CHIRALITY 258-64 (1990)
Absolute Configuration of &-5,6=Dihydrodiol Enantiomers Derived From Helical Conformers of 1,122-Dimethylbenz[a]anthracene SHEN K. YANG, MOHAMMAD MUSHTAQ, AND PETER P. FU Department of Pharmacology, F. Edward Hkbert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 208144799 ( S K Y . ,M.M.) and National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas (PPP.)
ABSTRACT
1,12-Dimethylbenz[alanthracene(1,12-DMBA) cis-5,6-dihydrodiol was synthesized by oxidation of 1,12-DMBAwith osmium tetroxide in pyridine in low yield ( ~ 3 %and ) was purified by sequential use of reversed-phase and normal-phase HPLC. Two pairs of 1,12-DMBA cis-5,6-dihydrodiol enantiomers, derived from P (right-handed helix) and M (left-handed helix) conformers, were eluted as a single chromatographic peak on both reversed-phase and normal-phase HPLC. However, these four enantiomers were resolved by sequential use of two chiral stationary phase (CSP) HPLC columns. CSP (Pirkle type I) columns were packed with either (R)-N-(3,5-dinitrobenzoyl)phenylglycineor (S)-N-(3,5dinitrobenzoyl)leucine, which is ionically bonded to y-aminopropylsilanized silica. Absolute configurations of enantiomers were determined by comparing their circular dichroism spectra with those of conformationally similar cis-5,6dihydrodiol enantiomers of 4-methylbenz[alanthraceneand 7,12-dimethylbenz[alanthracene with known absolute stereochemistry.
KEY WORDS: 1,12-dimethylbenz[alanthracene,helical conformers, cis-5,6-dihydrodiol, high-performance liquid chromatography, chiral stationary phase, optical isomers, resolution of enantiomers, absolute configuration, circular dichroism spectra INTRODUCTION
Due to steric crowding brought about by the two bay region methyl groups, 1,12-dimethylbenz[alanthracene (1,12-DMBA)should exist in two helical conformers: P and M conformers (Fig. 1).X-Ray crystallographic study of 1,lZDMBA indicated that the mutual inclination between A and C ring planes in 1,12-DMBA is 27.9, with BCD anthracene fragment remaining closely planar (for detailed X-ray crystallographic data, see ref. 1).In comparison, mutual inclinations between A and C ring planes in benz[alanthracene (BA) and other methyl-substituted BA derivatives are
Abbreviations: 1,12-DMBA, 1,12-dimethylbenz[a]anthracene;BA, benzblanthracene; 4-MBA, 4-methylbenz[a]anthracene;7-Br-127,12-DMBA, 7,12-diMBA, 7-bromo-12-methylbenz[alanthracene; methylbenz[a]anthracene; 7-Br-l-MBA, 'I-bromo-l-methylbenz[alanthracene;ee, enantiomeric excess; P conformer, the A ring and the BCD ring (anthracene moiety) in 1,lP-DMBA forms a righthanded helix, M conformer, the A ring and BCD ring in 1,lZ-DMBA forms a left-handed helix; e, quasiequatorial; a, quasiaxial; P5e6a indicates that the cis-5,6-dihydrodiolis derived from the P conformer of 1,lP-DMBA and the 5- and 6-hydroxyl groups adopt a quasiequatorial and quasiaxial conformation, respectively; M5a6e indicates that the cis-5,6-dihydrodiolis derived from the M conformer of 1,12DMBA and the 5- and 6-hydmxyl groups adopt a quasiaxial and quasiequatorial conformation, respectively; P5a6e and M5e6a are similarly defined. 0 1990 Wiley-Liss, Inc.
2.9" (BA), 18.7" (1-MBA), 19.2" (12-MBA), and 21.2" (7,12-DMBA),respectively (1 and references therein). Compared to that of 7,12-DMBA, the severe distortion of the A ring in 1,12-DMBA is also reflected in its UV absorption spectrum; it has less defined absorption peaks in the wavelength region of 270-310 nm and blue shift in the region of 310-400 nm (Fig. 2). In solution at ambient temperature, P and M conformers of 1,12-DMBA are expected to be in an equilibrium state and interconvertible. We thought that it should be possible to demonstrate the existence of two helical conformers by converting 1,12-DMBA to 1,12DMBA cisd,6-dihydrodiol. Two cis-5,6-dihydrodiols should be formed: one derived from the P conformer and the other from the M conformer. In this paper, we describe the results on the resolution of two enantiomeric pairs of cis-5,6-dihydrodiolderived from P and M conformers of 1,12-DMBA by chiral stationary-phase HPLC and elucidation of their absolute configurations by CD spectral analyses.
Received for publication October 30, 1989; accepted November 20, 1989. Mohammad Mushtaq's present address:Merck, Sharp and Dohme Research Laboratories, Three Bridges, NJ 08887. Address reprint requests to S.K.Yang at the address given above.
