Stereoselective formation and hydration of 12-methylbenz[a]anthracene 5,6-epoxide enantiomers by rat liver microsomal enzymes.

The K-region trans-5,6-dihydrodiols formed in the metabolism of 12-methylbenz[a]anthracene (12-MBA) by liver microsomal preparations from untreated, phenobarbital-treated and 3-methylcholanthrene-treated male Sprague-Dawley rats were found by chiral stationary-phase h.p.l.c. (c.s.p.-h.p.l.c.) analyses to contain (5S,6S)/(5R,6R) enantiomer ratios of 93:7, 88:12 and 97:3 respectively. The absolute stereochemistry of a 12-MBA trans-5,6-dihydrodiol enantiomer was elucidated by the exciton-chirality c.d. method. The 5,6-epoxides formed in the metabolism of 12-MBA by liver microsomal preparations from untreated, phenobarbital-treated and 3-methylcholanthrene-treated male Sprague-Dawley rats in the presence of the epoxide hydrolase inhibitor 3,3,3-trichloropropylene 1,2-oxide were isolated from a mixture of metabolites by normal-phase h.p.l.c., and their (5S,6R)/(5R,6S) enantiomer ratios were found by c.s.p.-h.p.l.c. analyses to be 73:27, 78:22 and 99:1 respectively. The absolute configurations of 12-MBA 5,6-epoxide enantiomers, resolved by c.s.p.-h.p.l.c., were determined via high-resolution (500 MHz) proton-n.m.r. and c.d. spectral analyses of the two isomeric methoxylation products derived from each of the 12-MBA 5,6-epoxide enantiomers. Enantiomeric pairs of the two methoxylation products were resolved by c.s.p.-h.p.l.c. The results indicate that enantiomeric 5S,6R-epoxide and 5S,6S-dihydrodiol were the major enantiomers preferentially formed in the metabolism at the K-region 5,6-double bond of 12-MBA by all three rat liver microsomal preparations. Optically pure 12-MBA 5S,6R-epoxide was hydrated predominantly at the C(6) position (R centre) to form 12-MBA trans-5,6-dihydrodiol with a (5S,6S)/(5R,6R) enantiomer ratio of 97:3. However, optically pure 12-MBA 5R,6S-epoxide was hydrated nearly equally at both C(5) and C(6) positions to form 12-MBA trans-5,6-dihydrodiol with a (5S,6S)/(5R,6R) enantiomer ratio of 57:43.     

Stereoselective formation and hydration of benzo[c]phenanthrene 3,4- and 5,6-epoxide enantiomers by rat liver microsomal enzymes

The K-region 5,6-epoxides, formed in the metabolism of benzo[c]phenanthrene (BcPh) in the presence of an epoxide hydrolase inhibitor 3,3,3-trichloropropylene 1,2-oxide (TCPO) by liver microsomes from untreated, phenobarbital-treated, 3-methylcholanthrene-treated, and polychlorinated biphenyls (Aroclor 1254)-treated rats of the Sprague-Dawley and the Long-Evans strains, were found by chiral stationary phase high-performance liquid chromatography analyses to be enriched (58-72%) in the 5S, 6R enantiomer. In the absence of TCPO, the metabolically formed BcPh trans-5,6-dihydrodiol was enriched (78-86%) in the 5S,6S enantiomer. The major enantiomer of the BcPh 3,4-epoxide metabolite was found to be enriched in the 3S,4R enantiomer which undergoes racemization under the experimental conditions. The major enantiomer of the 5,6-dihydrodiol metabolite was elucidated by the exciton chirality circular dichroism (CD) method to have a 5S,6S absolute stereochemistry. Absolute configurations of enantiomeric methoxylation products derived from each of the two BcPh 5,6-epoxide enantiomers. Optically pure BcPh 5S,6R-epoxide was enzymatically hydrated exclusively at the C6 position to form an optically pure BcPh 5S,6S-dihydrodiol. However, optically pure BcPh 5R,6S-epoxide was hydrated at both C5 and C6 positions to form a BcPh trans-5,6-dihydrodiol with a (5S,6S):(5R,6R) enantiomer ratio of 32:68.     

