Australasian Biotechnology, Vol. 11 No. 2, 2001, pp. 26-28
BIOPROCESSINGMICROBIAL BIOTRANSFORMATIONS: STEREOSELECTIVE SYNTHESIS OF PHARMACEUTICAL DRUG PRECURSORS
Shafiq Ahmad, Kylie Henderson, Geoff Dumsday and Michael Zachariou
Code Number: au01026
Biotransformations are becoming increasingly popular in the production of enantio- and regiopure- intermediates for synthesis of complex organic compounds. Whole microbial cells or enzymes can be used to carry out such specific chemical reactions that are otherwise difficult to achieve synthetically. Chemoenzymatic synthesis is of particular interest in the pharmaceutical industry where some steps can be carried out enzymatically to obtain chiral synthons for further enzymatic/chemical processing. In this report, we discuss some recent developments in the field together with a few examples.
A key area of industrial pharmaceutical research is the search for selective enzyme inhibitors and receptors. Improved knowledge of molecular interactions has led to an increased understanding of the importance of chirality to the efficacy of many products. For instance, in a chiral mixture, only one isomer may be biologically active whereas the other stereoisomer may be responsible for side effects. In an attempt to avoid such side effects the pharmaceutical industry is directing much effort in developing homochiral drugs.
Chiral compounds have at least one stereogenic (asymmetric) carbon atom, which means the molecule can exist as two different stereoisomers and which may have different biological activity. A readily available pool of chiral precursors is necessary and needs to be inexpensive and optically active. Chiral precursors can be prepared by different routes. One route is to obtain them from naturally occurring chiral synthons. A second route is to obtain them by racemic resolution of compounds, which can be either carried out by preferential crystallization or by kinetic resolution using chemical or microbial means. Finally, the chiral synthons can also be prepared by asymmetric synthesis using chemical or microbial systems. The advantages of microbial systems over chemical synthesis are numerous such as stereoselectivity of enzymes and reactions can be performed at ambient temperature and atmospheric pressure. Such advantages help minimize problems such as isomerisation, racemisation, epimerisation and rearrangement that may occur during chemical processing. Furthermore, microbial biotransformations reduce the requirement of harmful chemicals whilst introducing the possibility of reusing microbial cells/enzymes.
This report will highlight some of the latest developments and examples of stereoselective biotransformation to preparation of chiral precursors for chemical or biological synthesis of pharmaceuticals. For more detailed information the reader is referred to a number of recent reviews on the topic of stereoselective biotransformation (Johnson and Wells 1998; Andreas et al., 1999; Ward and Singh 2000; Azerad and Buisson 2000).
STEROIDS AS CHIRAL SYNTHONS
Steroids are widely used as anti-inflammatory, diuretic, anabolic, contraceptive and anticancer agents some of which have stereoselective requirements. Chemical modification of the steroid ring structure imposes many problems due to steric hindrance and the complexity of the molecule. An alternative approach that obviates the need for chemical modification of the pharmaceutically active steroids has been to use microbial systems (Ahmad et al., 1992) which dates back to the early 1950s (Murray and Peterson 1952). For example, microbial sterol side-chain cleavage for production of steroid drug precursors, such as the cyclopentanoper-hydrophenanthrene ring structure, from sterol raw materials such as cholesterol and stigmasterol, are well known and documented (Mahato and Banerjee 1985; Ahmad et al., 1991; 1992; Ahmad and Johri 1993; Naito 2000) (Fig 1.). This process (Fig 1) has always competed with chemical side-chain cleavage of steroids, however microbial processes (most of them are patented) are preferred because of their stereoselectivity. The products of partial or complete side-chain degradation are valuable for microbial or chemical synthesis of most of the steroid drugs or hormones.
Another important process for development of bioactive steroids is, stereoselective hydroxylations such as 11α-, 11ß- and 16α-hydroxylation, all of which are now exclusively achieved by microbial means (Mahato and Garai 1997). Yoshioka and Asada (1994) patented a process for 14α-hydroxylation of androst-4-ene-3, 17-dione (AD) (Fig. 1). A potent inhibitor of breast cancer cells 6ß, 14α-dihydroxy-androst-4-ene-3, 17-dione was achieved by microbial transformation of AD by the genus Myrothecium (Yoshioka et al., 1994). A novel steroid drug which has demonstrated strong antitumor (ovarian) activity 14α-hydroxyandrost-4-ene-3, 6, 17-trione was also produced by oxidation of 6ß, 14α-Dihydroxyandrost-4-ene-3, 17-dione (Yoshihama 1993) (Fig. 1).
The regio- and stereoselective hydroxylation of progesterone catalyzed by Rhizopus nigricans is a key step in the manufacture of steroid drugs with a market value of more then one billion dollars (Mahato and Garai 1997) (Fig. 1). Another approach used in the hydroxylation of progesterone may be the use of 16α-progesteronehydroxylase from Streptomyces roseochromogenes NCIB 10984 (Barrie et al., 1999). The enzyme could be tested on other steroid ring structures as well.