59
c~-~,~-DIHYDRODIOL ENANTIOMERS
MOD, Chadds Ford, PA), eluted with 5% (vol ratio) of ethanouacetonitrile (2:1, v/v) in hexane at 2 ml/min. The structure of purified 1,12-DMBA cis-5,6-dihydrodiol was confirmed by mass (molecular ions a t m/z 290) and UV absorption (Fig. 2) spectral analyses. Enantiomeric pairs of 1,12-DMBA cis-5,6-dihydrodiol were resolved on CSP columns (4.6 mm i.d. x P conformer M conformer 250 mm; Regis Chemical Co., Morton Grove, IL) packed (right-handed helix) (left-handed helix) with spherical particles of 5 p,m diameter of Fig. 1. Structures, numbering system, and abbreviations of P and M conformers of 1,12-DMBA. K-region is the 5,g-double bond, the y-aminoprophysilanized silica to which either (S)most electron-rich region of the molecule. Solid and hashed triangle N-(3,5-dinitrobenzoyl)leucine [(S)-DNBLI or (R)bonds at the 1 and 12 positions indicate that the methyl group with N-(3,5-dinitrobenzoyl)phenylglycine[(R)-DNBPGIwas the associated A ring (or BCD ring) is puckered upward (toward the either ionically (I)or covalently (C) bonded. The mobile viewer) or downward (away from the viewer), respectively. phase [5 or 10% of ethanol/acetonitrile (2:1, v/v) in hexanel was eluted at a flow rate of 2 mumin. Elution MATERIALS AND METHODS order of an enantiomeric pair on different CSPs was Materials established by areas under the chromatographic peaks 1,12-DMBAwas purchased from Alfred Bader Chem- with a sample containing a mixture of two resolved icals Division, Aldrich Chemical Co. (Milwaukee, WI). enantiomers a t a ratio of approximately 2:l. 1,12-DMBA cis-5,6-dihydrodiol (UV absorption specSpectral Analysis trum in Fig. 2) was prepared by reaction of 1,12-DMBA with OsO, (Aldrich Chemical Co., Milwaukee, WI) in Mass spectral analysis was performed on a Finnigan pyridine for 1 to 3 weeks. The yield of cis-5,6-dihy- Model 4000 gas chromatograph-mass spectrometer drodiol was highly variable; the highest yield was found, by normal-phase HPLC analysis, to be -3% among several experiments. This low yield was due to the instability of 1,12-DMBA cis-5,6-dihydrodiol, which was gradually converted to 1,12-DMBA upon storage in organic solvents (methanol or elution solvents used in CSP HPLC) for up to several weeks at 4°C. Enantiomers of 1,12-DMBA cis-5,6-dihydrodiol were stable (with negligible decomposition) on the day of CD spectral and chromatographic analyses. High-Performance Liquid Chromatography
HPLC was performed using a Waters Associates 1 (Milford, MA) liquid chromatograph consisting of a w Model 6000A solvent delivery system, a Model M45 0 solvent delivery system, a Model 660 solvent programz a mer, and a Kratos Analytical Instruments (Ramsey, m NJ) Model Spectraflow 757 UV-VIS variable waveK 0 length detector. Samples were injected via a Valco v) Model N60 loop injector (Valco Instruments, Houston, m a TX). Retention times and area under chromatographic peaks were determined with a Hewlett-Packard Model 3390A integrator. Eluent was monitored a t 254 nm. 1,12-DMBA cis-5,6-dihydrodiol formed by oxidation of 1,12-DMBA with OsO, was purified from the unreacted 1,12-DMBA and other reaction by-products by sequential use of reversed-phase HPLC and normalphase HPLC. The reaction product mixture was first applied to a Zorbax ODS column (6.2 mm i.d. x 80 mm, 3 p,m particle; MAC-MOD, Chadds Ford, PA), equilibrated and eluted with methanouwater (3:1, v/v) a t 1.5 0 ml/min. Once 1,12-DMBA cis-5,6-dihydrodiol (retention time 3.9 min) was collected, 1,12-DMBAand other reaction by-products were washed off the column with WAVELENGTH ( nm ) methanol. 1,12-DMBA czs-5,6-dihydrodiol (retention Fig. 2. W absorption spectra of 1,12-DMBA (---), 1,12-DMBA time 6.0 min) was further purified on a Zorbax Golden cis-5,6-dihydrodiol (-), and 7,12-DMBA (. dissolved in methSIL column (6.2 mm i.d. x 80 mm, 3 p,m particle; MAC- anol. 0)
60
YANG ET AL.
