C«-dihydrodiols

The application of di-arene dihydrodiols has been noted in Part 1 of this chapter, and it is sufficient to note here the application of a mutant of Alcaligenes eutrophus strain B9 that is blocked in the degradation of benzoate (and some halogenated benzoates). This produced the cA-l,2-dihydrodiol (Reiner and Hegeman 1971), and has been used as the source of ring B for the synthesis of a range of tetracyclines (Charest et al. 2005). A range of substituted c -dihydrodiols has been produced, and it has been shown that for 3-substituted benzoates both 3- and 5-substituted cA-dihydrodiols were formed (Reineke et al. 1978). 

The c -dihydrodiols have been prodnced from a nnmber of triflnoromethylbenzoates ... 

Table II. Successful conversions of aryl silanes to c -dihydrodiols using toluene dioxygenase (TDO). 

Althongh the prodnct from the transformation of toluene by mntants of Pseudomonas putida lacking dehydrogenase activity is the cis-2R,3S dihydrodiol, the cis-2S,3R dihydrodiol has been synthesized from 4-iodotoluene by a combination of microbiological and chemical reactions. P. putida strain UV4 was used to prepare both enantiomers of the di-dihydrodiol, and iodine was chemically removed nsing H2 -Pd/C. Incubation of the mixtnre of enantiomers with P. putida NCIMB 8859 selectively degraded the 2R,3S componnd to prodnce toluene cis-2S,3R dihydrodiol (Allen et al. 1995). 

FIGURE 8.7 Examples of chemical syntheses based on cyclohexadiene cis dihydrodiols (a) pinitol, (b) condnramine A, (c) (-)-laminitol, and (d) conduritol analogs. (From Neilson, A.H. and Allard, A.-S. The Handbook of Environmental Chemistry, Springer, 1998. With permission.)... 

Studies using either molecular oxygen-18 or oxygen-18 water indicated that the 4R,5R-dihydrodiol is derived by water attack at the C-4 position of the metabolically formed 4,5-epoxide intermediate (15.21), These results established that 4S,5R-epoxide is formed as the metabolic precursor of the 4R,5R-dihydrodiol. Hydration studies of the optically pure BaP 4,5-epoxide enantiomers indicated that the 4S,5R-epoxide is hydrated exclusively at the S-center (C-4 position) whereas 85% of the 4R,5S-epoxide is hydrated at the S-center (C-5 position) (22 and Figure 4). 

It was shown that microsomal epoxide hydrolase-catalyzed trans-addition of water to BaP 9,10-epoxide occurs stereospecifically at the C-9 position (15). Since BaP is metabolized essentially to an optically pure 9R,10R-dihydrodiol (13 and L5 Table I), the 9,10-epoxide formed in BaP metabolism must have 9S,10R absolute stereochemistry (Figure 1). Similarly, the 7,8-epoxide formed in BaP metabolism is hydrated specifically at the C-8 position to form the 7R,8R-dihydrodiol (14.21). Hence the enzymatically formed 7,8-epoxide intermediate has 7R,8S absolute stereochemistry (Figure 1). Although the 7R,8R-dihydrodiol is formed almost exclusively from BaP metabolism in rat liver microsomes (Table I) and in bovine bronchial explants (25). the 7S,8S-dihydrodiol is also formed from BaP metabolism in mouse skin epidermis in vivo (5). 

In contrast to the metabolism of BA and BaP, the 5,6-dihydrodiols formed in the metabolism of DMBA by liver microsomes from untreated, phenobarbital-treated, and 3-methylcholanthrene-treated rats are found to have 5R,6R/5S,6S enantiomer ratios of 11 89, 6 94, and 5 95, respectively (7.49 and Table II). The enantiomeric contents of the dihydrodiols were determined by a CSP-HPLC method (7.43). The 5,6-epoxide formed in the metabolism of DMBA by liver microsomes from 3MC-treated rats was found to contain predominantly (>97%) the 5R,6S-enantiomer which is converted by microsomal epoxide hydrolase-catalyzed hydration predominantly (>95%) at the R-center (C-5 position, see Figure 3) to yield the 5S,6S-dihydrodiol (49). In the metabolism of 12-methyl-BA, the 5S,6S-dihydrodiol was also found to be the major enantiomer formed (50) and this stereoselective reaction is similar to the reactions catalyzed by rat liver microsomes prepared with different enzyme inducers (unpublished results). Labeling studies using molecular oxygen-18 indicate that 5R,68-epoxide is the precursor of the 5S,6S-dihydrodiol formed in the metabolism of 12-methyl-BA (51). 

