Hydroxylation. microbial

Pleuromutilin (Scheme 17.9) is an antibiotic from Pleurotus or Clitopilus basidiomycete strains which kills mainly Gram-positive bacteria and mycoplasms. Metabolism of pleuromutilin and derivatives results in hydroxylation by microsomal cytochrome P-450 at the 2- or 8-position and inactivates the antibiotics. Modification of the 8-position of pleuromutilin and analogs was of interest as a means of preventing the metabolic hydroxylation. Microbial hydroxylation at the... 

Conventional chemical syntheses of metabolites are often difficult and can be time consuming e.g. remote hydroxylation. Microbial systems are particularly attractive where stereo or regiospecific transformations are required. 

Experimental procedures have been described in which the desired reactions have been carried out either by whole microbial cells or by enzymes (1—3). These involve carbohydrates (qv) (4,5) steroids (qv), sterols, and bile acids (6—11) nonsteroid cycHc compounds (12) ahcycHc and alkane hydroxylations (13—16) alkaloids (7,17,18) various pharmaceuticals (qv) (19—21), including antibiotics (19—24) and miscellaneous natural products (25—27). Reviews of the microbial oxidation of aUphatic and aromatic hydrocarbons (qv) (28), monoterpenes (29,30), pesticides (qv) (31,32), lignin (qv) (33,34), flavors and fragrances (35), and other organic molecules (8,12,36,37) have been pubflshed (see Enzyp applications, industrial Enzyt s in organic synthesis Elavors AND spices). 

A third advancement in microbial biotechnology of steroid production was the abiUty to introduce a 16a-hydroxyl group microbiologicaHy (163). Modifications of the liP-hydroxylation, 16a-hydroxylation 1,2-dehydrogenation microbial processes are used for the synthesis of hydrocortisone, prednisolone, triamcinolone, and other steroid pharmaceuticals. A few microbial transformations that have been used to manufacture steroids are Hsted in Table 1 (164). 

In another process, diosgenin is degraded to 16-dehydropregnenolone by chemical methods. Conversion of 16-dehydropregnenolone to 11-deoxycortisol (125) can be accompHshed in 11 chemical steps. These steps result in hydroxylations at C21 and C17, oxidation at C3, and to double-bond isomeri2ation (175). Microbial oxidation of (125) also produces cortisol (29). 

In the olivanic acid series of carbapenems the ( )-acetamidoethenyl grouping can be isomerised to the (Z)-isomer (19) (22) and reaction with hypobromous acid provides a bromohydrin that fragments to give a thiol of type (20) when R = H, SO H, or COCH. The thiol is not isolated but can react to provide new alkyl or alkenyl C-2 substituents (28). In the case of the nonsulfated olivanic acids, inversion of the stereochemistry at the 8(3)-hydroxyl group by way of a Mitsunobu reaction affords an entry to the 8(R)-thienamycin series (29). An alternative method for introducing new sulfur substituents makes use of a displacement reaction of a carbapenem (3)-oxide with a thiol (30). Microbial deacylation of the acylamino group in PS-5 (5) has... 

Biotransformations are carried out by either whole cells (microbial, plant, or animal) or by isolated enzymes. Both methods have advantages and disadvantages. In general, multistep transformations, such as hydroxylations of steroids, or the synthesis of amino acids, riboflavin, vitamins, and alkaloids that require the presence of several enzymes and cofactors are carried out by whole cells. Simple one- or two-step transformations, on the other hand, are usually carried out by isolated enzymes. Compared to fermentations, enzymatic reactions have a number of advantages including simple instmmentation reduced side reactions, easy control, and product isolation. 

Many examples of microbial hydroxylation of sterols/steroids have been reported. These hydroxylations usually involve mixed function oxidases which utilise molecular oxygen and cytochrome P-450. The reaction can be represented by ... 

A single crystallisation is unlikely to lead to the isolation of pure crystals. In practice the product recovered in this process contains about 90% 11 a-hydroxyprogeterone with low levels of other products (especially 5 a-pregnane-3,20-dione and 60, lla-dihydroxyprogesterone). An example of a manufacturer who uses microbial 11a hydroxylation is Upjohn progesterone is used as substrate. 

De Beyer A, F Lingens (1993) Microbial metabolism of quinoline and related compounds XVI. Quinaldine oxidoreductase from Arthrobacter spec. Rii 61a a molybdenum-containing enzyme catalysing the hydroxylation at C-4 of the heterocycle. Biol Chem Hoppe-Seyler 374 101-120. 

Archelas A, J-D Fonmeron, R Fnrstoss, M Cesario, C Pascard (1988) Microbial transformations. 8. First example of a highly enantioselective microbiological hydroxylation process J Amer Chem Soc 53 1797-1799. 

Watson GK, C Houghton, RB Cain (1974) Microbial metabolism of the pyridine ring. The hydroxylation of 4-hydroxypyridine to pyridine-3,4-diol (3,4-dihydroxypyridine) by 4-hydroxypyridine-3-hydroxylase. Biochem J 140 265-276. 

Smith RV, Rosazza JP (1974) Microbial models of mammalian metabolism. Aromatic hydroxylation. Arch Biochem Biophys 161(2) 551-558... 

Hartman RE et al. (1964) Microbial hydroxylation of indole alkaloids. Appl Microbiol 12 138-140... 

Peczyfiska-Czoch W et al. (1996) Microbial transformation of azacarbazoles X re-gioselective hydroxylation of 5,ll-dimethyl-5ff-indolo[2,3-l)]quinoline, a novel DNA topoisomerase 11 inhibitor, hy Rhizopus arrhizus. Biotechnol Lett 18(2) 123-128... 

MaursM et al. (1999) Microbial hydroxylation of natural drimenic lactones. Phytochemistry 52(2) 291-296... 

Lonza, for example, has commercialized processes for highly chemo- and regioselective microbial ring hydroxylation and side-chain oxidation of heteroaromatics (see Fig. 2.32 for examples) (Kiener, 1995, 1999). The pharmaceutical intermediate 5-methylpyrazine-2-carboxylic acid, for example, is manufactured by microbial oxidation of 2,5-dimethylpyrazine. Many conversions of the type shown in Fig. 2.32 would not be possible by conventional chemical means. 

Biotransformation with flasks can be used to make gram quantities of a desired product, as shown for the 21 -hydroxylation of epothilone B [75]. In cases when greater quantities of a metabolite are needed, microbial biotransformations can be carried out in a fermentor, which will allow better monitoring and control of fermentation conditions (such as pH, oxygen and glucose levels, etc.) for reaction optimization [76]. 

---