DEVELOPMENT OF CONTINUOUS-FLOW PROCESSES FOR REDOX BIOCATALYSIS AND APPLICATION IN THE CHEMO-ENZYMATIC SYNTHESIS OF ACTIVE PHARMACEUTICAL INGREDIENTS

The present thesis has the aim of develop and find new and more environmental friendly synthetic routes for the synthesis of active pharmaceutical ingredients (APIs) and pharmaceutically interesting intermediates, exploiting the advantages of the combination between flow chemistry and biocatalysis. Indeed, biocatalytic processes in continuous flow reactors have attracted attention in recent years for carrying out continuous manufacturing systems with high level of intensification. Flow processing has the potential to accelerate heterogeneous biotransformations due to biocatalyst high local concentration and enhanced mass transfer, making large-scale production more economically feasible in significantly smaller equipment with a substantial decrease in reaction time, from hours to a few minutes, and improvement in space–time yield, with increases of up to 650-fold as compared to batch processes. Moreover, biocatalyst stability is enhanced by working in an environment where harsh mixing is avoided. Overall, these features result in reduced inventory, waste and energy requirements of the flow biocatalytic process, as compared to the conventional batch mode. In particular, I focused my attention on redox reactions, since for these the traditional chemical procedures and reagents are far from being sustainable and environmental friendly. For example, for oxidative reactions the most used chemical reactives are Chromium VI (a well known cancerogenic agent), Dess-Martin periodinane (a potential explosive reagent) and the Swern reagent, a thiol based compound that produces dimethyl sulphide as co-product. Moreover, the traditional chemical methods are not able to reach the selectivity and specificity that is possible to achieve with biocatalytical systems. Briefly, the projects I was involved in during my PhD and that are present in the thesis are: 1. Development of a new synthetic route to obtain Captopril, using both chemical and biocatalyzed reactions and exploiting the advantages of flow chemistry, that allows to perform continuous synthesis; 2. Development of a flow based biocatalyzed oxidation with immobilized whole cells of Acetobacter aceti in order to obtain enantiomerically pure mono-carboxylic acids, starting from the corresponding diols; 3. Stereoselective reduction of ketones and di-ketones, in order to obtain enantiomerically pure mono-alcohol products, using together two enzymes (ketoreductase from Pichia glucozyma and a glucodehydrogenase from Bacillus megaterium) in a Flow Chemistry pcked bed reactor; 4. Stereoselective reduction of 2,2-disubstituted 1,3-cyclopenta- and 1,3 cyclohexanediones using both whole cells and a purified ketoreductase from Pichia glucozyma, to obtain enantiomerically pure mono-alcohols products, that can be important intermediates in the synthesis of various steroids. 5. Use of an immobilized transaminase from Halomonas elongata able to perform transaminations in both directions (from amine to aldehydes, and from aldehydes to amine) with the Flow Reactor technology; To reach the goal, I both used whole cells and purified enzymes as biocatalysts, either in a free or immobilized form, and I exploited many advantages of continuous flow technology, as for example downstream processes (i.e., in-line acidifications, extractions and purifications) that allowed me to in-line purify the products, thus avoiding the traditional work-up procedures, reducing the operational times and the amount of organic solvents used. In almost all cases, important results were achieved, as faster kinetics, cleaner procedures that required less purification steps, complete stereo- and regioselectivity, higher conversions and productivities compared to batch procedures, increased stability of the biocatalyst, that could be used for several cycles, thus reducing the waste.