R) 7.1 (llR,12R) 9.9 (llR,12R) 6.3 11.5 21.1 14.0 (5R,6S) 19.7 (5R,6S) 13.6 19.1 (5R,6S) 6.8 13.9 7.4 14.3 (5R,6S) 33.7 18.8 24.0 14.2 12.6 (5S,6S) 20.5 (5S,6S) 17.6 (5S,6S) 12.4 (5R,6R) 10.7 20.2 (5R,6R) 11.7 (5R,6R) 18.4 (5R,6R) 16.0 (5R,6R) 11.7 (5R,6R) 10.5 19.4 12.0 (4R,5S) 18.1 (4R,5S) 15.5 (4R,5S) 24.4 (4R,5S) 13.3 15.6 24.4 (4S,5S) 15.0 34.9 (4S,5S) 17.3 (4R,5R) 40.3 (4R,5R) 10.9 (4R,5R) 22.2 (4R,5R) 21.3 (4S,5S) 36.2 (4S,5S) 17.1 36.0 (4S,5S) 10.5 (4R,5R) 9.6 (4R,5R)

9.7 (llS,12R) 15.7 (11S,12R) 14.9 (llS,12R) 27.4 (llS,12R) 9.5 (llR,12S) 14.5 (llR,12S) 11.0 (llSJ2R) 17.0 (llS,12R) 21.2 ( l l S , l 2 S ) 53.2 (llS,12S) 17.8 (llS,12S) 24.5 (llS,12S) 12.1 26.9 33.7 (llR,12R) 63.6 (llR,12R) 13.4 ( l l S , l 2 S ) 32.7 ( l l S , l 2 S ) 15.7 (llR,12R) 30.4 (llR,12R) 11.7 (llR,12R) 13.1 (llS,12S) 22.4 (llS,12S) 11.8 (11S,l2S) 23.8 (llS,12S) 7.6 (llS,12S) 10.3 (llS,lZS) 6.3 11.5 21.1 14.5 (5S,6R) 20.5 (5S,6R) 13.6 19.3 (5S,6R) 6.8 13.9 7.6 14.9 (5S,6R) 33.7 18.8 24.0 14.2 13.1 (5R,6R) 22.6 (5R,6R) 18.5 (5R,6R) 13.4 (5S,6S) 10.7 21.5 (5S,6S) 12.4 (5S,6S) 19.8 (5S,6S) 17.8 (5S,6S) 12.2 (5S,6S) 10.5 19.4 12.5 (4S,5R) 18.8 (4S,5R) 15.9 (4S,5R) 25.0 (4S,5R) 13.3 15.6 25.2 (4R,5R) 15.0 35.3 (4R,5R) 17.6 (4S,5S) 41.2 (4S,5S) 11.1 (4S,5S) 23.6 (4S,5S) 21.4 (4R,5R) 38.6 (4R,5R) 17.1 36.7 (4R,5R) 11.2 (4S,5S) 9.9 (4S,5S)

RV' 1.4 2.4 0.1 0.7 0.1 0.4 0.3 0.4 0.8 1.1 2.1 1.3 0.0 0.0 0.6 1.8 3.3 3.4 0.5 1.4 1.0 2.8 4.2 3.9 6.1 2.5 1.0 0.0 0.0 0.0 0.8 1.0 0.0 0.1 0.0 0.0 0.2 0.9 0.0 0.0 0.0 0.0 1.0 2.5 1.2 1.8 0.0 0.8 1.6 1.7 2.3 1.2 0.0 0.0 0.7 1.0 0.5 0.5 0.0 0.0 0.8 0.0 0.2 0.3 0.5 0.3 0.6

-0.1 1.4 0.0 0.3 1.1 0.4

ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987 2883

Table I (Continued) Chemical"

CSPb

% A'

BP monomethyl ether 2

R-DNBPG-I R-DNBPG-C S-DNBGI 5'-DNBL-C

5.0 5.0 5.0 5.0

(4a5a)

retention timed enantiomer 1 enantiomer 2

RV'

19.0 (4R,5R) 15.4 (4R,5R) 10.3 (4R,5R) 9.4 (4R,5R)

2.0 2.4 0.2 -0.1

20.6 (4S,5S) 17.4 (4S,5S) 10.5 (4S,5S) 9.5 (4S,5S)

oMonomethyl ethers are designated as 1 and 2 according to their elution order on normal-phase HPLC (see Figures 2-4). Conformations (a for quasi-axial and e for quasi-equatorial)of dihydrodiols and monomethyl ethers are indicated in parentheses. Part of the data on CSP HPLC resolutions of epoxide and trans-dihydrodiol enantiomers were reported earlier (8, 14,15) and data shown in this table were updated from reanalysis. bCSPs are defined in Materials and Methods. 'Percentage of solvent A (ethanol/acetonitrile,2:1, v/v) in hexane. The flow rate was 2 mL/min with a void volume of 2.4 mL. dSee text for the assignments of absolute configurations of resolved enantiomers. Enantiomers are designated as 1and 2 according to their elution order on CSP HPLC and have CD spectra that are mirror images of each. 'RV = resolution value = 2 (Vz- Vl)/(W2+ Wl), where Vis retention volume and W is peak width at base.

