The development and improvement of biocatalytic reactions

Conventional chemical processes often require a series of complex synthesis steps that utilise a great deal of energy and raw materials. There is a huge commitment by the chemical industry to search for “green”, environmentally friendly processes. Biocatalysis, using enzymes to catalyse a chemical reaction, is emerging as an economical and ecological production route for a wide range of fine chemicals, pharmaceuticals and food ingredients. In addition to biocatalysis, miniaturised chemistry has also become of interest and significance to the chemical industry over the last decade. Microreactors allow for the synthesis of small volumes of compounds quickly and in high yield, with fast optimisation of conditions. Additionally, when used with immobilised biocatalysts, these miniaturised devices can enable the rapid throughput screening of a wide range of enzymes and their substrates. Despite this however, developments of applications in the field of miniaturised enzymatic processes are still in the initial stages. Using lipase catalysed, enzymatic esterification as a model reaction, Chapter 2 demonstrates the use of miniaturised technology for biocatalytic reactions. The work described within this chapter explored the feasibility of combining biocatalysis with miniaturisation and enabled us to gain familiarity with both the biocatalytic processes and miniaturised technology. Benchmarked again batch reactions, nine alkyl esters were successfully synthesised enzymatically, using Novozyme 435, in a pressure driven, packed-bed, miniaturised, continuous flow reactor. In some cases close to 100% ester conversions were achieved. Chapter 2 also demonstrates the ability to screen the enzyme for substrate specificity. In nature, many molecules exist as racemic mixtures. As discussed in Chapter 3, enantiomers of racemic compounds can often exhibit diverse pharmacological and therapeutic properties. In fact, different drugs usually have their biological activity based mainly on one enantiomer. Consequently, research into the preparation of enantiomerically pure chiral compounds is increasing in interest. Dynamic kinetic resolution is emerging as a potentially efficient process, whereby standard enzymatic kinetic resolution is coupled with in situ racemisation of the slow reacting starting substrate, using a suitable racemising catalyst. However, compatibility issues between the biocatalyst and racemising agent limit the number of applications to which this dynamic kinetic resolution set up can be applied. With this in mind, Chapter 3 reports the development of a dynamic kinetic resolution process. A thorough study into the kinetic resolution, racemisation and dynamic kinetic resolution batch reactions of a range of substrates, of interest to the pharmaceutical industry, are detailed in Chapter 3. Efficient methods for the enantioselective hydrolysis of a series of esters, using a range of lipases, were primarily developed. The D-forms of the racemic ester and thioester substrates were found to be substrates for lipase catalysed enantioselective hydrolysis to the corresponding chiral acids, in excellent optical purity (>99%eeD). Base catalysed racemisation studies showed that the ester substrates could not be racemised to any extent, under the reaction conditions studied. However, three organic bases were found to be suitable racemising catalysts for the L-enantiomers of the two thioester substrates under investigation. The combined reactions allowed for a dynamic kinetic resolution process to be established for the two thioesters with excellent to good conversions and enantioselectivies being achieved. The successful dynamic kinetic resolution processes were subsequently investigated with the aid of miniaturised reactor chemistry as a means of improving the technique further. Chapter 4 therefore, details a combination of the miniaturised reactor technology described in Chapter 2 and the biocatalytic resolution reactions presented in Chapter 3. The purpose here was to determine if the inherent advantages associated with miniaturisation, discussed in Chapter 2, could be observed for enzymatic resolution set ups. Additionally, in an attempt to overcome compatibility issues, with regards to the enzyme and racemising agent in the dynamic kinetic resolution process, as discussed in Chapter 3, Chapter 4 discusses the development of a separated dynamic kinetic resolution reaction. Unfortunately, issues surrounding the activity of the immobilised bases, used as racemising agents, did not allow for the full dynamic kinetic resolution reaction to be achieved separately. However, kinetic resolution reactions utilising an immobilised lipase were successfully conducted in the miniaturised system with inherent advantages over the equivalent batch process being observed.