Figure 1. Microbial sterol side-chain degradation and stereoselective oxidation.
Figure 2. Enantioselective hydroxylation of aliphatic caboxylic acids.
ALIPHATIC CARBOXYLIC ACIDS AS CHIRAL SYNTHONS
An example of microbial biotransformation of aliphatic carboxylic acids to produce a chiral synthon is the regio- and enantioselective ß-hydroxylation performed by Candida rugosa and C. parapsitosis. (R)-ß-Hydroxyisobutyric acid produced using this reaction is chiral synthon in the chemical synthesis of antihypertensive drugs. All angiotensin converting enzyme (ACE) inhibitors (antihypertensive drugs) are synthetic and chiral molecules and without exception are marketed as a single isomer. For instance, perindopril has five asymmetric centres and is marketed as one of 32 (25) possible isomers (Fig. 2).
CYCLOHEXADIENE-CIS-DIOLS AS CHIRAL SYNTHONS
The capacity to functionalize cis-diols chemically at every position in a stereocontrolled manner has served to produce enantiopure precursors, such as cyclohexadiene-cis-diols, of pharmaceutically important organic compounds (Fig. 3) (Hudlicky and Thorpe (1996). Stereoselective synthesis of pancratistatin (antitumor agent) using cyclohexadiene-cis-diol as a precursor has been described (Hudlicky et al., 1996).
Figure 3. Dioxygenase mediated cis-hydroxylation.
Gibson et al. (1968) reported dioxygenase mediated cis-hydroxylation of aromatic compounds to cyclohexadiene-cis-diols by Pseudomonas putida. The dioxygenase gene was cloned in to Escherichia coli to stop further metabolization of cis-diols and to over produce the enzyme. (Fig. 3). Recently, an interesting strain of Burkhoderia sp. was reported to utilize a wide range of carbon sources including toluene, cresol and a range of alkanes ranging in length from C8 to C25 (Ma and Herson 2000). The catechol 2,3-dioxygenase gene (catB) was also cloned and sequenced from this interesting strain (Ma and Herson 2000). This enzyme may have a different substrate specificity in a different reaction environment.
GENERATION OF CHIRAL SYNTHONS FROM RACEMIC SOLUTIONS USING BIOTRANSFORMATION
One of the major routes of chiral precursors for pharmaceutical drug synthesis is microbial or enzymatic resolution of racemic mixtures derived either from natural or chemical sources. A commercial mixture of nitropentanol isomers was separated using Hansenula subpelliculosa (ATCC 16766) (Morgan et al., 1999). Nitropentanol isomers are used in chemical synthesis of azole antifungal compounds. Isopropyl amine and 1,2-isopropylideneglycerol isomers were produced using Pseudomonas sp. and P. putida respectively (Fig.4A). Isopropylideneglycerol particularly R isomer is an industrially valuable precursor for a number of pharmaceutically active drugs, such as ß-blockers, antiviral agents and thromboxane synthase inhibitor. Isopropylamine is converted to an optically active L-alaninol, which is a precursor to an important antibacterial agent (Fig. 4B).
Figure 4. Enantioselective microbial oxidation.
A chemically difficult reaction to carry out is the Baeyer-Villinger (BV) oxidation of linear or cyclic ketones. The oxidation of 3-phenylcyclobutanone to γ-butyrolactone was achieved in a single step with the fungal strain Cunninghamella echinulata NRRL 3655 (Alphand and Furstoss 2000) (Fig. 5). An enantioselective synthesis of baclofen (muscle relaxant) has been described using optically active γ-butyrolactone (Mazzini et al., 1997).
Figure 5. Regioselective oxidation of ketones and resolution.
A number of new applications of stereoselective biotransformations are appearing in the literature. A small sample of these have been presented in this report to represent a form of bioprocessing used for many decades. This type of bioprocessing is finding an increasing use in the pharmaceutical industry as it strives for lower levels of toxicity in its products and, also, in the chemical industry as it begins to redefine itself as a more environmentally friendly and efficient sector. The ability of the ubiquitous and versatile microbe to rapidly adapt to changing environments has now made it a target as a synthetic tool for production of novel building blocks.
Microbial resolution of racemic mixtures of chemical precursors or final products has already been established. Microbial transformation or biocatalysis has proven to be a valuable tool in production of pharmaceutical intermediates, in particular enantiomerically pure compounds. In some of the cases, solubility of the reactants and or products in the reaction mixture employing enzymes or microbial cells remains a critical problem to be solved. Furthermore, stability of microbial enzyme(s) could be a problem in the chemical environment suited for chemical reaction.
Developments in microbial biotransformation technology, including new and more stable enzymes, improvement of existing enzyme systems using site-directed mutagenesis and search for novel microorganisms with desired enzymatic properties will further advance the application of chemoenzymatic synthesis in organic chemistry.
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