IB

OH

OH

llR,12R

d

UJ

11s,12s

(u

I-

a

w

= -16 10

o

0

z

-Julr

R-DNBPG-C ,1 O % A I

0

l

l

12s

10

16

-201

;'"I

I l I I l I 250

4

1

~

1

1

1

1

1

1

1

1

1

1

35 0

WAVELENGTH ( nm

1

RETENTION TIME (mid Flgure 2. NormaCphase HPLC separation of monomethyl ethers derived from methoxylation of racemic MC ll,l2-epoxide (upper chromatogram, A), CSP HPLC separation of trans-1l-methoxy-l2-hydroxy-l1,12dihydro-MC enantiomers on R-DNBPG-C column and 10% solvent A (ethanol:acetonW, 2:1, vlv) in hexane (middle chromatogram, A), CSP HPLC separation of trans-l l-hydroxy-12-methoxy-l1,12dihydro-MCenantiomers on RDNBPGC column and 10% solvent A In hexane (lower chromatogram,A), and CD spectra of MC-ll(R),12(R)dhydrod!uI A; enantiomeric excess 95%, @ 2 5 0 / A 2 7 4 = -13.7 mdeg, ref 14), ~ 1 1 ( R ~ ~ t h o x y - 1 2 ( ~ ~ y d r o ~ 1 1 , 1 2 d h y d(-, r o - MBCthe less strongly retained enantiomer In the middle chromatogram in A; enantiomerically pure, @250/A274 = -18.6 mdeg), MC-1 1(R~hydroxy-l2(R)-methoxy-l1,12dihydro-MC (- - , B the less strongly retained enantiomer in the lower chromatogram In A; enantiomericaiiy pure, @250/A274 = -23.4 mdeg), and MC-cis-11(S),12(R)-dihydrodiol(---, B; enantiomerically pure, @ 2 5 0 / A 274 = -18.7 mdeg; ref 15).

-

Absolute Configurations of Enantiomeric MC 11,12Epoxides. A racemic MC 11,12-epoxide was methoxylated to form a pair of monomethyl ethers in a 32:68 ratio (Figure 2A, upper chromatogram). Both monomethyl ethers had UV-vis absorption spectra similar to that of MC-trans11,12-dihydrodiol, indicating that both monomethyl ethers are saturated a t the 11,12 positions. MC monomethyl ether 1(M' at m/z 316 with characteristic fragment ions at m/z 299 (loss of OH), 298 (loss of H,O), 285 (loss CH30), and 284 (loss of CH30H)) was dehydrated with HC1 in acetone to a major product in addition to several unidentified minor products as revealed by reversed-phase HPLC analysis. This major product (M+ a t m / z 298 with characteristic fragment ions at m/z 283 (low of CHJ, 267 (loss of CH30H), and 255 (loss of CH3CO)) was identical with the authentic 11-methoxy-MC with respect to its retention time

on reversed-phase HPLC, UV-vis absorption spectra in methanol and in alkaline methanol, and mass spectrum. Unlike the hydroxyl proton in 11-hydroxy-MC, the 11-methyl group in 11-methoxy-MC is not ionizable. Consequently 11methoxy-MC (or other monomethoxy-PAH) has similar absorption spectra in both methanol and in alkaline methanol. To further confirm the position of methoxy (or hydroxyl) group, MC monomethyl ether 1was ethoxylated with C2H51 in NaH-treated THF,resulting in a methoxy-ethoxy product (M+ at m / z 344 with characferistic fragment ions at m / z 312 (loss of CH30H), 299 (loss of C2H50), and 298 (loss of CH30H)). This methoxy-ethoxy derivative was refluxed in sodium-treated diethyl ether (16)containing BF3 etherate, resulting in a product which was found to be 11-methoxy-MC by reversed-phase HPLC, UV-vis absorption, and mass spectral analyses. Hence the methoxy group of MC mono-

2884

ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987

,

A

@@&$OCH,/!,

(*I+

&OH

-B

OCH,

1

(fW2

50%

50%

dOH

t -.-.-J'lLl NP HPLC

I

4-

m ,,,

,

20

10

55,6'

R-DNBPG-C,

R-DNBPG-C, 2 . 5 % A

10

20

RETENTION TIME ( min

1

Figure 3. NormaCphase HPLC separation of monomethylethers derlved from methoxylatlon of racemic BA 5,6-epoxide (upper chromatogram, A), CSP HPLC Separation of trans-5-methoxy-6-hydrox5,6dihydro-BA enantiomers on an R-DNBPG-C column and 2.5% solvent A in hexane (middle chromatogram,A), CSP HPLC separation of trans -5-hydroxy-6methoxy-5,6dlhydro-BA enantiomers on R-DNBPGC column and 2.5 % solvent A in hexane (lower chromatogram, A), and CD spectra of BA 5(R),6(R)dihydrodioi (---, A; enantiomeric excess -60%, +26,1A 266 = -3.95 mdeg; ref 7 and 22), 5(S)-methoxy~(S~ydroxy-5,6d~y~o-BA (-, B; the less strongly retained enantiomer In the middle chromatogram In A; optically pure, +,,,/A 286 = +7.7 rndeg), BA-5(R)-hydroxy-6(R)-methoxy-5,6dihydro-BA (- - -, B; the less strongly retained enantiomer in the lower chromatogram in A; optically pure, @,,,/A 286 = -8.8 mdeg), and 7-methyl-BA-5(R),6(R)-dlhydrodiol (. .,enantiomeric excess 70.6%; aZ3,/A ,*, = -16.6 mdeg; ref 24; elllptlclty scale is reduced by 50%).