The effect of the bay-region 12-methyl group on the stereoselective metabolism at the K-region of 7,12-dimethylbenz[a]anthracene by rat liver microsomes.

The enantiomers of a trans-5,6-dihydrodiol formed in the metabolism of 7,12-dimethylbenz[a]anthracene by rat liver microsomes (microsomal fractions) were resolved by chiral stationary-phase high-performance liquid chromatography. The major 7,12-dimethylbenz[a]anthracene trans-5,6-dihydrodiol enantiomer and its hydrogenation product 5,6,8,9,10,11-hexahydro-trans-5,6-diol were found to have 5S,6S absolute configurations by the exciton chirality c.d. method. The R,R/S,S enantiomer ratios of 7,12-dimethylbenz[a]anthracene trans-5,6-dihydrodiol formed in the metabolism of 7,12-dimethylbenz[a]anthracene by liver microsomes from untreated, 3-methylcholanthrene-treated and phenobarbital-treated male Sprague-Dawley rats were found to be 11:89, 6:94, and 5:95 respectively. These findings and those reported previously on the metabolic formations of trans-5,6-dihydrodiols from 7-methylbenz[a]anthracene and 12-methylbenz[a]anthracene suggest that the 12-methyl group in 7,12-dimethylbenz[a]anthracene plays an important role in determining the stereoselective metabolism at the K-region 5,6-double bond. Furthermore, the finding that formation of 5S,6S-dihydrodiol as the predominant enantiomer was not significantly affected by the isoenzymic composition of cytochrome P-450 present in microsomes prepared from the livers of the rats pretreated with the different inducing agents indicates that the stereoselectivity depends on the substrate metabolized rather than on the precise nature of the metabolizing-enzyme system.     

Stereoselective fungal metabolism of 7,12-dimethylbenz[a]anthracene: identification and enantiomeric resolution of a K-region dihydrodiol.

Syncephalastrum racemosum UT-70 and Cunninghamella elegans ATCC 36112 metabolized 7,12-dimethylbenz[a]anthracene (7,12-DMBA) to hydroxymethyl metabolites as well as 7-hydroxymethyl-12-methylbenz[a]anthracene trans-3,4-, -5,6-, -8,9-, and -10,11-dihydrodiols. The 7,12-DMBA metabolites were isolated by reversed-phase high-performance liquid chromatography and identified by their UV-visible absorption, mass, and nuclear magnetic resonance spectral characteristics. A comparison of the circular dichroism spectra of the K-region (5,6-position) dihydrodiol of both fungal strains with those of the 7,12-DMBA 5S,6S-dihydrodiol formed from 7,12-DMBA by rat liver microsomes indicated that the major enantiomer of the 7-hydroxymethyl-12-methylbenz[a]anthracene trans-5,6-dihydrodiol formed by both fungal strains had a 5R,6R absolute stereochemistry. Direct resolution of the fungal trans-5,6-dihydrodiols by chiral stationary-phase high-performance liquid chromatography indicated that the ratios of the R,R and S,S enantiomers were 88:12 and 77:23 for S. racemosum and C. elegans, respectively. These results indicate that the fungal metabolism of 7,12-DMBA at the K region (5,6-position) is highly stereoselective and different from that reported for mammalian enzyme systems.     

