Microbial arene oxidation

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Microbial arene oxidation (MAO) refers to the process by which microbial enzymes convert aromatic compounds into more highly oxidized products. Although a number of oxidized products are possible, the most commonly employed for organic synthesis are cis-1,2-dihydroxy-cyclohexa-3,5-dienes ("dihydrodiols").[1]

Introduction

Microbial enzymes are able to catalyze chemical transformations that would otherwise be prohibitively slow or require harsh conditions. The oxidation of aromatic compounds to dearomatized products is an example of one such transformation, and the dihydrodiol products that result are useful synthetic intermediates. Since a seminal study by Gibson of Pseudomonas putida in 1968,[2] four classes of enzymes have been identified that accomplish arene oxidation to dihydrodiols:

  • Toluene dioxygenases (TDs)
  • Naphthalene dioxygenases (NDs)
  • Biphenyl dioxygenases (BPDs)
  • Benzoic acid dioxygenases (BZDs)

The substrate specificity of these enzymes is lower than their names suggest. Enantiomeric purities in excess of 90% are regularly obtained for reactions of prochiral substrates (although enantiomeric purity has rarely bben measured for these reactions, and homochirality of the products is often assumed). However, accessing the "unnatural" enantiomer of product is often difficult without tailored enzymes.

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Mechanism and Stereochemistry

Prevailing Mechanism

Oxidations by bacterial dioxygenases stereospecifically give cis-dihydrodiols. This outcome sets the mechanism of bacterial oxidation apart from mammalian and fungal versions of the process, which yield trans-dihydrodiols.[3] The cis configuration of the product together with isotopic labeling studies implicate a dioxetane intermediate;[4] however this intermediate has not been observed directly.

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Stereochemical Model

A reliable model has been developed that accounts for the stereo- and site selectivity of the reaction.[5] With the large substituent of the arene pointing up and other substituents pointed leftward, approach of dioxygen occurs to the top face of the arene, on the right-hand side. This model breaks down for some highly substituted substrates, such as phenanthrene and 2-naphthalenes, and does not apply to BZDs.

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Scope and Limitations

Toluene dioxygenase oxidizes toluene to 1,2-dihydroxyl-6-methylcyclohexa-3,5-diene.[6] Aromatic esters are also good substrates for these enzymes, and give dihydrodiols in moderate yields (along with some other oxidation products; see equation (8) below).

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Naphthalene dioxygenase is found in a variety of Pseudomonas organisms. It catalyzes the oxidation of other polyclic aromatic compounds as well, although yields tend to be low for substrates other than naphthalene.[4]

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Biphenyl dioxygenase oxidizes a relatively wide array of aromatic substrates and exhibits low substrate specificity.[7] Biphenyl oxidation can also be accomplished using TDOs or NDOs.

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The site selectivity of BZDs differs from that of the other three classes. Oxidation takes place in an ipso-cis fashion, independent of the substitution pattern of the arene.[8]

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Undesirable oxidized side products are often observed during microbial arene oxidations, particularly for "unnatural" substrates. Benzylic oxidation has been noted in a number of cases. Sulfides are always oxidized to sulfoxides.[9]

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An important limitation of the reaction is that only a single enantiomer of product is available when the wild type enzyme is used. Enzymes that generate "unnatural" enantiomers must be engineered via site-directed mutagenesis or other biochemical techniques. The development of organisms and enzymes that exhibit "unnatural" stereoselectivity is an ongoing research activity.[10]

Synthetic Applications

Because of concerns about the efficiency and selectivity of oxidation of more complex substrates, MAO is usually carried out early in synthetic sequences. However, simple dihydrodiols may be manipulated to give complex products through a variety of methods. In addition, the microbial oxidation process is compatible with a number of functional groups.

For instance, thioether-containing dihydrodiols may be accessed by the oxidation of iodobenzene followed by cross-coupling in the presence of tin sulfides.[11]

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Dihydrodiols have been elaborated to a variety of alkaloid natural products. Two examples are shown below.[12][13]

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Experimental Conditions and Procedure

Typical Conditions

The conditions of MAO reactions require the use of specialized techniques for growing and handling microbes in an aseptic environment. Often, specialized bacterial strains will be needed to effect particular transformations, and these can usually only be obtained from the laboratories in which they were first cultured. Techniques for the preparation of bacterial media and handling of microbes are well documented in the biochemical literature.