data system by electron impact with a solid probe a t 70 eV and 250°C ionizer temperature. Ultraviolet-visible absorption spectra of samples in methanol were determined a t ambient temperature 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 1cm path length at room temperature (23°C) were measured using a Jasco Model 500A spectropolarimeter equipped with a Model DP500 data processor. The concentration of the sample is indicated byAA21ml(absorbance units at wavelength X2 per ml of solvent). CD spectra are expressed by ellipticity (QAllAh2,in millidegrees) for solutions that have an absorbance of Ah2unit per ml of solvent at wavelength A2 (usually the wavelength of maximal absorption). Under conditions of measurements indicated above, the molar ellipticity ([@IA1, in deg cm2 dmol-l) and ellipticity (QA1/AA2, in millidegrees) are related to the molar extinction coefficient (€A29 in cm-I M-l) as follows: [el,,
= 0.1 eA2 (QAiIAAA
It is apparent from the equation above that molar ellipticity [03 of an enantiomer must be reported along with its molar extinction coefficient. Unfortunately, molar ellipticities of enantiomers are often reported in the literature in the absence of molar extinction coefficients. Molar extinction coefficients in a specified solvent of most chemicals are either not available or difficult to determine accurately. For the purpose of comparing data obtained in different laboratories, we strongly urge investigators to express CD spectral data by ellipticity for a solution of 1.0 absorbance unit at a specified wavelength per ml of solvent, instead of molar
A
(S)-DNBL-I
ellipticity [el. In this study, it was not possible to obtain sufficient quantity of the unstable 1,12-DMBA cis5,6-dihydrodiolto determine its molar extinction coefficient. RESULTS AND DISCUSSION CSP HPLC Separation and CD Spectra of 1,12-DMBA cis-5,6-DihydmdiolEnuntiomers
The enantiomers of 1,12-DMBA cisd,6-dihydrodiol, which are eluted as a single chromatographic peak on both normal-phase and reversed-phase HPLC, are separated into three peaks on an (S)-DNBL-I column (peaks C1, C2, and C3 in Fig. 3A). Peaks C1 and C3 are a pair of enantiomers and have CD spectra that are mirror images of each other (Fig. 4A). Peak C2 was collected and was subsequently separated into a pair of enantiomers (see CD spectra in Fig. 4B) on an (R)DNBPG-I column (peaks C2a and C2b in Fig. 3B). Thus, the chromatograms in Figure 3 revealed that there are four stereoisomers contained in 1,12-DMBA cis-5,6-dihydrodiol. All four enantiomers were confirmed by mass spectral analyses (molecular ions a t mlz 290 and characteristic fragment ions a t mlz 272 due to loss of water) and by CD spectral analyses (Fig. 4). Elution order of enantiomers for each enantiomeric pair was determined on four different CSP columns (Table 1). Results in Table 1 revealed that the covalently bonded CSP columns were less useful in the separation of the four enantiomers of 1,12-DMBA czs5,6-dihydrodiol. The sum of areas under the chromatographic peaks C1 and C3 to that of peak C2 is 1.27 (Fig. 3A). Thus two pairs of enantiomers constitute 56 and 44%, respec-
c2
I
c1
B
(R)-DNBPG-I C2a C2b
c3 OH
5R,6S
.
(P5e6a)
"OH
5R,6S (M5a6e)
5S,6R (PSa6e)
I
I
I
6
I
I
10
-
I
I
I
i I
l
l
30 RETENTION TIME (min)
14
l
I
l
l
l
I
40
Fig. 3. CSP HPLC resolution of two enantiomeric pairs of cisd,b-dihydrodiolsderived from P and M conformers of 1,12-DMBA on (S)-DNBL-I(A) and (R)-DNBPG-I(B) columns. Mobile phase was 10%(A) and 5%(B) of ethano/acetonitrile (21, v/v) in hexane, respectively, at 2 mumin. In A, peaks C1 and C3 are enantiomericto each other. Peak C2 (A) is collectedand further resolved into peaks C2a and C2b (B).
&-5,6-DIHYDRODIOL ENANTIOMERS
t
61
5S,6R (M5e6a)
250 300 WAVELENGTH ( nm ) Fig. 4. CD spectra of two enantiomeric pairs of cis-5,6-dihydrodiolderived from 1,lP-DMBA.Peaks C1, C3, C2a, and C2b are separated as described in Figure 3. See text for the assignment of absolute configurations. Spectral data and optical purity of samples are C1, concentration 1.0 A253/ml;a2%= - 50.1 millidegrees, ee 100%;C3, concentration 1.0 panel AZ5.Jml; , ,@, = + 50.7 millidegrees, ee 100%; C2a, concentration 1.0 AZ5,/ml,+234 = +33.5 millidegrees, ee -98%; C2b, concentration 1.0 A 2 & d , Qza4 = - 26.7 millidegrees, ee -76%. 250
300
tively, of the chemically synthesized 1,12-DMBA cis5,6-dihydrodiol.