-

methyl ether 1 was confirmed to be at the Cll position. On the basis of foregoing data, MC monomethyl ether 1was established to be trans-ll-methoxy-12-hydroxy-ll,l2-dihydro-MC. MC monomethyl ether 2 (M+ at m/z 316 with characteristic fragment ions at m/z 299 (loss of OH), 298 (loss of HzO), 285 (loss of CH,O), and 284 (loss of CH,OH)) was dehydrated with HCl in acetone to a 11-hydroxy-MC (M' at m / z 284 with fragment ions at m / z 269 (loss of CHB), 255 (loss of CHO), and 252 (loss of CH,OH)) as the predominant product, which was identical with the authentic 11-hydroxy-MC with respect to its retention time on reversed-phase HPLC and UV-vis absorption spectra in both methanol and alkaline methanol and mass spectrum. MC monomethyl ether 2 was also ethoxylated with CZHJ in NaH-treated THF, resulting in a methoxy-ethoxy product (M+ at m/z 344 with fragment ions a t m/z 312 (loss of CHaOH), 299 (loss of CzH,O), and 298 (loss of C,H,OH)). This methoxy-ethoxy derivative was refluxed in sodium-treated diethyl ether (16)containing BFB etherate, resulting in a product which was found to be 11-ethoxy-MC by reversedphase HPLC, UV-vis absorption, and mass spectral analyses (M+ at m / z 312). Hence the methoxy group of MC monomethyl ether 2 was confirmed to be at the C12position. On the basis of the results described above, MC monomethyl ether 2 was established to be trans-ll-hydroxy-12methoxy- 1l112-dihydro-MC. Enantiomers of MC monomethyl ethers 1 and 2 were resolved by CSP HPLC (Figure 2A and Table I). CD spectra of the less strongly retained enantiomes on R-DNBPG-C are shown in Figure 2B. The characteristics of CD Cotton effects on both enantiomeric monomethyl ether 1 and 2 shown in

Figure 2B are identical with those of MC-ll(R),l2(R)-dihydrodiol. The absolute configuration of an enantiomeric MC trans-1 1,12-dihydrodiol has previously been determined by the exciton chirality CD method (14). Furthermore, CD Cotton effects of enantiomeric MC cis-11,12-dihydrodiob (15) are different from those of the trans enantiomers (Figure 2B). An enantiomerically pure MC-trawl l(S),l2(S)-dihydrodiol was methylated with CH31in NaH-treated T H F and the resulting two monomethyl ethers were separated by normalphase HPLC (Figure 2A, upper chromatogram). Both moand nomethyl ethers derived from MC-trans-ll,l2-dihydrodiol 11,12-epoxidehad identical retention times on normal-phase HPLC; monomethyl ethers derived from MC-cis-11,12-dihydrodiol had different retention times. The CD spectrum of monomethyl ether 1 was identical with that of the more strongly retained enantiomer shown in the middle chromatogram of Figure 2A. The CD spectrum of monomethyl ether 2 was identical with that of the more strongly retained enantiomer shown in the lower chromatogram of Figure 2A. On the basis of the data described above, the less strongly retained enantiomers of both MC monomethyl ethers 1and 2 on R-DNBPG-C are deduced to have ll(R),12(R)absolute configurations. An enantiomeric MC 11,12-epoxide less strongly retained (enantiomer 1)on R-DNBPG-I was reacted with NaOCH, in methanol. The resulting monomethyl ethers were separated by normal-phase HPLC (upper chromatogram, Figure 2A). Monomethyl ethers 1 and 2 have CD spectra identical with those of 11(S)-methoxy-12(S)-hydroxy-ll,l2-dihydro-MC and 11@)-hydroxy-12(R)-methoxy-11,la-dihydro-MC, respectively. The foregoing results thus established that an MC 11,12epoxide enantiomer less strongly retained by R-DNBPG-I has

ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987

a llR,12S absolute stereochemistry. Absolute Configurations of Enantiomeric BA 5,6-Epoxides. A racemic BA 5,6-epoxide was methoxylated to form a pair of monomethyl ethers in an approximately 1:l ratio (Figure 3A, upper chromatogram). Both monomethyl ethers have UV-vis absorption spectra similar to that of BAtrans-5,6-dihydrodioldio1, indicating that both monomethyl ethers are saturated at the 5,6 positions. BA monomethyl ether 1 (M+ at m / z 276 with characteristic fragment ions at m/z 259 (loss of OH), 258 (loss of H20), 245 (loss of CH30),and 244 (loss of CH30H))was dehydrated with HC1 in acetone to a major product in addition to several unidentified minor products as revealed by reversed-phase HPLC analysis. The major product (M+ at m / z 244) was identical with the authentic 6-hydroxy-BA with respect to its retention time on reversed-phase HPLC, UV-vis absorption spectrum in methanol and in alkaline methanol, and mass spectrum. BA monomethyl ether 2 (M+ at m/z 276 and characteristic fragment ions at m/z 259 (loss of OH), 258 (loss of H,O), 245 (loss of CH30),and 244 (loss of CH3OH)) was dehydrated with HCl in acetone to a 5-hydroxy-BA (M+ at m f z 244) as the predominant product, which was identical with the authentic 5-hydroxy-BA with respect to its retention time on reversed-phase HPLC and UV-vis absorption spectra in both methanol and alkaline methanol and mass spectrum. Taken together, the results indicated that BA monomethyl ethers 1and 2 are truns-5-methoxy-6-hydroxy-5,6-dihydro-BA and trulzs-5-hydroxy-6-methoxy-5,6-dihydro-BA7 respectively. Enantiomers of BA monomethyl ethers 1 and 2 were resolved by CSP HPLC (Figure 3A and Table I). CD spectra of the less strongly retained enantiomers on R-DNBPG-C are shown in Figure 3B. The characteristics of CD Cotton effects of both enantiomeric monomethyl ether 1 and 2, which are less strongly retained on R-DNBPG-C, are similar to those and 7-methyl-BA-5of 7-methyl-BA-5(S),6(S)-dihydrodiol (R),G(R)-dihydrodiol, respectively. The absolute confiiations and BAof enantiomeric 7-methyl-BA-truns-5,6-dihydrodiol truns-5,6-dihydrodiol have been determined by the exciton chirality CD method (23,24). The CD Cotton effects of BA-5(R),6(R)-dihydrodio17 which has a quasi-diequatorial conformation, change signs when it is converted to a preferentially quasi-diaxial diacetate derivative (23). The CD Cotton effects of BA-5(R),G(R)-dihydrodioldiacetate are also similar to those of 7-MBA-5(R),6(R)-dihydrodiol(23).Furthermore, CD Cotton effects of an enantiomeric 8-methyl-BA (or 10methyl-BA)-cis-5,6-dihydrodiol(15) are characteristically different from those of trans dihydrodiol enantiomers shown in Figure 3B. An enantiomerically pure BA-trans-5(R),G(R)-dihydrodiol was methylated with CHJ in NaH-treated THF and the resulting two monomethyl ethers were separated by normalphase HPLC (Figure 2A, upper chromatogram). Both moand nomethyl ethers derived from BA-truns-5,6-dihydrodiol 5,6-epoxide had identical retention times on normal-phase HPLC; monomethyl ethers derived from BA-cis-5,6-dihydrodiol had different retention times. The CD spectrum of monomethyl ether 1 was identical with that of the more strongly retained enantiomer shown in the middle chromatogram of Figure 3A. The CD spectrum of monomethyl ether 2 was identical with that of the less strongly retained enantiomer shown in the lower chromatogram of Figure 3A. Bis(methy1ether) of BA-truns-5(R),6(R)-dihydrodiolwas also isolated and had a CD spectrum similar to that of BAtruns-5(R),6(R)-dihydrodioldiacetate (23). On the basis of the data described above, the less strongly retained enantiomer of both BA monomethyl ethers 1and 2 on R-DNBPG-C are deduced to have 5S,6S and 5R76Rab-

2885

solute configurations, respectively. An enantiomeric BA 5,6-epoxide less strongly retained (enantiomer 1) on R-DNBPG-I was reacted with NaOCH, in methanol. The resulting monomethyl ethers were separated by normal-phase HPLC (upper chromatogram, Figure 3A). Monomethyl ethers 1 and 2 have CD spectra identical with those of 5(S)-methoxy-G(S)-hydroxy-5,6-dihydro-BA and 5(R)-hydroxy-5(R)-methoxy-5,6-dihydro-BA, respectively. The foregoing results thus established that the BA 5,6-epoxide enantiomer less strongly retained by R-DNBPG-I has the 5R,6S absolute stereochemistry. Absolute Configurations of Enantiomeric BP 4,5-Epoxides. A racemic BP 4,5-epoxide was methoxylated to form a pair of monomethyl ethers in a 53:47 ratio (Figure 4A, upper chromatogram). Both monomethyl ethers had UV-vis absorption spectra similar to that of BP-trans-4,5-dihydrodio17 indicating that both monomethyl ethers are saturated at the 4,5 positions. BP monomethyl ether 1 (M+ at m / z 300 (base peak) with characteristic fragment ions at m/z 283 (loss of OH), 282 (loss of H,O), 269 (loss of CH30), and 268 (loss of CH30H), 255, 252,239, and 226) was dehydrated with HCl in acetone to both 4-methoxy-BP (major product; M+ at rnlz 282) and 4hydroxy-BP (minor product; M+ at m/z 268), as revealed by reversed-phase HPLC analysis; 5-hydroxy-BP was not detectable. The dehydration products were identical with the authentic compounds with respect to their retention times on reversed-phase HPLC, UV-vis absorption spectra in methanol and in alkaline methanol, and mass spectra. BP monomethyl ether 2 (M+ a t m / z 300 (base peak) with characteristic fragment ions at m/z 283 (loss of OH), 282 (loss of H20) 269 (loss of CH30), 268 (loss of CH30H), 255, 252, 239, and 226) was dehydrated with HCl in acetone to both 4-hydroxy-BP (major product) and 5-methoxy-BP (minor product) which were identical with the authentic compounds with reaped to their retention times on reversed-phase HPLC, UV-vis absorption spectra in both methanol and alkaline methanol, and mass spectra. Taken together, the results indicated that BP monomethyl ethers 1and 2 are trans-4-methoxy-5-hydroxy-4,5-dihydro-BP and trans-4-hydroxy-5-methoxy-4,5-dihydro-BP, respectively. Enantiomeric pairs of BP monomethyl ethers 1and 2 were resolved by CSP HPLC (Figure 4A and Table I). CD spectra of the less strongly retained enantiomers on R-DNBPG-C are shown in Figure 4B. The characteristics of CD Cotton effects of both enantiomeric monomethyl ether 1and 2 are identical with those of enantiomeric 6-brorno-BP-truns-4,5-dihydrodiol (25) and different from those of BP-I(R),5(R)-dihydrodiol(26,

