Dihydrodiols

Benzene dioxygenase is a complex enzyme consisting of three protein components, that catalyse the conversion of benzene to benzene cis-dihydrodiol. Give two reasons why this biotransformation should be carried out using whole cells as opposed to using enzyme preparations. 

Metabolic pathways containing dioxygenases in wild-type strains are usually related to detoxification processes upon conversion of aromatic xenobiotics to phenols and catechols, which are more readily excreted. Within such pathways, the intermediate chiral cis-diol is rearomatized by a dihydrodiol-dehydrogenase. While this mild route to catechols is also exploited synthetically [221], the chirality is lost. In the context of asymmetric synthesis, such further biotransformations have to be prevented, which was initially realized by using mutant strains deficient in enzymes responsible for the rearomatization. Today, several dioxygenases with complementary substrate profiles are available, as outlined in Table 9.6. Considering the delicate architecture of these enzyme complexes, recombinant whole-cell-mediated biotransformations are the only option for such conversions. E. coli is preferably used as host and fermentation protocols have been optimized [222,223]. 

Dioxygenases that are exploited at present do not provide general access to antipodal cis-diols and a combined chemoenzymatic strategy is required. While oxidation of bromobenzene (and other mono- and dihalogenated precursors) with TDO expressing cells gives the corresponding (—)-dihydrodiol in excellent... 

In the rabbit, the nonplanar PCB 2,2, 5,5 -tetrachlorobiphenyl (2,2, 5,5 -TCB) is converted into the 3, 4 -epoxide by monooxygenase attack on the meta-para position, and rearrangement yields two monohydroxymetabolites with substitution in the meta and para positions (Sundstrom et al. 1976). The epoxide is also transformed into a dihydrodiol by epoxide hydrolase attack (see Chapter 2, Section 2.3.2.4). This latter conversion is inhibited by 3,3,3-trichloropropene-l,2-oxide (TCPO), thus providing strong confirmatory evidence for the formation of an unstable epoxide in the primary oxidative attack (Forgue et al. 1980). 

McMillan DC, PP Fu, CE Cerniglia (1987) Stereoselective fungal metabolism of 7,12-dimethylbenz[a]anthra cene identification and enantiomeric resolution of a K-region dihydrodiol. Appl Environ Microbiol 53 2560-2566. 

Sutherland JB, JP Freeman, AL Selby, PP Fu, DW Miller CE Cerniglia (1990) Stereoselective formation of a K-region dihydrodiol from phenanthrene by Streptomyces flavovirens. Arch Microbiol 154 260-266. 

Initially formed polar metabolites such as phenols and amines may be conjugated to water-soluble terminal metabolites that are excreted into the medium and function as an effective mechanism of detoxification. For example, pentachlorophenol and pentachlorothiophenol produced from pentachloronitrobenzene conjugated represented the major metabolites. Although the naphthalene dihydrodiol was the major metabolite produced from naphthalene, the further transformation... 

The aryl hydroxylase of Saccharomyces cerevisiae that transforms benzo[a]pyrene to the 3- and 9-hydroxy compounds, and the 7,8-dihydrodiol (King et al. 1984). 

Generally the di-dihydrodiols are dehydrogenated to the corresponding catechol. In some, dehydrogenation is not reqnired and elimination of CO2H, NHj, Hal, NOj", and SOj or exceptionally OH prodnces catechols directly. These are assigned to group II. 

Boyd DR, McMordie RAS, Porter HP, Dalton H, Jenkins RO, Howarth OW (1987) Metabolism of bicyclic aza-arenes by Pseudomonas to yield vicinal ci -dihydrodiols and phenols. J Chem Soc Chem Commun 1722-1724. 

The bacterial aerobic degradation of pyrene is initiated by the formation of cfi-pyrene-4,5-dihydrodiol. Analysis for this metabolite was used to demonstrate the biodegradability of pyrene in an environment in which there was continuous input of the substrate, when it was not possible to use any diminution in its concentration as evidence for biodegradation (Li et al. 1996). The corresponding metabolite from naphthalene—cfi-naphthalene-1,2-dihydrodiol—has been used to demonstrate biodegradation of naphthalene both in site-derived enrichment cultures and in leachate from the contaminated site (Wilson and Madsen 1996). 

The use of H-labeled substrates has been used to determine details of the dehydrogenation of ciT-dihydrodiols produced by dioxygenases from aromatic substrates (Morawski et al. 1997), and it was possible to demonstrate the specificity of hydrogen transfer from the dihydrodiol substrates to NAD. 

For dihydrodiols derived from substituted benzenes, the key to their significance lies in the availability of two adjacent chiral centers with an established absolute stereochemistry. The dihydrodiol from benzene is, of conrse, the meso compound, although enantiomers produced by subsequent reaction with a chiral reagent are readily separated. There are useful reviews containing nnmerous applications (Carless 1992 Ribbons et al. 1989), many of which involve, in addition, the nse of di-flnoro-, di-chloro-, or di-bromobenzene-2,3-dihydrodiols. 

Only a few illnstrative syntheses nsing benzene and toluene di-dihydrodiols are given below ... 

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). 

Racemic pinitol from benzene di-dihydrodiol benzoate by snccessive epoxidation and osmylation (Fignre 8.7a) (Ley et al. 1987). 

Laminitol from toluene di-dihydrodiol by snccessive epoxidations (Figure 8.7c) (Carless and Oak 1991). 

Analogs of conduritols from toluene cA-dihydrodiol by reaction with singlet oxygen followed by scission with thiourea (Figure 8.7d) (Carless et al. 1989). 

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.)... 

There is clearly enormous potential using other cis dihydrodiols produced from benzocyclo-alkenes, or from the numerous dihydrodiols produced from polycyclic carbocyclic and heterocyclic substrates. 

Boyd DR, ND Sharma, R Boyle, RAS McMordie, J Chuna, H Dalton (1992) A h NMR method for the determination of enantiomeric excess and absolute configuration of ciT-dihydrodiol metabolites of polycyclic arenes and heteroarenes. Tetrahedron Lett 33 1241-1244. 

Rogers JE, DT Gibson (1977) Purification and properties of cw-toluene dihydrodiol dehydrogenase from Pseudomonas putida. J Bacterial 130 1117-1124. 

Ziffer H, K Kabuta, DT Gibson, VM Kobal, DM Jerina (1977) The absolute stereochemistry of several cis dihydrodiols microbially produced from substituted benzenes. Tetrahedron 33 2491-2496. 

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