Studies of biosynthetic pathways

In the antraquinone part of the molecule, the only atom without a coupling partner was C2, which revealed the site of decarboxylation. Combined with the fact that the first ( western ) ring contained two and not three intact [13C2] units, 

Further examples of the use of INADEQUATE in biosynthetic studies include biosynthesis of naphthylisoquinoline alkaloids,69 blepharismin C, toxic pigments of the ciliate Blepharisma japonicum,70 lambertellols A and B and lambertellin and metabolites of Ascomycete fungi.71 

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The ultimate aim of a biosynthetic investigation is not only the identification of the intermediates involved in the elaboration of the final product but also the identification of the enzymes which catalyze the various transformations between these intermediates. A complete understanding of the biosynthesis of a particular metabolite would require investigation of the mechanism and regulation of each of the individual enzymes involved and the relationship of the pathway to other cellular processes. In this context it would be fair to say that with the exception of some primary pathways which we shall consider later, the ideal criteria mentioned above have not as yet been met for most natural products. The wide range of questions which must be answered, however, does raise the point that the study of biosynthetic pathways is a multidisciplinary exercise and that no single technique or approach can be envisaged which will answer all the questions. 

Table 5 reveals the products of substrates which have been deuterated in H20. In other cases deuterated enoates were reduced in H2O. The reductions in H20 lead to acylates with two chiral carbon atoms due to stereospecific deuteration of methylene groups. Most of these have been used in studies of biosynthetic pathways of natural products. For examples see (29,30). Reactions in H20 proceed usually slower than in H2O. The differences may be less than a factor 2 if optimal pH and p H values are determined and applied. The p H and pH value for optimal reaction rates are not the same (31). 

Recent developments in plant tissue culture and the need to gain control on bean quality and yield turns our aitemiou to this method as iinolhcr approach to solve some of the problems associated with the agronomic production of vanilla (5). Plant tissue culture can be used for three different objectives 1. micropropagaiion 2. production of secondary products and 3. study of biosynthetic pathways. 

Few areas of natural products chemistry have seen as many major advances in the study of biosynthetic pathways as have occurred in polyketide compounds. Birch and Donovan (1953) demonstrated that a wide range of structural types are derived from acetate (later shown to be acetate and malonate). In experiments witfi deuterated precursors, acetate serves preferentially as a starter unit for the formation of 6-methylsalicylic acid in Penicillium griseofulvum (Simpson, 1983). Thus, polyketides are derived from the same precursors as fatty acids and the initial step seems to be similar (Fig. 5.1). Extensive purifrcation of 6-methylsalicylate synthetase from Penicillium patulum has been performed. This enzyme system is distinct and separable from the co-occurring fatty acid synthetase and has a molecular weight approximately half that of the former enzyme. NADPH is required as a coenzyme for methylsalicylate synthetase from this source (O Hagan, 1990 Packter, 1980). 

Employment of mutants for the study of biosynthetic pathways is often complicated and is usually feasible only for microorganisms or plants in tissue culture. The mutant must be unable to grow in the absence of the products of the pathway of interest (Weiss and Edwards, 1981). 

The use of stable isotopes in the elucidation of pathways of biosynthesis is char-actised by two particular features. In many cases, NMR, and in particular C-NMR, has been the method of isotope detection employed. Furthermore, in many cases the use of radioactive isotopes and their ease of detection has been combined with the structural dehnition achievable by the use of stable isotopes in conjunction with NMR. Although much of the reported work has utilised carbon-13 in combination with carbon-14 labelling, there are, nevertheless many instances of the use of nitrogen-15 and oxygen-18 in the study of biosynthetic pathways. 

Synthesis is in an excellent state of health and constitutes a marvellous tool, providing us with substances with which we can test mechanistic aspects of biologically important processes. In the first part of the panel discussion I would like to tackle this role of organic synthesis as a tool. We need not discuss the synthesis of labelled substrates since these only require the modification or extension of existing syntheses. Nevertheless, I remind you that, for the study of biosynthetic pathways, total synthesis is often required to prepare putatative intermediates which display a specific label, a heavy isotope, at a give position. Discussions on previous days has made us all aware of the importance of total synthesis for the obtention of mimics of cofactors or enzymes. 

In principle, use of tissue cultures in well defined media under controlled conditions (illumination, temperature, etc.) represents a technique ideal for obtaining reproducible information on plant biochemistry, undisturbed by climatic and ecological variables. One important question is whether undifferentiated cells produce the same enzyme systems and the same secondary metabolites as intact plants. The answer probably depends on the compound(s) of interest and on the culture conditions. Recent work illustrates the importance of this research area, whose successes promise to provide new tools for the study of biosynthetic pathways leading to polythiophenes. 

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