Dihydrodiols themselves must be stored under basic conditions (pH > 9) to keep them from undergoing acid-catalyzed dehydration. A convenient procedure for preparing diols for storage involves centrifugation of the crude reaction mixture followed by adjustment of the supernatant pH and concentration. Despite the acidity issues that benzoic acid dihydrodiols present, they have been isolated and characterized and may be stored as salts.

Example Procedure[14]

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A flask of inoculum (50 mL in a 250-mL flask) of Pseudomonas putida 39/D was prepared in advance of the fermentation. Mineral salt broth (MSB) medium (0.5 L) and L-arginine hydrochloride (10 g) were placed in a 2.8-L Fernbach flask fitted with an air inlet tube and a vapor bulb extended through the closure of the flask mouth. The flask, fittings, and contents were sterilized in an autoclave. After cooling, the previously prepared flask of inoculum was transferred to the Fernbach flask using aseptic technique. The vapor bulb was charged with chlorobenzene (10 mL), and the flask was shaken on a rotary shaker at 150 rpm and 30° for 48 hours. The vapor bulb with excess chlorobenzene was removed, and the pH of the aqueous contents of the flask was measured and adjusted to pH ~9 if necessary. The aqueous mixture was divided equally into centrifuge tubes and the solids were separated by centrifugation for 30 minutes at ~8,000 rpm. The aqueous supernatant was decanted, combined, saturated with NaCl, and extracted with EtOAc (4 × 100 mL). The combined organic extracts were dried (Na2SO4 or MgSO4), filtered, and concentrated under reduced pressure, giving 190 mg of a tan colored solid. Centrifugation was used to aid breaking of any emulsions observed with the organic extracts. Recrystallization of the solid from CH2Cl2-hexane gave an off-white solid, 0.160 g, mp 82–84°; 1H NMR (CDCl3) δ 6.12 (m, 1H), 5.87 (m, 2H), 4.48 (m, 1H), 4.19 (t, J = 7.3 Hz, 1H), 2.74 (d, J = 7.3 Hz, 1H), 2.63 (d, J = 8.4 Hz, 1H); 13C NMR (CDCl3) δ 134.9 (C), 128.0 (CH), 123.4 (CH), 122.7 (CH), 71.4 (CH), 69.1 (CH).

References

  1. Johnson, R. A. Org. React. 2004, 63, 117. doi: (10.1002/0471264180.or063.02)
  2. Gibson, D. T.; Koch, J. R.; Kallio, R. E. Biochemistry 1968, 7, 2653
  3. Walker, N.; Wiltshire, G. H. J. Gen. Microbiol. 1953, 8, 273.
  4. a b Jeffrey, A. M.; Yeh, H. J. C.; Jerina, D. M.; Patel, T. R.; Davey, J. F.; Gibson, D. T. Biochemistry 1975, 14, 575.
  5. Boyd, D. R.; Sharma, N. D.; Hand, M. V.; Groocock, M. R.; Kerley, N. A.; Dalton, H.; Chima, J.; Sheldrake, G. N. J. Chem. Soc., Chem. Commun. 1993, 974.
  6. Gibson, D. T.; Hensley, M.; Yoshioka, H.; Mabry, T. J. Biochemistry 1970, 9, 1626.
  7. Gibson, D. T.; Roberts, R. L.; Wells, M. C.; Kobal, V. M. Biochem. Biophys. Res. Commun. 1973, 50, 211.
  8. Knackmuss, H.-J.; Beckmann, W.; Otting, W. Angew. Chem., Int. Ed. Engl. 1976, 15, 549.
  9. Boyd, D. R.; McMordie, R. A. S.; Sharma, N. D.; Dalton, H.; Williams, P.; Jenkins, R. O. J. Chem. Soc., Chem. Commun. 1989, 339.
  10. Yu, C.-L.; Parales, R. E.; Gibson, D. T. J. Indust. Microbiol. Biotech. 2001, 27, 94.
  11. Boyd, D. R.; Hand, M. V.; Sharma, N. D.; Chima, J.; Dalton, H.; Sheldrake, G. N. J. Chem. Soc., Chem. Commun. 1991, 1630.
  12. Butora, G.; Hudlicky, T.; Fearnley, S. P.; Gum, A. G.; Stabile, M. R.; Abboud, K. Tetrahedron Lett. 1996, 37, 8155.
  13. Gonzalez, D.; Martinot, T.; Hudlicky, T. Tetrahedron Lett. 1999, 40, 3077.
  14. Hudlicky, T.; Stabile, M. R.; Gibson, D. T.; Whited, G. M. Org. Synth. 1999, 76, 77.