Sadiq, Rizwana (2015) Enzymatic reactions and coordination complexes of steroids. Doctoral thesis, University of Huddersfield.

Research in this thesis investigates a simple, rapid, reproducible and specific liquid chromatography-mass spectroscopy (LC-MS) method for the simultaneous detection, confirmation and quantitation of beclomethasone dipropionate (BDP) and its metabolites beclomethasone monopropionate (BMP) and beclomethasone (BOH) which has been developed and validated. The method was validated over a concentration range from 0.001-100 mg/L for BDP and 0.01-10 mg/L for BOH. The calibration graphs exhibited excellent linearity with typical R2 values for BDP of 0.997 and for BOH of 0.999. The analytes stability was investigated and they were found to be stable for many weeks. BDP was introduced as the first inhaled glucocorticoid for the treatment of asthma in the 1970s and is still a major treatment for asthma nowadays. BDP is an inactive prodrug; for BDP to be effective, it needs to be activated to its active form, beclomethasone-17-monopropionate (17-BMP) and beclomethasone-21-monopropionate (21-BMP). The method involved in vitro metabolism of the BDP, incubated with esterase enzyme at 37° C for 2 hrs. The product was analysed by LC-MS. A liquid-liquid extraction procedure was used to purify the products, with four different solvents and five sequential extractions, for initial extraction steps. The extracts were analysed by LC-MS. Extraction with ether gave very good results with 99 % extraction of the required products. For the first time new methods of labelling steroids, including progesterone and testosterone, with rhenium-containing luminescent labels. For this purpose rhenium complex with 3,3’-diamino-2,2’-bipyridine (complex 1) was used as a fluorescent label for various biomolecules. Preparative HPLC, LC-MS, NMR spectroscopy and X-ray crystallography have been used to identify and characterize the products formed. The timed reaction of complex 1 with progesterone was run using the newly developed chromatographic method (55 % MeCN, 0.5 mL/min). A solution of complex 1 and progesterone in acetonitrile was heated in the dark for 3 hrs followed by a timed NMR experiment for 6 hours, collecting data at regular intervals. The sample was separated using the preparative HPLC which showed some side products and two overlapping peaks for the product isomers. The single-crystal X-ray structure of the Re-progesterone product was obtained and measurements of selected bond lengths showed a rearrangement of the double bond between C4 and C5 had occurred at the conjugated end of the progesterone. The refined structure further confirmed the shifting of the double bond. For the analagous product of complex 1 and testosterone attempts were made to crystallise the product both by slow evaporation and by refrigeration of an acetonitrile solution of fraction 2. To date these attempts have been unsuccessful. Finally, the research work was conducted to combine the work presented above by using enzymatic reactions to modify a biomolecule for reaction with the rheniumcontaining complex 1. Galactose oxidase catalyses the oxidations of primary alcohols to the corresponding aldehyde with the reduction of dioxygen to hydrogen peroxide. Research presented in this part of the thesis investigates different methodologies for the oxidation of methyl �-D-galactopyranoside with the help of galactose oxidase to form an aldehyde-containing sugar which will react with the diamino moieties of complex 1. The best method to work with was found to be method 2 using catalase suspension, galactose oxidase and HRP in deionised water. The reaction mixture gave a fairly pure product in good yields.

Rizwana Final PhD Thesis.pdf - Accepted Version
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