27). An enantiomerically pure BP-truns-4(R),5(R)-dihydrodiol was methylated with CH31in NaH-treated T H F and the resulting two monomethyl ethers were separated by normalphase HPLC (Figure 4A, upper chromatogram). Monomethyl ether 1had identical CD spectrum and retention time to that of the more strongly retained enantiomer shown in the middle chromatogram of Figure 4A. Monomethyl ether 2 had identical CD spectrum and retention time to that of the less strongly retained enantiomer shown in the lower chromatogram of Figure 4A. On the basis of the results described above, the less strongly retained enantiomers of BP monomethyl ethers 1 and 2 on R-DNBPG-I are deduced to have 4S,5S and 4R,5R absolute configurations, respectively. An enantiomeric B P 4,5-epoxide less strongly retained (enantiomer 1) on R-DNBPG-I (8) was obtained and was reacted with NaOCH3in methanol. The resulting monomethyl ethers were separated by normal-phase HPLC (upper chromatogram, Figure 4A). Monomethyl ethers 1 and 2 have CD

2686

ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987

A

B 10 53%

"QCH, OH

4s,5s

(*:)a2

t

---- @@

-

OCH,

n

47%

NP HPLC

F

20

l2

28

v

50

38

I

I

I

I

I

I

I

I

20

10

I

26

RETENTION TIME ( min ) Flgure 4. Normal-phase HPLC separation of monomethyl ethers derived from methoxylation of racemic BP 4,5-epoxide (upper chromatogram, A), CSP HPLC separation of trans -4-methoxy-5-hydroxy-4,5dihydro-BP enantiomers on an R-DNBPGI column and 2.5 % solvent A (ethanol: acetonlrile, 2: 1, v/v) in hexane (middle chromatogram,A), CSP HPLC separation of trans -4hydroxy-5-methoxy-4,5dihydro-BP enantiomers on R-DNBPGI column and 5 % solvent A in hexane (lower chromatogram, A), and CD spectra of BP 4(R),5(R)-dihydrodioI( - e -, A; enantiomeric excess 88%, @.270/A274 = -6.3 mdeg; ref 7), 4(S)-methoxy-5(S)-hydroxy-4,5dihydroBP (- - -, B; the less strongly retained enantiomer in the B; the middle chromatogram in A; enantiomeric excess -92%, @,,/A 272 = +7.6 mdeg), BP 4(R)-hydroxy-5(R)-methoxy-4,5dhydro-BP (-, less strongly retained enantiomer in the lower chromatogram in A; enantiomeric excess -95%, @,,,/A 272 = -9.3 mdeg), and 6-bromo-BP-4(R),5(R)-dihydrodiol .,enantiomeric excess >98%; @ 2 3 7 / A 273 = -8.5 mdeg; ref 25). (.e

spectra identical with those of 4(S)-methoxy-5(S)-hydroxy4,5-dihydro-BP and 4(R)-hydroxy-5(R)-methoxy-4,5-dihydro-BP, respectively. The foregoing results thus established that BP 4,5-epoxide enantiomer less strongly retained by R-DNBPG-I has a 4R,5S absolute stereochemistry. The enantiomers of K-region epoxides studied to date are either not resolved at all or poorly resolved by the S-DNBL (Table I and ref 8, 9, and 11);however, they are more efficiently resolved on the ionically bonded R-DNBPG (Table I and ref 8, 9, and 11).

DISCUSSION Absolute configurations of non-K-region PAH epoxide enantiomers can be determined by the position of epoxide hydrolase catalyzed water attack and the absolute configuration of the resulting trans dihydrodiol(5,6,10). In this approach, an label is incorporated into the non-K-region trans dihydrodiol which is obtained either by incubation of the parent hydrocarbon under 1 8 0 2 with rat liver microsomes or by incubation of the epoxide in H2180with microsomal epoxide hydrolase (5,6,10). The **O-containingtrans dihydrodiol is then converted by acid-catalyzed dehydration to two isomeric phenolic products which usually can be separated by reversed-phase HPLC (5,6,10,28).The l9-containing phenols can then be determined by mass spectral analyses. However, this approach cannot be applied to determine the absolute configurations of K-region epoxide enantiomers. The reasons are mainly 2-fold: (i) the 180-label is associated with both isomeric phenols resulting from acid-catalyzed dehydration

