values were determined by a combination of methods to show their enantiopurity. The cis‐dihydroxylation of meta‐substituted phenols (m‐phenols) catalyzed by TDO, an arene dioxygenase from mutant strain of P. putida UV4, yields the corresponding cyclohexenone cis‐diol metabolites and several of their cyclohexene and cyclohexane cis‐triol derivatives [68]. Using the same bacterium strain and p‐xylene as the substrate, the major biotransformation product was the corresponding cis‐1,2‐dihydrodiol (95% relative yield) along with the two minor products, catechol (1% relative yield) and cyclohexenone cis‐diol (4% relative yield) via the intermediate p‐xylenol (Scheme 2.17). Another metabolite of p‐xylenol with TDO present in P. putida UV4 was o‐quinol dimmer (Scheme 2.17) [69]. However, p‐xylene was first catalyzed to cis‐p‐xylene dihydrodiol and further undergone dehydration to give 2,5‐dimethylhydroquinone by a novel o‐xylene dioxygenase from Rhodococcus sp. strain DK17 [70]. cis‐Dihydroxylation of aromatic carboxylic acid such as benzoic acid with mutants of Alcaligenes eutrophus B9 or P. putida U103 produces a diol containing a tertiary alcohol moiety is another interesting case for enantioselective synthesis, which demonstrates two stereogenic centers can be introduced simultaneously during organic synthesis [71]. A series of benzoate esters have been studied for the enzymatic dihydroxylation by whole‐cell fermentation with E. coli JM109 (pDTG601) to produce their corresponding cis‐cyclohexadienediols except for n‐butyl and t‐butyl benzoates [72]. The diols obtained from the enzymatic dihydroxylation of benzoate esters serve as intermediates for the synthesis of pseudo‐sugars, amino cyclitols, and bicyclic ring systems.
Scheme 2.15 Chemoenzymatic preparation of pancratistatin analogs.
Scheme 2.16 Whole‐cell fermentation of methyl 2‐iodobenzoate for organic synthesis.
Naphthalene dioxygenase (NDO), a Rieske dioxygenase [73, 74], from Pseudomonas strain NCIB9816‐4 catalyzes the cis‐hydroxylation of naphthalene to cis‐(1R,2S)‐dihydroxy‐1,2‐dihydronaphthalene (naphthalene cis‐dihydrodiol) (Scheme 2.18) [75, 76]. Incubation of naphthalene with E. coli BL21(DE3) harbored with a recombinant expression plasmid of the Rhodococcus sp. strain DK17 o‐xylene dioxygenase also produces cis‐1,2‐naphthalene dihydrodiol [70]. In the same manner, the recombinant E. coli JM109(DE3)(pDTG141) expressing the NDO from Pseudomonas sp. NCIB9616‐4 has been used for the whole‐cell biotransformation of a series of azaarene compounds. The study showed that several bicyclic azaarenes can be catalyzed well by NDO to give cis‐dihydrodiol derivatives [77]. In addition, the DK17 o‐xylene dioxygenase catalyzes the cis‐dihydroxylation of biphenyl to form cis‐2,3‐biphenyl dihydrodiol (Scheme 2.19) [70]. BPDOs from mutant bacterial strains that lack the corresponding diol dehydrogenase enzymes can be utilized to transform polycyclic arenas such as anthracene and benz[a]anthracene to cis‐hydrodiols and acetonide derivatives of the cis‐dihydrodiols of biphenyl and phenanthrene to enantiopure bis(cis‐dihydrodiol) metabolites [78]. The produced cis‐diols in turn have been widely used for organic synthesis.
Scheme 2.17 Toluene dioxygenase catalyzed cis‐dihydroxylation of phenols toward catechol, cyclohexenone cis‐diol, and o‐quinol dimmer metabolites.
Scheme 2.18 Naphthalene dioxygenase catalyzed cis‐dihydroxylation of naphthalene.
Scheme 2.19 Regioselective oxidation of biphenyl by the Rhodococcus sp. DK17 o‐xylene dioxygenase.
2.1.5 Epoxidation
The cytochromes P450 (CYPs) catalyzed a wide variety of reactions including the oxidation of alkenes to form reactive epoxides. The oxidations of propene, cyclohexene, and styrene for producing their corresponding epoxides have been reported by P450cam from P. putida and P450 BM‐3 variant 139‐3 [79, 80]. A mutant, T252A P450cam, prepared from the water‐soluble camphor‐hydroxylating P450cam of P. putida has been used to catalyze the epoxidation of more complex 1R‐camphor [81]. CYP153 enzymes expressed in P. putida GPo12 also showed the ability of epoxidation for styrene, octene, and cyclohexene in a large scale [40]. Except for one CYP153 produced (R)‐epoxide, others yielded (S)‐epoxide with up to 80% e.e. The CYP450 was further used for the epoxidation of tea tree oil ingredient p‐cymene to form thymol by a postulated enzymatic reaction mechanism that involves the epoxidation of one of the π‐double bonds in the benzene moiety followed by an National Institutes of Health (NIH) shift as shown in Scheme 2.20 [82]. Dihydrochalcomycin (DHC) is a 16‐membered macrolide antibiotic that is clinically applied in the treatment of bacterial infections against gram‐positive as well as gram‐negative bacteria except against E. coli. Two CYP450 enzymes, gerPI and gerPII, were characterized for the hydroxylation at the C8 position by the gerPII P450 followed by the epoxidation reaction by gerPI P450 at the C12–C13 position in the biosynthetic pathway for DHC from Streptomyces sp. KCTC 0041BP [83].
Scheme 2.20 Postulated reaction mechanism for the formation of thymol from p‐cymene through arene epoxidation.
Scheme 2.21 The epoxidation reaction catalyzed by squalene epoxidase (SE).
Squalene monooxygenase (squalene epoxidase, SE, E.C. 1.14.99.7) converts the squalene by regio‐ and stereospecifically forming an epoxide on the C–C double bond to yield (3S)2,3‐oxidosqualene that requires molecular oxygen, FAD, and depending on the organism, either