Stereoselective fungal metabolism of 7,12-dimethylbenz[a]anthracene: identification and enantiomeric resolution of a K-region dihydrodiol

Syncephalastrum racemosum UT-70 and Cunninghamella elegans ATCC 36112 metabolized 7,12-dimethylbenz[a]anthracene (7,12-DMBA) to hydroxymethyl metabolites as well as 7-hydroxymethyl-12-methylbenz[a]anthracene trans-3,4-, -5,6-, -8,9-, and -10,11-dihydrodiols. The 7,12-DMBA metabolites were isolated by reversed-phase high-performance liquid chromatography and identified by their UV-visible absorption, mass, and nuclear magnetic resonance spectral characteristics. A comparison of the circular dichroism spectra of the K-region (5,6-position) dihydrodiol of both fungal strains with those of the 7,12-DMBA 5S,6S-dihydrodiol formed from 7,12-DMBA by rat liver microsomes indicated that the major enantiomer of the 7-hydroxymethyl-12-methylbenz[a]anthracene trans-5,6-dihydrodiol formed by both fungal strains had a 5R,6R absolute stereochemistry. Direct resolution of the fungal trans-5,6-dihydrodiols by chiral stationary-phase high-performance liquid chromatography indicated that the ratios of the R,R and S,S enantiomers were 88:12 and 77:23 for S. racemosum and C. elegans, respectively. These results indicate that the fungal metabolism of 7,12-DMBA at the K region (5,6-position) is highly stereoselective and different from that reported for mammalian enzyme systems.     

Regio- and stereoselective metabolism of 7,12-dimethylbenz[a]anthracene by Mycobacterium vanbaalenii PYR-1

The degradation of 7,12-dimethylbenz[a]anthracene (DMBA), a carcinogenic polycyclic aromatic hydrocarbon, by cultures of Mycobacterium vanbaalenii PYR-1 was studied. When M. vanbaalenii PYR-1 was grown in the presence of DMBA for 136 h, high-pressure liquid chromatography (HPLC) analysis showed the presence of four ethyl acetate-extractable compounds and unutilized substrate. Characterization of the metabolites by mass and nuclear magnetic resonance spectrometry indicated initial attack at the C-5 and C-6 positions and on the methyl group attached to C-7 of DMBA. The metabolites were identified as cis-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a]anthracene (DMBA cis-5,6-dihydrodiol), trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a]anthracene (DMBA trans-5,6-dihydrodiol), and 7-hydroxymethyl-12-methylbenz[a]anthracene, suggesting dioxygenation and monooxygenation reactions. Chiral stationary-phase HPLC analysis of the dihydrodiols showed that DMBA cis-5,6-dihydrodiol had 95% 5S,6R and 5% 5R,6S absolute stereochemistry. On the other hand, the DMBA trans-5,6-dihydrodiol was a 100% 5S,6S enantiomer. A minor photooxidation product, 7,12-epidioxy-7,12-dimethylbenz[a]anthracene, was also formed. The results demonstrate that M. vanbaalenii PYR-1 is highly regio- and stereoselective in the degradation of DMBA.     

Absolute configuration of the 5,6-oxide formed from 7,12-dimethylbenz[a]anthracene by cytochrome P450c

The absolute configurations of the enantiomeric 5,6-arene oxides of 7,12-dimethylbenz[a]anthracene (DMBA) were recently assigned such that the late eluting enantiomer from a chiral HPLC column has 5R,6S absolute configuration. [Mushtaq et al. (1984) BBRC 125, 539]. The authors further concluded that the 5R,6S-enantiomer predominates on metabolism of DMBA by cytochrome P450c in liver microsomes from 3-methylcholanthrene-treated rats. Their chemical assignment of absolute configuration is incorrect. Thus, metabolism of DMBA by these microsomes as well as by homogeneous cytochrome P450c produces 5,6-oxide highly enriched (95%) in the 5S,6R-enantiomer in accord with theoretical predictions.     

