In the Introduction the importance of N-dealkylation in pharmaceutical studies is summarised and the scope and potential advantages of microbial transformations in effecting N-dealkylation are examined. Possible applications of chromatographic and spectroscopic techniques to the study of microbial transformations are discussed. Chapter 2 describes the development of quantitative HPLC techniques for the determination of a model substrate codeine, and its N-demethylated transformation product, norcodeine, from microbial transformation mixtures. Extraction procedures applicable to 7 litre fermenter cultures are also investigated. The effect of carbon source and substrate concentration on the growth and N-demethylation of codeine by Cunninghamella sp. are investigated in Chapter 3. Chapter 4 describes the development of procedures for the preparation of a cell-free extract from Cunninghamella sp. with optimal N-demethylase activity. Reaction conditions for N- demethylation of codeine by cell-free extracts of C. bainieri are optimised in terms of substrate concentration and co-factor requirements. Kinetic data for the N-demethylation of codeine and codeine N-oxide by C. bainieri are presented in Chapter 5. The effect of selected inducers and specific enzyme inhibitors is also examined. A reaction mechanism for the N-demethylation of codeine by cell-free extracts of C. bainieri is proposed. Chapter 6 describes the development and application of a 13C NMR spectroscopic technique to detect the possible intermediates in the N-demethylation of codeine by cell-free extracts of C. bainieri, and provides further evidence in support of the proposed reaction mechanism. In Chapter 7 a HPLC technique is employed to estimate the lipid solubility of a selected series of 1,4-benzodiazepines from retention data. The dependence on substrate lipophilicity, of N-dealkylation of these compounds by C. bainieri, is examined. The data are considered with reference to current microbial transformation literature. The proposed mechanism of microbial N-demethylation is compared to mammalian transformations of similar substrates and suggestions for future study are emphasised.
|Date of Award||1984|