Methods for the synthesis of the biologically active dihydrodiol and diol epoxide metabolites of both carcinogenic and noncarcinogenic polycyclic aromatic hydrocarbons are reviewed. Four general synthetic routes to the trans-dihydrodiol precursors of the bay region anti and syn diol epoxide derivatives have been developed. Syntheses of the oxidized metabolites of the following hydrocarbons via these methods are described benzo(a)pyrene, benz(a)anthracene, benzo-(e)pyrene, dibenz(a,h)anthracene, triphenylene, phen-anthrene, anthracene, chrysene, benzo(c)phenanthrene, dibenzo(a,i)pyrene, dibenzo(a,h)pyrene, 7-methyl-benz(a)anthracene, 7,12-dimethylbenz(a)anthracene, 3-methylcholanthrene, 5-methylchrysene, fluoranthene, benzo(b)fluoranthene, benzo(j)fluoranthene, benzo(k)-fluoranthene, and dibenzo(a,e)fluoranthene. 

Methylcholanthrene (3-MC) is a potent carcinogen, intermediate in activity between DMBA and BP (27,77). It was first prepared in 1925 by Wieland from desoxycholic acid (89). Biological studies have tentatively identified the 9,10-dihydrodiol (24a) and/or its 1- or 2-hydroxy derivatives (24b and 24c) and the corresponding diol and triol epoxides (25 -c) as the proximate and ultimate carcinogenic forms, respectively, of 3-MC (90-93). 

The 9,10-dihydrodiol of 3-MC (24a) was synthesized from 9-hy-droxy-3-MC by Method IV (86). Oxidation of this phenol with Fremy s salt in the presence of Adogen 464, a quaternary ammonium phase transfer catalyst, furnished 3-MC 9,10-dione. Reduction of the qui-none with NaBH -C gave pure 24a in good yield. Treatment of 24a with m-chloroperbenzoic acid was monitored by HPLC in order to optimize the yield of the anti diol epoxide (25 ) and minimize its decomposition. 

The rates of hydrolysis and binding to DNA of anti-DE-I, syn-DE-I, anti-DE-II, syn-DE-II, and anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrochrysene (anti-chrysene-DE) were studied in order to relate the chemical reactivity of these dihydrodiol epoxides to their biological activities. The half-lives of the dihydrodiol epoxides in cacodylate buffer at pH 7.0 and 37°C are summarized in Table III and their relative extents of binding to DNA in Table IV. It is clear that the rates of hydrolysis of the dihydrodiol epoxides do not correlate with their DNA binding properties. 

Table III. Half-Lives of Dihydrodiol Epoxides of 5-MeC and Chrysene at pH 7.0 and 37 °C in the Absence and Presence of Native and Denatured Calf Thymus DNA...

Bowden, G.T., Hsu, I.C., and Harris, C.C. (1979). The effect of caffeine on cytotoxicity, mutagenesis and sister chromatid exchanges in Chinese hamster cells treated with dihydrodiol epoxide derivatives of benzo(a)pyrene. Mutation Res. 63 361-370. 

Roubal et al. (8) applied TLC to the separation and identification of metabolites of l c-iabeled naphthalene administered to coho salmon fingerlings via intraperitoneal injection. 1-Naphthol, a dihydrodiol, mercapturic acid, 1-naphthyl glucuronic acid and a glycoside/sulfate fraction were identified in brain, liver, gall bladder, and muscle 1-naphthol, a dihydrodiol, and 1-naphthyl glucuronic acid were the only metabolites found in heart. 

Conversion of epoxides (arene oxides) into phenols is spontaneous. The conversion of epoxides into dihydrodiols is catalyzed by EH (EC 4.2.1.63). Hydroxyl containing PAHs can act as substrates for conjugases (C) (UDP glucuronsyl transferase (EC 2.4.1.17) and phenol sulphotransferase (EC 2.8.2.1)). This pathway usually leads to inactive excretable products. Epoxides are scavenged by GSH and the reaction is catalyzed by GSHt (EC 2.5.1.18). When GSH is depleted and/or the other pathways are saturated, epoxides of dihydrodiols (particularly 7,8-diol-9,10-epoxides in the case of BP) and phenol metabolites react with cellular macromolecules such as DNA, RNA, and protein. If repair mechanisms are exceeded the detrimental effects of PAH may result. 

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