Biotransformation of Steroids and alkaloids.pdf

Biotransformation of Steroids and alkaloids Steroids (stereos = solids) are organic compounds derived from alcohols, which are widely distributed in the animal and plant kingdoms. Their base skeleton has 17 carbon atoms in a tetracyclic ring system known as cyclopentanoperhydrophenanthrenes (gonane...

Biotransformation of Steroids and alkaloids Steroids (stereos = solids) are organic compounds derived from alcohols, which are widely distributed in the animal and plant kingdoms. Their base skeleton has 17 carbon atoms in a tetracyclic ring system known as cyclopentanoperhydrophenanthrenes (gonane and estrane). Sterols are hydroxylated steroids – that is, they are alcohols derived from steroids. The hydroxyl (OH) group of sterols is usually substituted at position C3. In this group of substances, life-vital compounds are categorized, such as cholesterol, bile acids, sex hormones, vitamin D, corticosteroids, cardiac aglycones, and antibiotics, among others. Some of the most potent toxins are steroid alkaloids. Steroids are responsible for important biological functions in the cell; for example, the steroids derived from androstane, pregnane, and estrane have hormonal activity; bile acids are important for the digestion and absorption of fats; and cardiotonic aglycones are used for the treatment of heart disease. Sterols are constituents of the cell membrane, essential for cell stability and development; also, they are precursors of bile acids and steroid hormones. The physiological activity of steroids depends on their structure, the type, number, spatial orientation, and reactivity of the different functional groups present in the tetracyclic core as well as the oxidation state of the rings. For example, the presence of an oxygenated function in C-11β is crucial for the anti-inflammatory activity; the hydroxyl function in C-17β determines androgenic properties; the aromatization of ring A confers estrogenic effect; and corticosteroids have the 3-keto-4-ene group and the pregnane side chain at C-17. The introduction of a hydroxyl group “biohydroxylation” in the steroid skeleton is an important step in the synthesis of new steroids used physiologically as hormones and active drugs. Microbiological transformations are an effective tool for the preparation of various compounds, which can be difficult to obtain by conventional chemical methods and have been widely used in the bioconversion of steroids. In 1950, the pharmacological effects of cortisol and progesterone were reported, in addition to the hydroxylation of the latter in C- 11α using Rhizopus species. This began a very important stage in the development of the synthesis of steroids with biological activity. The microbiological transformations of steroids have been an essential chemical tool used for the preparation of many intermediaries and in the generation of new drugs, where chemical functionalization-hydroxylation, Baeyer-Villiger oxidation, reduction, isomerization, Michael additions, and condensation reactions can be carried out in different positions of the steroid skeleton in chemo-, regio-, and stereoselective ways, being very complicated or even impossible by the classic chemical methods. Manufacture of steroids In 1937, the first microbial transformation of steroids was carried out. Testerone was produced from dehydroepiandrosterone using Corynebacterium sp. Subsequently, cholesterol was produced from 4-dehydroeticholanic acid and 7-hydroxycholesterol using Nocardia spp. These developments were virtually unexploited until 1949, when the dramatic curative effect of cortisone on rheumatoid arthritis, a disease in which painful swellings occur at the joints of the body, was announced. The cortisone used in this work had been prepared by complex and tedious chemical synthesis beginning with deoxycholic acid, a bile acid. So tedious was this that it took 32 chemical steps and two years to produce only 11 gm of cortisone acetate. Additionally, it was difficult to find enough of the starting materials. The use of biological agents to transform readily available steroids by introducing oxygen at carbon C11, a process which took 12 steps in chemical synthesis. The use of biological agents originally consisted of the use of ground or homogenized adrenal tissues and fungi. Two of the earliest such microbial transformations were the conversion of progesterone to 11– a hydroxy progesterone by the introduction of – OH at the position 11 using Rhizopus nigricans and the conversion of cortisol to prednisolone by Corynebacterium simplex. The microbial transformation of steroids