Stereoselective formation of benz[a]anthracene (+)-(5S,6R)-oxide and (+)-(8R,9S)-oxide by a highly purified and reconstituted system containing cytochrome P-450c

The principal oxidative metabolites formed from benz[a]anthracene (BA) by the rat liver microsomal monooxygenase system are the 5,6- and 8,9-arene oxides. In order to determine the enantiomeric composition and absolute configuration of these metabolically formed arene oxides, an HPLC procedure has been developed to separate the six isomeric glutathione conjugates obtained synthetically from the individual enantiomeric arene oxides. Both (+)- and (?)-BA 5,6-oxide gave the two possible positional isomers, but only one positional isomer was formed in each case from (+)- and (?)-BA 8,9-oxide. When [¹⁴C]-BA was incubated with a highly purified and reconstituted monooxygenase system containing cytochrome P-450c, and glutathione was allowed to react with the arene oxides formed, radio-active adducts were formed predominantly (>97%) from the (+)-(5S,6R) and (+)-(8R,9S) enantiomers. The present results are in accord with theoretical predictions of the steric requirements of the catalytic binding site of cytochrome P-450c.     

Effects of a fluoro substituent on the fungal metabolism of 1-fluoronaphthalene.

The metabolism of 1-fluoronaphthalene by Cunninghamella elegans ATCC 36112 was studied. The metabolites were isolated by reverse-phase high-pressure liquid chromatography and characterized by the application of UV absorption, 1H nuclear magnetic resonance, and mass spectral techniques. C. elegans oxidized 1-fluoronaphthalene predominantly at the 3,4- and 5,6-positions to form trans-3,4-dihydroxy-3,4-dihydro-1-fluoronaphthalene and trans-5,6-dihydroxy-5,6-dihydro-1-fluoronaphthalene. In addition, 1-fluoro-8-hydroxy-5-tetralone, 5-hydroxy-1-fluoronaphthalene, and 4-hydroxy-1-fluoronaphthalene as well as glucoside, sulfate, and glucuronic acid conjugates of these phenols were formed. Circular dichroism spectra of the trans-3,4- and trans-5,6-dihydrodiols formed from 1-fluoronaphthalene indicated that the major enantiomers of the dihydrodiols have S,S absolute stereochemistries. In contrast, the trans-5,6-dihydrodiol formed from 1-fluoronaphthalene from 3-methylcholanthrene-treated rats had Cotton effects that are opposite in sign (R,R) to those formed by C. elegans. The results indicate that the fungal monooxygenase-epoxide hydrolase systems are highly stereoselective in the metabolism of 1-fluoronaphthalene and that a fluoro substituent blocks epoxidation at the fluoro-substituted double bond, decreases oxidation at the aromatic double bond that is peri to the fluoro substituent, and enhances metabolism at the 3,4- and 5,6-positions of 1-fluoronaphthalene.     

Cytochrome P-450-catalyzed stereoselective epoxidation at the K region of benz[a]anthracene and benzo[a]pyrene

The enantiomers of K-region benz[a]anthracene (BA) 5,6-epoxide and benzo[a]pyrene (BP) 4,5-epoxide were resolved by chiral stationary-phase high-performance liquid chromatography (CSP-HPLC). The K-region epoxides formed in the metabolism of BA by liver microsomes from untreated (control), phenobarbital (PB)-treated, and 3-methylcholanthrene (MC)-treated male Sprague-Dawley rats were determined by CSP-HPLC to have a 5R,6S/5S,6R enantiomer ratio of 25:75, 21:79, and 4:96, respectively. The K-region 4,5-epoxide formed in the metabolism of BP by the same rat liver microsomal preparations contained a 4R,5S/4S,5R enantiomer ratio of 48:52 (control), 40:60 (PB), and 5:95 (MC), respectively. The results indicate that various cytochrome P-450 isozymes of rat liver exhibit different stereoselective properties in catalyzing the epoxidation reactions at the K region of BA and of BP.     

