| dc.description.abstract | Exploration of microbes in carrying out transformations of natural products has been an important and fascinating method in synthetic organic chemistry. Microbes offer the opportunity to effect selective transformations-a feature of great utility in organic syntheses-that otherwise would be difficult to carry out by conventional chemical methods. It is now becoming widely appreciated that, compared with chemical methods, reactions catalyzed by microbes often offer significant advantages including efficiency, regiospecificity, stereoselectivity, etc. The conditions under which microbial reactions take place are mild and hence compounds which are sensitive to heat, acid and base become amenable to such transformations. Thus, microbes serve as an important tool in the hands of organic chemists to achieve selective transformations of complex natural products.
Considerable progress has been made in the microbial transformations of various classes of organic compounds such as steroids, carbohydrates and aromatic compounds. Although acyclic terpenes are one of the widely distributed classes of natural products, progress in the field of microbial transformations of these compounds lagged behind considerably as compared to the advances made with regard to other classes of compounds. This is partly due to their antimicrobial nature and due to the experimental difficulties involved in working with these volatile substrates.
The main aim of the present investigation was:
To find a suitable fungal system for carrying out the selective oxidation of various acyclic isoprenoids and to investigate the enzyme system(s) involved in the transformation.
To explore the possibility of transforming acyclic terpenes by microbial means into useful intermediates and to study some of the properties of the corresponding enzymes after purification.
Earlier, we isolated a strain of Aspergillus niger from garden soil which was found to carry out regiospecific methyl hydroxylation of acyclic monoterpene alcohols. Since methyl hydroxylation is one of the important reactions in organic synthesis, we explored the potential of this fungal system to effect such a selective transformation in the case of higher acyclic isoprenoids. To ascertain the versatility and specificity of the organism, we subjected nerolidol, farnesol and several synthetically modified acyclic isoprenoids (I–VII) to biotransformation using Aspergillus niger. Large scale fermentations were carried out in each case. The metabolites formed were isolated and purified by chromatographic methods. Characterization was performed by conventional spectroscopic methods (IR, UV, NMR & GC MS) and further confirmation of the structure was done by comparison with literature data and, in some cases, by chemical synthesis.
When higher acyclic isoprenoids were used as substrates, the organism showed its unique ability to carry out the oxidation of not only the methyl group but also the remote double bond (Chart 1). However, these two activities appear to have preferential structural requirements; the ratio of the products formed depends upon the nature of the R group. Oxidation of the remote double bond in compounds I to VII resulted in the formation of optically active vicinal diols (IB to VIIB). Besides, the organism also carried out asymmetric reduction of VI to optically active V. In the case of III, small amounts of IIIB were also isolated, whereas VI gave minor amounts of VIA and VIB in addition to the other hydroxylated products. An attempt was made to study the enzyme system involved in these selective hydroxylation reactions, but the activity could not be detected at the cell free level. All the aforementioned studies constitute PART I of the thesis.
PART II of the thesis deals with the metabolism of nerolidol and related terpenoids using Alcaligenes eutrophus. As compared to the extensive work accomplished in the field of microbial transformations of monoterpenes, only limited data are available on the microbial metabolism of sesquiterpenes. Most of the reports in the literature show that sesquiterpenes as a group yield relatively fewer products than monoterpenes. While this factor ought to have made the study of their microbial metabolism easier, the problem of getting an organism to grow on sesquiterpene substrates seems to be a major deterrent. Hence, in the present investigation, the study of the bacterial metabolism of the acyclic sesquiterpene alcohol nerolidol was undertaken.
In an attempt to isolate an organism which can grow on nerolidol as sole source of carbon and energy, a bacterial strain identified as Alcaligenes eutrophus was isolated from garden soil by enrichment culture technique. Large scale fermentation of nerolidol was carried out using this strain. Degradation of nerolidol (1) by A. eutrophus yielded two major and a couple of very minor metabolites. The major metabolites formed have been identified as geranylacetone (2) and an optically active alcohol, (S)-(+)-geranylacetol (3). The characterization was based on spectral data and also direct comparison of the metabolite with authentic samples. On the basis of the metabolites isolated, a new pathway for the formation of 3 from 1 was proposed, involving epoxidation of the 1,2 double bond of 1 to 4 followed by hydrolysis to yield a triol (5). Then cleavage of the C2–C3 bond leads to the formation of 2 and glycolaldehyde (6). Compound 2 and glycolaldehyde (6), upon reduction, afforded 3 and ethylene glycol (7), respectively (Chart 2). Further support for the proposed pathway comes from the in vivo transformation of synthetically prepared probable intermediates (4) and (5) to 2 and also from manometric studies which strongly support the postulated pathway. However, the cell free extract prepared from nerolidol grown A. eutrophus failed to convert 1, 4 and 5 to 2 in the presence of an exogenous cofactor. To the best of our knowledge, this is the first report on the bacterial degradation of nerolidol.
Our studies on the bacterial degradation of nerolidol revealed the ability of the organism to carry out the asymmetric reduction of geranylacetone (2). We were interested to purify, characterize and establish the substrate specificity of this enzyme. This constitutes the subject matter of the next chapter.
The concluding portion (PART III) of the thesis describes the enzyme purification and characterization. The cell free extract (105,000 × g supernatant) prepared from nerolidol grown Alcaligenes eutrophus converted 2 to an optically active alcohol. | |