freedom about the axis of the single bond between phenyl and naphthyl rings. Owing to the steric hindrance brought about by the peri methyl substituent, 4-MBA Relationship Between Conformation, Absolute cis-5,6-dihydrodiol adopts only a 5a6e conformation, Configuration, and CD Cotton Effects whereas cis-5,6-dihydrodiols of 7-Br-l-MBA, 7,12A sufficient amount of an enantiomeric 1,12-DMBA DMBA, and 7-Br-12-MBA adopt only a 5e6a conformacis-5,6-dihydrodiolcould not be obtained (due to its in- tion. Although 4-MBA (5R,6S)-dihydrodio14 and 7,12stability and low yield in synthesis) for the determination of its absolute configuration by the exciton chirality DMBA (5R,6S)-dihydrodio16have identical absolute CD method.2The exciton chirality CD method has been configurations, their major CD Cotton effects centered successfully used in our laboratory to determine the around 240 nm have opposite signs (Fig. 5). It is apparabsolute configuration of trans and cis dihydrodiols of a ent that the difference in the signs of major CD Cotton large number of polycyclic aromatic hydro~arbons.~-~effects is due to their difference in conformation; 4Although two pairs of 1,12-DMBA cis-5,6-dihy- MBA (5R,GS)-dihydrodioladopts the 5a6e conformadrodiol enantiomers have characteristically different tion and 7,12-DMBA (5R,GS)-dihydrodiol adopts the CD , ~ Cotton effects of (5R,6S)CD Cotton effects (Fig. 41, absolute configurations of 5e6a c ~ n f o r m a t i o n . ~ these enantiomers cannot be readily established based dihydrodiol enantiomers of 7-Br-1-MBA4 and 7solely on their CD spectral data. Before the assignment Br-12-MBA3 are similar to those of 7,12-DMBA of absolute configurations is presented, it is necessary (5R,GS)-dihydrodiol shown in Figure 5. 4-MBA cisd,6-dihydrodiol is the only example of BA to review available information on the relationship of conformation, absolute configuration, and CD Cotton derivatives studied to date which adopts a 5a6e conforeffects of cisd,6-dihydrodiol enantiomers of 4-MBA mation. Unlike 1-MBA, 12-MBA, and 7,12-DMBA, and 7,12-DMBA,each of which has a methyl group peri there is no distortion of A ring relative to BCD ring in 4-MBA. Thus, the CD spectrum of 4-MBA (5R,6S)to the K-region. In the absence of steric and/or electronic factors, each dihydrodiol is not the ideal example for the purpose of hydroxyl group of a K-region cis-5,6-dihydrodiol of un- comparing its CD Cotton effects with those of 1,12substituted or alkylhalogen-substitutedBA adopts ei- DMBA cis-5,6-dihydrodiolenantiomers. The ideal refther a quasiequatorial or a quasiaxial conformation. erence compounds relevant to this study would have For example, the hydroxyl groups of cisd,6-dihydrodiol been (BR,GS)-dihydrodiols of 1,4-DMBA and/or 4,12of BA, 1-MBA, and 12-MBA adopt both 5a6e and 5e6a DMBA. Other important features pertinent to this discussion conformations. This is due to the limited rotational
62
YANG ET AL.
TABLE 1. CSP-HPLC resolution of enantiomeric cis-5,6dihydrodiols of 1,12-DMBA, 4-MBA, 7-Br-l-MBA, ?-Br-12-MBA, and 7,12-DMBA Retention time (min)" cis-5,6-Dihydrodiol 4-MBA (5a,6e)
1,12-DMBA (5a,6e) 1,12-DMBA (5e,6a)
7-Br-1-MBA (5e,6a)
csp"
%Ab
Enantiomer 1
Enantiomer 2
RVd
(R)-DNBPG-I (R)-DNBPG-C (S)-DNBL-I (S)-DNBL-C (R)-DNBPG-I (R)-DNBPG-C (S)-DNBL-I (S)-DNBL-C (R)-DNBPG-I
10 10 10 10 10 10 10 10 10
(R)-DNBPGC (S)-DNBL-I (S)-DNBL-C (R)-DNBPG-I (R)-DNBPG-C (S)-DNBL-I
10 10 10 10 10 10
(S)-DNBL-C
10
(R)-DNBPG-I (R)-DNBPG-C (S)-DNBL-I (S)-DNBL-C (R)-DNBPG-I (R)-DNBPG-C (S)-DNBL-I (S)-DNBL-C
10 10 10 10 10 10 10 10
17.2 (5S,6R) 12.6 (5S,6R) 14.7 (5R,6S) 9.4 (5R,6S) 10.5 (5S,6R)-F 8.7 (5S,6R)-P 10.3 (5R,6S)-M 7.5 (5R,6S)-M 12.1 (5R,6S)-P 32.5 (5R,6S)-P 9.1 11.2 7.5 14.9 (5R,6S) 10.5 (5R,6S) 11.8 (5R,6S) 24.4 (5R,6S) 8.5 17.3 15.2 (5R,6S) 10.9 (5R,6S) 16.2 (5R,6S) 8.8 (5R,6S) 20.9 13.2 (5R,6S) 10.3 (5R,6S) 15.6 (5R,6S)
19.1 (5R,6S) 13.6 (5R,6S) 15.3 (5S,6R) 10.0 (5S,6R) 12.2 (5R,6S)-Me 9.4 (5R,6S)-M 12.2 (5S,6R)-P 8.4 (5S,6R)-P 12.4 (5S,6R)-M 33.7 (5S,6R)-M 9.2 11.2 7.5 15.1 (5S,6R) 10.6 (5S,6R) 12.1 (5S,6R) 27.4 (5S,6R) 8.5 17.3 16.0 (5S,6R) 11.6 (5S,6R) 17.7 (5S,6R) 9.1 (5S,6R) 20.9 (5S,6R) 13.8 (5S,6R) 10.6 (5S,6R) 17.2 (5S,6R)
2.3 1.5 0.8 2.1 2.9 1.5 3.5 1.8 0.5 0.8
Absolute Configuration of &-5,6=Dihydrodiol Enantiomers Derived From Helical Conformers of 1,122-Dimethylbenz[a]anthracene SHEN K. YANG, MOHAMMAD MUSHTAQ, AND PETER P. FU Department of Pharmacology, F. Edward Hkbert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 208144799 ( S K Y . ,M.M.) and National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas (PPP.)