Microbial metabolism and detoxification of 7,12-dimethylbenz[a]anthracene

Summary Six strains of fungi grown on Sabouraud dextrose broth in the presence of 7,12-dimethylbenz[a]anthracene (DMBA) were surveyed for their ability to metabolize DMBA. Experiments with [¹⁴C]DMBA indicated that the extent of formation of organic-soluble metabolites ranged from 6 to 28% after 5 days of incubation, depending on the organism tested. The yields of water-soluble metabolites also varied, and ranged from 1 to 33% after 5 days.Cunninghamella elegans ATCC 36112 andSyncephalastrum racemosum UT-70 exhibited the highest DMBA-metabolizing activity among the organisms surveyed.S. racemosum metabolized DMBA primarily to 7-hydroxymethyl-12-methylbenz[a]anthracene (7-OHM-12-MBA)_ and 7,12-dihydroxymethylbenz[a]anthracene (7,12-diOHMBA). Minor metabolites included 7-OHM-12-MBA-trans-5,6-, 8,9- and 10,11-dihydrodiols, and glucuronide and sulfate conjugates of phenolic derivatives of DMBA. In contrast, the major DMBA metabolites produced byC. elegans were water-soluble. The predominant organic-soluble metabolites produced byC. elegans included 7-OHM-12-MBA-trans-5,6-, 8,9- and 10,11-dihydrodiols. DMBA-trans-3,4-dihydrodiol was also detected. Circular dichroism spectral analysis revealed that the major enantiomer of the 7-OHM-12-MBA-trans-8,9-dihydrodiol formed by each organism has anS,S absolute configuration, while the major enantiomers of the 5,6-, 10,11- and 3,4-dihydrodiols had anR,R configuration. The mutagenic activity of extracts fromS. racemosum exposed to DMBA were determined inSalmonella typhimurium TA98. The mutagenicity of DMBA decreased by 36% over a period of 5 days as 33% of the compound was metabolized. Comparison of these results with previously reported results in mammalian systems suggests that there are similarities and differences between the fungal and mammalian oxidation of DMBA and that the overall balance of fungal metabolism is towards a detoxification rather than a bioactivation pathway.     

Absolute configuration of cis-5,6-dihydrodiol enantiomers derived from helical conformers of 1,12-dimethylbenz[a]anthracene

1,12-Dimethylbenz[a]anthracene (1,12-DMBA) cis-5,6-dihydrodiol was synthesized by oxidation of 1,12-DMBA with osmium tetroxide in pyridine in low yield (less than or equal to 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)phenylglycine or (S)-N-(3,5-dinitrobenzoyl)leucine, which is ionically bonded to gamma-aminopropylsilanized silica. Absolute configurations of enantiomers were determined by comparing their circular dichroism spectra with those of conformationally similar cis-5,6-dihydrodiol enantiomers of 4-methylbenz[a]anthracene and 7,12-dimethylbenz[a]anthracene with known absolute stereochemistry.     

Stereoselective formation of benzo(c)phenanthrene (+)-(3S,4R) and (+)-(5S,6R)-oxides by cytochrome P450c in a highly purified and reconstituted system

The principal oxidative metabolites formed from benzo(c)phenanthrene (B(c)Ph) by the cytochromes P450 in liver microsomes from control and treated rats are the 3,4- and 5,6-arene oxides. A procedure is described which allows determination of the enantiomer composition and absolute configuration of these arene oxides based on HPLC separation of isomeric thiolate adducts formed with N-acetyl-L-cysteine in base. Incubation of [3H]-B(c)Ph with highly purified cytochrome P450c in a reconstituted monooxygenase system followed by trapping of the metabolically formed arene oxides as above indicated that the 3,4-oxide was predominantly the (+)-(3S,4R)-enantiomer (90%) and that the 5,6-oxide consisted mainly of the (+)-(5S,6R)-enantiomer (76%). The results are discussed in terms of their implications about the catalytic binding site of cytochrome P450c.     

Regio- and stereo-selective metabolism of 4-methylbenz[a]anthracene by the fungus Cunninghamella elegans.