of an l80-containing K-region trans dihydrodiol enantiomer, although only one of the two hydroxyl groups in the trans dihydrodiol contains the l80,and (ii) the isomeric K-region phenols are difficult, if not impossible, to separate by reversed-phase HPLC (28). These difficulties have previously been addressed in the determination of the absolute configurations of K-region trans dihydrodiol and epoxide enantiomers of BA and BP (29-31). The conclusions reached in this study on the absolute configurations of K-region epoxide enantiomers of BA and BP are in agreement with those reported by other investigators (29-31), who used considerably more complex procedures. The results of this study show that analysis of methoxylation products derived from a K-region epoxide successfully leads to the determination of the absolute stereochemistry of epoxide enantiomers. In this and previous studies (20-22), the technique was applied to synthetic racemic epoxides. However, the method has been applied to the determination of the absolute configurations of K-region epoxides formed in the metabolism of benzo[c]phenanthrene (9) and 12-methyl-BA (11). Thus racemic as well as limited quantities of metabolically formed epoxides, which are often enriched in one of two enantiomers, can be utilized in stereochemical elucidation, especially where synthesis of enantiomeric epoxides may be difficult to achieve. The enantiomeric pairs of MC-trams-ll,12-dihydrodiolcan be efficiently resolved by at least one of four CSP columns utilized (Table I). The enantiomers of BP-trans-4,B-dihydrodiol were partially resolved. However, the enantiomers were not resolved by anyone of of BA-trans-5,6-dihydrodiol

ANALYTICAL CHEMISTRY, VOL. 59, NO. 22, NOVEMBER 15, 1987

4S,5R

5S,6R

5S,6R

2687

0;

"7 0 1 0

phenanthrene Flgure 6. Structure and numbering of phenanthrene.

nine PAHs (Figure 5) have been established (8,9,11,18,20-22, 23, 31, and this paper). Positions of PAH molecules are numbered according to the IUPAC rules (33). Structures of PAH K-region epoxide enantiomers are drawn so that the phenanthrene portion of the molecules is similarly situated (Figure 5). In Figure 5, chiral centers are designated clockwise. The S,R enantiomers of K-region epoxides of planar PAHs (BA, BP, MC, 7-MBA, and chrysene) are more strongly retained by the ionically bonded R-DNBPG (Figure 5; ref 8 and 16). Hence dibenz[a,h]anthracene 5(S),G(R)-epoxideenan6R,5S (llR,12S) (12R,llS) (5R,6S) tiomer is predicted to be more strongly retained by the ionically bonded R-DNBPG (8). The R,S enantiomers of nonFlgwe 5. Structures of epoxide enantiomers that are (or are predicted planar PAHs (1-methyl-BA,12-methyl-BA,7,12-dimethyl-BA, to be) more strongly retained by an ionically bonded R-DNBPG. Epoxides in upper half are derived from planar molecules: epoxides in and benzo[c]phenanthrene (34-36)) are more strongly retained lower half are derived from nonpianar molecules. The predicted abby the ionically bonded R-DNBPG (Figure 5; ref 8,9,11,22). solute configurationsof more strongly retained enantiomers are shown Based on these results, it appears that there exist some comin parentheses. mon rules in the relationship between elution order of enanthe four CSP columns tested. It is of interest to note that tiomers resolved by the ionically bonded R-DNBPG and abthe enantiomers of monomethyl ether derivatives of BAsolute configurations. can be truns-5,6-dihydrodiol and BP-trans-4,5-dihydrodiol The smallest PAH with a K-region is phenanthrene (Figure efficiently resolved (resolution value 1 2 ) by either ionically 6). The C4 and C5 positions of phenanthrene are in the bay region. Because of its molecular symmetry, phenanthrene or covalently bonded R-DNBPG columns (Table I). The 9,lO-epoxide is achrial. A substituent (methyl, halogen, benzo enantiomers of monomethyl ether derivatives of MC-transring, etc.) at C1, Cz, Cs, and/or C4 position or the 1,2- or 3,411,12-dihydrodiol are also more efficiently resolved than the enantiomers of nonmethylated dihydrodiols (Table I). Thus double bond of phenanthrene results in an asymmetric molecule; the K-region epoxide of each substituted phenanK-region trans dihydrodiols, either not resolved at all or poorly resolved by CSP HPLC, can be resolved by converting them threne molecule has a pair of enantiomers. Substitution at to monomethyl ether derivatives. We also found that nonnonbay region positions does not significantly perturb the K-region trans dihydrodiol enantiomers that are poorly replanarity of phenanthrene. As long as the PAH molecule solved or not resolved a t all can be efficiently resolved as retains its planarity (e.g., BA and 7-methyl-BA), the Sa enantiomer of K-region epoxide is more strongly retained by the monomethyl ether derivatives (32). The S,S enantiomers of monomethyl ether 2 derived from ionically bonded R-DNBPG (if the epoxide enantiomers are K-region trans dihydrodiols of MC, BA, and BP are more actually resolved). Substitution at one or both of the bay strongly retained by both ionically and covalently bonded region positions (C4and C5) results in a nonplanar molecule R-DNBPG and S-DNBL columns (Table I). The R,R enandue to steric crowding in the bay region; the R,S enantiomer tiomers of monomethyl ether 1derived from BA and BP, when is more strongly retained by the ionically bonded R-DNBPG resolved, are more strongly retained by R-DNBPG and are (e.g., benzo[c]phenanthrene, 1-methyl-BA,and 12-methyl-BA). It should be noted that, except BP-I(S),5(R)-epoxide,the less strongly retained by S-DNBL. The llR,12R enantiomer of MC monomethyl ether 1is more strongly retained by both (S,R)-epoxide enantiomers derived from planar PAH moleionically and covalently bonded S-DNBL. However, this cules do not have a substituent at the bay-region position (the llR,12R enantiomer is more strongly retained on the ionically position equivalent to the C4 position of phenanthrene) on the benzo ring. In comparison, (R,S)-epoxide enantiomers of bonded R-DNBPG but is less strongly retained on the covanonplanar PAHs all have a substituent at the bay-region lently bonded R-DNBPG. This is similar to that observed for the enantiomers of 7,12-dimethyl-BA-truns-5,6-dihydrodiol; position (the position equivalent to the C5 position of phenthe 5R,6R enantiomer is more strongly retained on the ionanthrene) of the benzo ring. At the present time, K-region ically bonded R-DNBPG but is less strongly retained on the epoxide enantiomers of only tetracyclic or larger PAHs are covalently bonded R-DNBPG (18). The underlying reason(s) known to be resolvable by CSP such as R-DNBPG. Based for the observed elution orders of enantiomeric dihydrodiols on the analysis presented above, it is possible to predict that and their monomethyl ethers is not clearly understood in dibenz[a,h]anthracene 5(S),G(R)-epoxide, 4-methyl-BA 5terms of CSP-solute recognition mechanisms. (S),G(R)-epoxide, 3,6-dimethylcholanthrene(or 6-methylcholanthrene) 11(R),12(S)-epoxide, 5-methylchrysene 12Enantiomeric pairs of ten PAH K-region epoxides can be (R),ll(S)-epoxide,and 14-methyldibenz[a,h]anthracene5directly resolved by ionically bonded R-DNBPG. These are (R),G(S)-epoxideare each more strongly retained by the ionK-region epoxides of MC, BA, BP, 1-methyl-BA,7-methyl-BA, ically bonded R-DNBPG (Figure 5). The DB[a,h]A 5,6-ep12-methyl-BA, chrysene, benzo[c]phenanthrene, 7,12-dioxide enantiomer which is more strongly retained by Rmethyl-BA, and dibenz[a,h]anthracene (8, 9). Except the enantiomers of dibenz[a,h]anthracene 5,g-epoxide (8), the DNBPG-I has been definitively established to have the 5S,6R absolute configurations of enantiomeric K-region epoxides of absolute stereochemistry by a method similar to that described