ABSTRACT
1,12-Dimethylbenz[alanthracene(1,12-DMBA) cis-5,6-dihydrodiol was synthesized by oxidation of 1,12-DMBAwith osmium tetroxide in pyridine in low yield ( ~ 3 %and ) was purified by sequential use of reversed-phase and normal-phase HPLC. Two pairs of 1,12-DMBA cis-5,6-dihydrodiol enantiomers, derived from P (right-handed helix) and M (left-handed helix) conformers, were eluted as a single chromatographic peak on both reversed-phase and normal-phase HPLC. However, these four enantiomers were resolved by sequential use of two chiral stationary phase (CSP) HPLC columns. CSP (Pirkle type I) columns were packed with either (R)-N-(3,5-dinitrobenzoyl)phenylglycineor (S)-N-(3,5dinitrobenzoyl)leucine, which is ionically bonded to y-aminopropylsilanized silica. Absolute configurations of enantiomers were determined by comparing their circular dichroism spectra with those of conformationally similar cis-5,6dihydrodiol enantiomers of 4-methylbenz[alanthraceneand 7,12-dimethylbenz[alanthracene with known absolute stereochemistry.
KEY WORDS: 1,12-dimethylbenz[alanthracene,helical conformers, cis-5,6-dihydrodiol, high-performance liquid chromatography, chiral stationary phase, optical isomers, resolution of enantiomers, absolute configuration, circular dichroism spectra INTRODUCTION
Due to steric crowding brought about by the two bay region methyl groups, 1,12-dimethylbenz[alanthracene (1,12-DMBA)should exist in two helical conformers: P and M conformers (Fig. 1).X-Ray crystallographic study of 1,lZDMBA indicated that the mutual inclination between A and C ring planes in 1,12-DMBA is 27.9, with BCD anthracene fragment remaining closely planar (for detailed X-ray crystallographic data, see ref. 1).In comparison, mutual inclinations between A and C ring planes in benz[alanthracene (BA) and other methyl-substituted BA derivatives are
Abbreviations: 1,12-DMBA, 1,12-dimethylbenz[a]anthracene;BA, benzblanthracene; 4-MBA, 4-methylbenz[a]anthracene;7-Br-127,12-DMBA, 7,12-diMBA, 7-bromo-12-methylbenz[alanthracene; methylbenz[a]anthracene; 7-Br-l-MBA, 'I-bromo-l-methylbenz[alanthracene;ee, enantiomeric excess; P conformer, the A ring and the BCD ring (anthracene moiety) in 1,lP-DMBA forms a righthanded helix, M conformer, the A ring and BCD ring in 1,lZ-DMBA forms a left-handed helix; e, quasiequatorial; a, quasiaxial; P5e6a indicates that the cis-5,6-dihydrodiolis derived from the P conformer of 1,lP-DMBA and the 5- and 6-hydroxyl groups adopt a quasiequatorial and quasiaxial conformation, respectively; M5a6e indicates that the cis-5,6-dihydrodiolis derived from the M conformer of 1,12DMBA and the 5- and 6-hydmxyl groups adopt a quasiaxial and quasiequatorial conformation, respectively; P5a6e and M5e6a are similarly defined. 0 1990 Wiley-Liss, Inc.
2.9" (BA), 18.7" (1-MBA), 19.2" (12-MBA), and 21.2" (7,12-DMBA),respectively (1 and references therein). Compared to that of 7,12-DMBA, the severe distortion of the A ring in 1,12-DMBA is also reflected in its UV absorption spectrum; it has less defined absorption peaks in the wavelength region of 270-310 nm and blue shift in the region of 310-400 nm (Fig. 2). In solution at ambient temperature, P and M conformers of 1,12-DMBA are expected to be in an equilibrium state and interconvertible. We thought that it should be possible to demonstrate the existence of two helical conformers by converting 1,12-DMBA to 1,12DMBA cisd,6-dihydrodiol. Two cis-5,6-dihydrodiols should be formed: one derived from the P conformer and the other from the M conformer. In this paper, we describe the results on the resolution of two enantiomeric pairs of cis-5,6-dihydrodiolderived from P and M conformers of 1,12-DMBA by chiral stationary-phase HPLC and elucidation of their absolute configurations by CD spectral analyses.
Received for publication October 30, 1989; accepted November 20, 1989. Mohammad Mushtaq's present address:Merck, Sharp and Dohme Research Laboratories, Three Bridges, NJ 08887. Address reprint requests to S.K.Yang at the address given above.