Metabolism of 4-methylbenz[a]anthracene by the fungus Cunninghamella elegans was studied. C. elegans metabolized 4-methylbenz[a]anthracene primarily at the methyl group, this being followed by further metabolism at the 8,9- and 10,11-positions to form trans-8,9-dihydro-8,9-dihydroxy-4-hydroxymethylbenz[a]anthracene and trans-10,11-dihydro-10,11-dihydroxy-4-hydroxymethylbenz[a]anthracene. There was no detectable trans-dihydrodiol formed at the methyl-substituted double bond (3,4-positions) or at the 'K' region (5,6-positions). The metabolites were isolated by reversed-phase high-pressure liquid chromatography and characterized by the application of u.v.-visible-absorption-, 1H-n.m.r.- and mass-spectral techniques. The 4-hydroxymethylbenz[a]anthracene trans-8,9- and -10,11-dihydrodiols were optically active. Comparison of the c.d. spectra of the trans-dihydrodiols formed from 4-methylbenz[a]anthracene by C. elegans with those of the corresponding benz[a]anthracene trans-dihydrodiols formed by rat liver microsomal fraction indicated that the major enantiomers of the 4-hydroxymethylbenz[a]anthracene trans-8,9-dihydrodiol and trans- 10,11-dihydrodiol formed by C. elegans have S,S absolute stereochemistries, which are opposite to those of the predominantly 8R,9R- and 10R,11R-dihydrodiols formed by the microsomal fraction. Incubation of C. elegans with 4-methylbenz[a]anthracene under 18O2 and subsequent mass-spectral analysis of the metabolites indicated that hydroxylation of the methyl group and the formation of trans-dihydrodiols are catalysed by cytochrome P-450 mono-oxygenase and epoxide hydrolase enzyme systems. The results indicate that the fungal mono-oxygenase-epoxide hydrolase enzyme systems are highly stereo- and regio-selective in the metabolism of 4-methylbenz[a]anthracene.     

Synthesis, absolute configuration and biological activity of both enantiomers of 2-(5,6-dichloro-3-indolyl)propionic acid: new dichloroindole auxins

Racemic 2-(5,6-dichloro-3-indolyl)propionic acid (5,6-Cl2-2-IPA) was synthesized from 5,6-dichloroindole-3-acetic acid (5,6-Cl2-IAA) by successive esterification, methoxycarbonylation, methylation, and double hydrolysis. The racemate was converted to the diastereomeric esters of (S)-(-)-1-phenylethyl alcohol. These were separated by HPLC into two optically active diastereomers and then hydrolyzed with p-TsOH to the optically active enantiomers of 5,6-Cl2-2-IPA. The absolute configurations of both the 5,6-Cl2-2-IPA enantiomers were determined by comparing the 1H-NMR spectra of their diastereomeric (S)-(-)-1-phenylethyl esters with those of the diastereomeric (S)-(-)-1-phenylethyl esters of 2-(3-indolyl)propionic acid (2-IPA) whose absolute configurations are already known. There was no essential difference between (S)-(+)- and (R)-(-)-5,6-Cl2-2-IPA in hypocotyl growth-inhibiting activity toward Chinese cabbage, but their inhibitory activities were stronger than that of the potent mother auxin, 5,6-Cl2-IAA. No essential difference in the coleoptile elongating activity of Avena sativa was apparent for the enantiomers, this activity being about one-third that of 5,6-Cl2-IAA.     