2688

Anal. Chem. 1987, 59, 2688-2691

in this report (37). Work is in progress to ascertain if a more definitive between the Order Of enantiomers and absolute configurations can be found.

LITERATURE CITED Sims, P.; Grover, P. L. Adv. CancerRes. 1974.2 0 , 165-274. Jerina, D. M.; Daly, J. W. Science 1974, 185, 573-581. Gelboin, H. V. Physiol. Rev. 1980, 6 0 , 1107-1166. Conney, A. H. Cancer Res. 1982, 42, 4875-4917. Yang. S . K.; McCourt, D. W.; Leutz, J. C.; Gelboin, H. V. Science 1977, 196. 1199-1201. Yang, S . K.; Roller, P. P.; Gelboin, H. V. Biochemistry 1977, 16, 3680-3686. Yang. S. K.; Chiu, P.-L. Arch. Biochem. Siophys. 1985, 240, 546-552. Weems, H. B.; Mushtaq, M.; Yang, S. K. Anal. Biochem. 1985, 148, 328-338. Yang, S. K.; Mushtaq, M.; Weems. H. B. Arch. Biochem. Biophys. 1987, 255, 48-63. Mushtaq, M.; Weems, H. B . ; Yang, S . K. Arch. Biochem, Biophys 1988, 246, 478-487. Yang, S. K.; Mushtaq, M.; Weems, H. B.; Miller, D. W.; Fu, P. P. Biochem. J . 1987,245, 191-204. Weems, H. B.; Yang, S. K. Anal. Biochem. 1982. 125, 156-161. Yang, S. K.; Weems, H. 6.;Mushtaq. M.; Fu. P. P. J. Chromatogr. 1984,316, 569-584. Yang, S . K.; Mushtaq, M.; Weems, H. B.; Fu, P. P. J. Liq. Chromatogr. 1988, 9 , 473-492. Yang, S . K.; Mushtaq, M.; Fu, P. P. J. Chromatogr. 1986, 371, 195-209. Weems, H. B.; Fu. P. P.; Yang, S. K. Carcinogenesis, 1986. 7, 1221-1230. Harada, N.; Nakanishi, K. Acc. Chem. Res. 1972.5 , 257-263. Yang, S . K.; Weems. H. B. Anal. Chem. 1984,5 6 , 2658-2662. Wong, L. K.; Kim, W. H.; Witiak. D. T. Anal. Biochem. 1980, 101,

-RA-RR . --.