59
c~-~,~-DIHYDRODIOL ENANTIOMERS
MOD, Chadds Ford, PA), eluted with 5% (vol ratio) of ethanouacetonitrile (2:1, v/v) in hexane at 2 ml/min. The structure of purified 1,12-DMBA cis-5,6-dihydrodiol was confirmed by mass (molecular ions a t m/z 290) and UV absorption (Fig. 2) spectral analyses. Enantiomeric pairs of 1,12-DMBA cis-5,6-dihydrodiol were resolved on CSP columns (4.6 mm i.d. x P conformer M conformer 250 mm; Regis Chemical Co., Morton Grove, IL) packed (right-handed helix) (left-handed helix) with spherical particles of 5 p,m diameter of Fig. 1. Structures, numbering system, and abbreviations of P and M conformers of 1,12-DMBA. K-region is the 5,g-double bond, the y-aminoprophysilanized silica to which either (S)most electron-rich region of the molecule. Solid and hashed triangle N-(3,5-dinitrobenzoyl)leucine [(S)-DNBLI or (R)bonds at the 1 and 12 positions indicate that the methyl group with N-(3,5-dinitrobenzoyl)phenylglycine[(R)-DNBPGIwas the associated A ring (or BCD ring) is puckered upward (toward the either ionically (I)or covalently (C) bonded. The mobile viewer) or downward (away from the viewer), respectively. phase [5 or 10% of ethanol/acetonitrile (2:1, v/v) in hexanel was eluted at a flow rate of 2 mumin. Elution MATERIALS AND METHODS order of an enantiomeric pair on different CSPs was Materials established by areas under the chromatographic peaks 1,12-DMBAwas purchased from Alfred Bader Chem- with a sample containing a mixture of two resolved icals Division, Aldrich Chemical Co. (Milwaukee, WI). enantiomers a t a ratio of approximately 2:l. 1,12-DMBA cis-5,6-dihydrodiol (UV absorption specSpectral Analysis trum in Fig. 2) was prepared by reaction of 1,12-DMBA with OsO, (Aldrich Chemical Co., Milwaukee, WI) in Mass spectral analysis was performed on a Finnigan pyridine for 1 to 3 weeks. The yield of cis-5,6-dihy- Model 4000 gas chromatograph-mass spectrometer drodiol was highly variable; the highest yield was found, by normal-phase HPLC analysis, to be -3% among several experiments. This low yield was due to the instability of 1,12-DMBA cis-5,6-dihydrodiol, which was gradually converted to 1,12-DMBA upon storage in organic solvents (methanol or elution solvents used in CSP HPLC) for up to several weeks at 4°C. Enantiomers of 1,12-DMBA cis-5,6-dihydrodiol were stable (with negligible decomposition) on the day of CD spectral and chromatographic analyses. High-Performance Liquid Chromatography
HPLC was performed using a Waters Associates 1 (Milford, MA) liquid chromatograph consisting of a w Model 6000A solvent delivery system, a Model M45 0 solvent delivery system, a Model 660 solvent programz a mer, and a Kratos Analytical Instruments (Ramsey, m NJ) Model Spectraflow 757 UV-VIS variable waveK 0 length detector. Samples were injected via a Valco v) Model N60 loop injector (Valco Instruments, Houston, m a TX). Retention times and area under chromatographic peaks were determined with a Hewlett-Packard Model 3390A integrator. Eluent was monitored a t 254 nm. 1,12-DMBA cis-5,6-dihydrodiol formed by oxidation of 1,12-DMBA with OsO, was purified from the unreacted 1,12-DMBA and other reaction by-products by sequential use of reversed-phase HPLC and normalphase HPLC. The reaction product mixture was first applied to a Zorbax ODS column (6.2 mm i.d. x 80 mm, 3 p,m particle; MAC-MOD, Chadds Ford, PA), equilibrated and eluted with methanouwater (3:1, v/v) a t 1.5 0 ml/min. Once 1,12-DMBA cis-5,6-dihydrodiol (retention time 3.9 min) was collected, 1,12-DMBAand other reaction by-products were washed off the column with WAVELENGTH ( nm ) methanol. 1,12-DMBA czs-5,6-dihydrodiol (retention Fig. 2. W absorption spectra of 1,12-DMBA (---), 1,12-DMBA time 6.0 min) was further purified on a Zorbax Golden cis-5,6-dihydrodiol (-), and 7,12-DMBA (. dissolved in methSIL column (6.2 mm i.d. x 80 mm, 3 p,m particle; MAC- anol. 0)
60
YANG ET AL.