differs from the „traditional‟ fermentations such as that of penicillin thus: (i) In many cases steroid transformations are one-step-processes which bring about relatively minor structural changes in the substrate, i.e. the steroid molecule. This differs from the synthesis of penicillin and many other fermentation products in which the product is synthesized entirely from the substrate offered in the medium. (ii) Whereas in much industrial fermentation, the process of production is completed in the fermentor, in the case of steroid transformations, readily available steroids are micro- biologically transformed into important intermediates which are then converted chemically to the final product. Alternately, the chemical syntheses are first performed and the products transformed microbiologically later. Types of microbial transformations in steroids and sterols Transformations by microorganisms affecting various positions in a wide range of steroids and sterols have been carried out. Although steroid hormones have been most widely studied, the transformation of bile acids, plant and animal sterols, steroid alkaloids have also occurred. The transformation reaction include: hydroxylation, dehydrogenation, reduction, side chain degradation, lactone formation, aromatization, isomerization, epoxidation, hydrolysis, esterification, halogenation, and cleavage of the steroid skeleton. Fermentation Conditions Used in Steroid Transformation The media used are highly variable, but in the main are not very complex. They are basically mineral salts media containing some carbon source such as glucose, dextrin or glycerol. Nitrogen sources may be ammonium salts, corn steep liquor, soybean, or a protein digest. In some cases yeast extract is added. Steroid and sterols are lipids; they are not water soluble and therefore must be dissolved in a water-miscible lipid-solvent. Acetone, ethanol, propylene glycol, and methanol are suitable because they dissolve a reasonable amount of the steroid while being relatively non-inhibitory to the enzymes; dimethyl formamide dissolves a reasonable amount of the steroids but has only a minimum of toxicity. Sometimes the steroid is added in small amounts at a time. In this way, any toxic effect of the solvent is minimized. The level of steroid added is variable and depends both on the transforming ability of the organisms as well as its susceptibility to the toxic effects of the steroid. Normally 200-800 mg/litre are added. To solve the problem of the insolubility of steroids in water, non-ionic surface-acting agents which reduce surface tension e.g. Tween 80 are often added to the medium. Some polysaccharides in the medium e.g. yeast cell wall mannan, bind to the steroids and cause them to be more available to the organism. A wide range of microorganisms, mainly fungi and bacteria, are used in the transformation of steroids. Some of these include the fungi Rhizopus nigricans, Curvularia lunata, Fusarium spp. Cylindrocarpon radicicola as well as the bacteria Mycobacterium spp., Corynedbacterium simplex, and Streptomyces spp. As has been mentioned, there are organisms to perform just about any conceivable transformation of the steroid molecule. The transformation may occur at different stages of the growth and the steroid may be added to the growing cultures either simultaneously with the inoculation of the culture or the resting or stationary stage of the organism. Fungal spores may sometimes be inoculated as the steroid is introduced into the medium. In recent times immobilized cells have been employed in the transformations of steroids. Reference: Michael J. Waites, Neil L. Morgan, John S. Rockey and Gary Higton. 2001. Industrial Microbiology: An Introduction. Blackwell Science Ltd., Paris, France. Industrial Microbiology: (By Casida L. E.New Age international (P) ltd publications) A Text Book of Industrial Microbiology: (2nd edition By Wulf Crueger & Anneliese Crueger) Manual of Industrial Microbiology and Biotechnology: (2nd Edition by Arnold L. Demain and Julian E. Davies, Ronald M. Atlas, Gerald Cohen, Charles L. Hershberger, Wei-Shou Hu, David H. Sherman, Richard C. Willson and J. H. David Wu) Industrial Microbiology-An introduction: By Michael J. Waites, Neil L. Morgan, John S. Rockey and Gary Higton) Comprehensive Biotechnology-The Principles, Applications and Rugulations of Biotechnology in Industry, Agriculture and Medicine: (By Mrray Moo Young) Fermentation Technology: Up Stream Fermentation Technology- Vol-I: (By H. A. Modi- Pointer Publications) Fermentation Technology: Down Stream Fermentation Technology- Vol-II: (By H. A. Modi- Pointer Publications)