Stereoselective metabolism of 7-methylbenz[a]anthracene: absolute configuration of five dihydrodiol metabolites and the effect of dihydrodiol conformation on circular dichroism spectra

7-Methylbenz[a]anthracene (7-MBA) was metabolized stereoselectively by rat liver microsomes to form five optically active dihydrodiols as the predominant metabolites. The dihydrodiols were purified by a combination of reversed-phase and normal-phase high performance liquid chromatography (HPLC). By comparison of their circular dichroism (CD) spectra with the corresponding benz[a]anthracene (BA) dihydrodiols of known absolute stereochemistry, the major dihydrodiol enantiomers of 7-MBA have been determined to have 1R,2R-, 3R,4R- and 10R , 11R - absolute configurations, respectively. Due to their quasi- diaxial conformations, the absolute configuration of trans-5,6- and trans-8,9-dihydrodiols, the two most abundant metabolites of 7-MBA, could not be determined by simple comparisons of their circular dichroism spectra with those of the quasidi -equatorial BA 5R, 6R - and 8R , 9R -dihydrodiols. The major enantiomers of the quasi- diaxial trans-5,6- and trans-8,9-dihydrodiol metabolites of 7-MBA were determined by comparison to the CD spectrum of 7-bromo-BA 5R, 6R -dihydrodiol and by the exciton chirality method to have R,R absolute stereochemistry. This study also revealed that the circular dichroism Cotton effects of an enantiomeric dihydrodiol of polycyclic aromatic hydrocarbons can be drastically altered if the conformation (quasi- diaxial vs. quasi di-equatorial ) of the dihydrodiol is changed.     

Synthesis and characterization of stereoisomers of 5,6-dihydro-5,6-dihydroxy-thymidine

Six products were isolated by reverse phase HPLC from the reaction of thymidine with osmium tetroxide. Four of the products were identified as stereoisomers of 5,6-dihydro-5,6-dihydroxy-thymidine (TG). The absolute configurations of these four compounds (from the shortest to the longest HPLC retention times) were determined by two-dimensional nuclear magnetic resonance spectroscopy to be (-)-trans-5S,6S-, (+)-trans-5R,6R-, (-)-cis-5R,6S-, and (+)-cis-5S,6R-5,6-dihydro-5,6-dihydroxy-thymidine. The other two products were dimers with unknown linking sites. Parameters of the mass and nuclear magnetic resonance spectra are reported and discussed.     

Resolution of racemic mixtures of carbocyclic analogues of nucleosides and assignment of their absolute configuration

Racemic trans-6-chloro-9-[2-(hydroxymethyl)cyclopentyl]purine was resolved using HPLC with a chiral column. The absolute configurations of the enantiomers were determined by NMR studies of their (R)- and (S)-methoxyphenylacetates.     

Regio- and Stereoselective Metabolism of 7,12-Dimethylbenz[a]anthracene by Mycobacterium vanbaalenii PYR-1

The degradation of 7,12-dimethylbenz[a]anthracene (DMBA), a carcinogenic polycyclic aromatic hydrocarbon, by cultures of Mycobacterium vanbaalenii PYR-1 was studied. When M. vanbaalenii PYR-1 was grown in the presence of DMBA for 136 h, high-pressure liquid chromatography (HPLC) analysis showed the presence of four ethyl acetate-extractable compounds and unutilized substrate. Characterization of the metabolites by mass and nuclear magnetic resonance spectrometry indicated initial attack at the C-5 and C-6 positions and on the methyl group attached to C-7 of DMBA. The metabolites were identified as cis-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a]anthracene (DMBA cis-5,6-dihydrodiol), trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a]anthracene (DMBA trans-5,6-dihydrodiol), and 7-hydroxymethyl-12-methylbenz[a]anthracene, suggesting dioxygenation and monooxygenation reactions. Chiral stationary-phase HPLC analysis of the dihydrodiols showed that DMBA cis-5,6-dihydrodiol had 95% 5S,6R and 5% 5R,6S absolute stereochemistry. On the other hand, the DMBA trans-5,6-dihydrodiol was a 100% 5S,6S enantiomer. A minor photooxidation product, 7,12-epidioxy-7,12-dimethylbenz[a]anthracene, was also formed. The results demonstrate that M. vanbaalenii PYR-1 is highly regio- and stereoselective in the degradation of DMBA.