data system by electron impact with a solid probe a t 70 eV and 250°C ionizer temperature. Ultraviolet-visible absorption spectra of samples in methanol were determined a t ambient temperature 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 1cm path length at room temperature (23°C) were measured using a Jasco Model 500A spectropolarimeter equipped with a Model DP500 data processor. The concentration of the sample is indicated byAA21ml(absorbance units at wavelength X2 per ml of solvent). CD spectra are expressed by ellipticity (QAllAh2,in millidegrees) for solutions that have an absorbance of Ah2unit per ml of solvent at wavelength A2 (usually the wavelength of maximal absorption). Under conditions of measurements indicated above, the molar ellipticity ([@IA1, in deg cm2 dmol-l) and ellipticity (QA1/AA2, in millidegrees) are related to the molar extinction coefficient (€A29 in cm-I M-l) as follows: [el,,
= 0.1 eA2 (QAiIAAA
It is apparent from the equation above that molar ellipticity [03 of an enantiomer must be reported along with its molar extinction coefficient. Unfortunately, molar ellipticities of enantiomers are often reported in the literature in the absence of molar extinction coefficients. Molar extinction coefficients in a specified solvent of most chemicals are either not available or difficult to determine accurately. For the purpose of comparing data obtained in different laboratories, we strongly urge investigators to express CD spectral data by ellipticity for a solution of 1.0 absorbance unit at a specified wavelength per ml of solvent, instead of molar
A
(S)-DNBL-I
ellipticity [el. In this study, it was not possible to obtain sufficient quantity of the unstable 1,12-DMBA cis5,6-dihydrodiolto determine its molar extinction coefficient. RESULTS AND DISCUSSION CSP HPLC Separation and CD Spectra of 1,12-DMBA cis-5,6-DihydmdiolEnuntiomers
The enantiomers of 1,12-DMBA cisd,6-dihydrodiol, which are eluted as a single chromatographic peak on both normal-phase and reversed-phase HPLC, are separated into three peaks on an (S)-DNBL-I column (peaks C1, C2, and C3 in Fig. 3A). Peaks C1 and C3 are a pair of enantiomers and have CD spectra that are mirror images of each other (Fig. 4A). Peak C2 was collected and was subsequently separated into a pair of enantiomers (see CD spectra in Fig. 4B) on an (R)DNBPG-I column (peaks C2a and C2b in Fig. 3B). Thus, the chromatograms in Figure 3 revealed that there are four stereoisomers contained in 1,12-DMBA cis-5,6-dihydrodiol. All four enantiomers were confirmed by mass spectral analyses (molecular ions a t mlz 290 and characteristic fragment ions a t mlz 272 due to loss of water) and by CD spectral analyses (Fig. 4). Elution order of enantiomers for each enantiomeric pair was determined on four different CSP columns (Table 1). Results in Table 1 revealed that the covalently bonded CSP columns were less useful in the separation of the four enantiomers of 1,12-DMBA czs5,6-dihydrodiol. The sum of areas under the chromatographic peaks C1 and C3 to that of peak C2 is 1.27 (Fig. 3A). Thus two pairs of enantiomers constitute 56 and 44%, respec-
c2
I
c1
B
(R)-DNBPG-I C2a C2b
c3 OH
5R,6S
.
(P5e6a)
"OH
5R,6S (M5a6e)
5S,6R (PSa6e)
I
I
I
6
I
I
10
-
I
I
I
i I
l
l
30 RETENTION TIME (min)
14
l
I
l
l
l
I
40
Fig. 3. CSP HPLC resolution of two enantiomeric pairs of cisd,b-dihydrodiolsderived from P and M conformers of 1,12-DMBA on (S)-DNBL-I(A) and (R)-DNBPG-I(B) columns. Mobile phase was 10%(A) and 5%(B) of ethano/acetonitrile (21, v/v) in hexane, respectively, at 2 mumin. In A, peaks C1 and C3 are enantiomericto each other. Peak C2 (A) is collectedand further resolved into peaks C2a and C2b (B).
&-5,6-DIHYDRODIOL ENANTIOMERS
t
61
5S,6R (M5e6a)
250 300 WAVELENGTH ( nm ) Fig. 4. CD spectra of two enantiomeric pairs of cis-5,6-dihydrodiolderived from 1,lP-DMBA.Peaks C1, C3, C2a, and C2b are separated as described in Figure 3. See text for the assignment of absolute configurations. Spectral data and optical purity of samples are C1, concentration 1.0 A253/ml;a2%= - 50.1 millidegrees, ee 100%;C3, concentration 1.0 panel AZ5.Jml; , ,@, = + 50.7 millidegrees, ee 100%; C2a, concentration 1.0 AZ5,/ml,+234 = +33.5 millidegrees, ee -98%; C2b, concentration 1.0 A 2 & d , Qza4 = - 26.7 millidegrees, ee -76%. 250
300
tively, of the chemically synthesized 1,12-DMBA cis5,6-dihydrodiol.