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(25) Fu, P. P.; Yang. S. K. Biochem. Biophys. Res. Commun. 1982, 109, 927-934. (26) Kedzierski, B,; Thakker, D, R,; Armstrong, R , N,; Jerina, D, M, Tetrahedron Lett. 1981. -~.2 2 . 405-408. (27) Yang, S.K.; Mushtaq, M.;-Chiul P.-L. I n Po/ycyclic Hydrocarbons and Cancer; Harvey. R. G., Ed.; ACS Symposium Series 283; American Chemical Society: Washlngton, DC, 1985; pp 19-34. (28) Mushtaq. M.; Bao. Z.;Yang, S. K. J. Chromatogr. 1987, 385, 293-298. (29) Armstrong, R. N.; Kedzierski, B.; Levin, W.; Jerina, D. M. J. Biol. Chem. 1981, 256, 4726-4733. (30) Thakker, D. R.; Yagi, H.; Levin. W.; Lu, A. Y. H.; Conney, A. H.; Jerina, D. M. J. Bioi. Chem. 1977,252, 6328-6334. (31) Armstrong, R . N.; Levin, W.; Ryan, D.; Thomas, P. E.; Mah, H. D.; Jerina, D. M. Biochem. Siophys. Res. Commun. 1981, 100, 1077- 1084. (32) Bao, 2.; Yang. S. K. Pharmacologist 1986, 28, 240 (abstract no. 793). (33) Dipple, A.; Moschel, R. C.; Bigger, C. A. H. Chemical Carclnogens, Second Edition, Revisedand Expanded; Searle, C. E., Ed.; ACS Symposium Series 182, American Chemical Society, Washington, DC, 1984; Vol. 2, pp 19-34. (34) Herbstein, F. H.; Schmidt, G. M. J. J . Chem. SOC.1954,3302-3313. (35) Briant, C. E.; Jones, D. W.; Shaw, J. D. J. Mol. Struct. 1985, 130, 167-176. (36) Kashino, S.;Zacharias, D. E.; Prout, C. K.; Carrell, H. L.; Glusker, J. P.; Hecht, S. S. Acta Crystallogr. Sect. C : Cryst. Strucf. Commun. I984 C40, 536-540. (37) Unpublished work. 1987. ~

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RECEIVED for review May 18,1987. Accepted August 4,1987. This work was supported by Uniformed Services University of the Health Sciences Protocol R07502 and U. S. Public Health Service Grant CA29133. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences. The experiments reported herein were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Animal Resources, National Research Council, DHEW Publication No. (NIH) 78-23.

Reversibly Immobilized Glucose Oxidase in the Amperometric Flow-Injection Determination of Glucose W. Uditha de Alwis, Brian S. Hill, Bruce I. Meiklejohn, and George S. Wilson* Department of chemistry, University of Arizona, Tucson, Arizona 85721 Glucose oxidase (EC 1.1.3.4) is immobilized in a reactor coupled to a flow-lnjectlon analysls system by using an hmunological reactlon. This reactor can then be used to determlne efucose in serum and other samples by electrochemlcal monltorlng of H,02 produced as a result of the reaction of the glucose oxidase catalyzed reactlon of glucose wlth oxygen. The reactor Is packed with a support on whlch an antibody Is immobilzed. An enzyme-labeled antibody conjugate or an Immune complex of antiglucose oxldase-glucose oxidase Is passed over the reactor packing, whlch Immoblilzes the enzyme as a result of the lmmunoioglcal reaction. The reactor can be eluted and reloaded with the conjugate to within f5% of the original actlvlty. The llnear dynamic ranges for the reactors are 1.1 X 10-lo-l.l X IO-' and 1.1 X lO-'-l.l X IO-' mol for the immune complex and conJugate loaded reactors, respectively, for a 20-pL sample. On the basis of these llnear detectlon ranges a triplicate analysis on a 1-FL serum sample can be carrled out.

*To whom correspondence should be addressed. Present address: Department of Chemistry, U n i v e r s i t y of Kansas, Lawrence, K S 66045. 0003-2700/87/0359-2688$01.50/0

The use of immobilized enzymes in the determination of substrates is well-documented (1-3). This includes the use of enzyme electrodes ( 4 , 5 )and immobilized enzyme reactors of both open-tube and packed-bed types (6, 7). These latter methods are extremely desirable because they are highly sensitive and low in reagent consumption and the reactors can handle low-volume (microliter) samples. In most reactors where electrochemical sensors are employed, sample pretreatment is necessary to remove proteins, which would otherwise foul the electrodes. This is achieved by the interposition of dialysis membranes or by use of precolumns. The use of dialysis membranes (8)brings about an attenuation of the signal, which can seriously decrease the detection limit, and precolumns (9) can fail due to overloading. In highperformance applications where high specific activity of the immobilized enzyme must be maintained, regeneration or replacement of the reactor may be frequently required. The new reactor may have very different characteristics, hence requiring time-consuming calibrations and equilibration intervals, thus increasing downtime. In addition, separate enzyme reactor columns are required for each assay. In this publication we wish to discuss the indirect immobilization of the enzyme such that regeneration of the system in the case G 1987 American Chemical Society