freedom about the axis of the single bond between phenyl and naphthyl rings. Owing to the steric hindrance brought about by the peri methyl substituent, 4-MBA Relationship Between Conformation, Absolute cis-5,6-dihydrodiol adopts only a 5a6e conformation, Configuration, and CD Cotton Effects whereas cis-5,6-dihydrodiols of 7-Br-l-MBA, 7,12A sufficient amount of an enantiomeric 1,12-DMBA DMBA, and 7-Br-12-MBA adopt only a 5e6a conformacis-5,6-dihydrodiolcould not be obtained (due to its in- tion. Although 4-MBA (5R,6S)-dihydrodio14 and 7,12stability and low yield in synthesis) for the determination of its absolute configuration by the exciton chirality DMBA (5R,6S)-dihydrodio16have identical absolute CD method.2The exciton chirality CD method has been configurations, their major CD Cotton effects centered successfully used in our laboratory to determine the around 240 nm have opposite signs (Fig. 5). It is apparabsolute configuration of trans and cis dihydrodiols of a ent that the difference in the signs of major CD Cotton large number of polycyclic aromatic hydro~arbons.~-~effects is due to their difference in conformation; 4Although two pairs of 1,12-DMBA cis-5,6-dihy- MBA (5R,GS)-dihydrodioladopts the 5a6e conformadrodiol enantiomers have characteristically different tion and 7,12-DMBA (5R,GS)-dihydrodiol adopts the CD , ~ Cotton effects of (5R,6S)CD Cotton effects (Fig. 41, absolute configurations of 5e6a c ~ n f o r m a t i o n . ~ these enantiomers cannot be readily established based dihydrodiol enantiomers of 7-Br-1-MBA4 and 7solely on their CD spectral data. Before the assignment Br-12-MBA3 are similar to those of 7,12-DMBA of absolute configurations is presented, it is necessary (5R,GS)-dihydrodiol shown in Figure 5. 4-MBA cisd,6-dihydrodiol is the only example of BA to review available information on the relationship of conformation, absolute configuration, and CD Cotton derivatives studied to date which adopts a 5a6e conforeffects of cisd,6-dihydrodiol enantiomers of 4-MBA mation. Unlike 1-MBA, 12-MBA, and 7,12-DMBA, and 7,12-DMBA,each of which has a methyl group peri there is no distortion of A ring relative to BCD ring in 4-MBA. Thus, the CD spectrum of 4-MBA (5R,6S)to the K-region. In the absence of steric and/or electronic factors, each dihydrodiol is not the ideal example for the purpose of hydroxyl group of a K-region cis-5,6-dihydrodiol of un- comparing its CD Cotton effects with those of 1,12substituted or alkylhalogen-substitutedBA adopts ei- DMBA cis-5,6-dihydrodiolenantiomers. The ideal refther a quasiequatorial or a quasiaxial conformation. erence compounds relevant to this study would have For example, the hydroxyl groups of cisd,6-dihydrodiol been (BR,GS)-dihydrodiols of 1,4-DMBA and/or 4,12of BA, 1-MBA, and 12-MBA adopt both 5a6e and 5e6a DMBA. Other important features pertinent to this discussion conformations. This is due to the limited rotational
62
YANG ET AL.
TABLE 1. CSP-HPLC resolution of enantiomeric cis-5,6dihydrodiols of 1,12-DMBA, 4-MBA, 7-Br-l-MBA, ?-Br-12-MBA, and 7,12-DMBA Retention time (min)" cis-5,6-Dihydrodiol 4-MBA (5a,6e)
1,12-DMBA (5a,6e) 1,12-DMBA (5e,6a)
7-Br-1-MBA (5e,6a)
csp"
%Ab
Enantiomer 1
Enantiomer 2
RVd
(R)-DNBPG-I (R)-DNBPG-C (S)-DNBL-I (S)-DNBL-C (R)-DNBPG-I (R)-DNBPG-C (S)-DNBL-I (S)-DNBL-C (R)-DNBPG-I
10 10 10 10 10 10 10 10 10
(R)-DNBPGC (S)-DNBL-I (S)-DNBL-C (R)-DNBPG-I (R)-DNBPG-C (S)-DNBL-I
10 10 10 10 10 10
(S)-DNBL-C
10
(R)-DNBPG-I (R)-DNBPG-C (S)-DNBL-I (S)-DNBL-C (R)-DNBPG-I (R)-DNBPG-C (S)-DNBL-I (S)-DNBL-C
10 10 10 10 10 10 10 10
17.2 (5S,6R) 12.6 (5S,6R) 14.7 (5R,6S) 9.4 (5R,6S) 10.5 (5S,6R)-F 8.7 (5S,6R)-P 10.3 (5R,6S)-M 7.5 (5R,6S)-M 12.1 (5R,6S)-P 32.5 (5R,6S)-P 9.1 11.2 7.5 14.9 (5R,6S) 10.5 (5R,6S) 11.8 (5R,6S) 24.4 (5R,6S) 8.5 17.3 15.2 (5R,6S) 10.9 (5R,6S) 16.2 (5R,6S) 8.8 (5R,6S) 20.9 13.2 (5R,6S) 10.3 (5R,6S) 15.6 (5R,6S)
19.1 (5R,6S) 13.6 (5R,6S) 15.3 (5S,6R) 10.0 (5S,6R) 12.2 (5R,6S)-Me 9.4 (5R,6S)-M 12.2 (5S,6R)-P 8.4 (5S,6R)-P 12.4 (5S,6R)-M 33.7 (5S,6R)-M 9.2 11.2 7.5 15.1 (5S,6R) 10.6 (5S,6R) 12.1 (5S,6R) 27.4 (5S,6R) 8.5 17.3 16.0 (5S,6R) 11.6 (5S,6R) 17.7 (5S,6R) 9.1 (5S,6R) 20.9 (5S,6R) 13.8 (5S,6R) 10.6 (5S,6R) 17.2 (5S,6R)
2.3 1.5 0.8 2.1 2.9 1.5 3.5 1.8 0.5 0.8
