In this context, the aim of my PhD project was to study the combination of flow chemistry and biocatalysis in order to develop greener and scalable routes for the synthesis of high values chemicals and active pharmaceutical ingredients (APIs), e.g., Captopril, an angiotensin-converting enzyme (ACE) inhibitor widely used for the treatment of hypertension. In particular, my attention was focused on continuous flow biocatalyzed redox reactions.
During my PhD, I performed heterogeneous reactions using immobilized biocatalysts (either whole microbial cells or enzymes) in packed bed reactors, also introducing a gas inlet when necessary. In many cases, the products were purified in-line, using scavengers or exploiting catch and release strategies, or performing in-line extractions and liquid/liquid separations. In this way, traditional work-up and chromatographic procedures were not necessary, the operational times were reduced and less amount of organic solvents was used, thus obtaining faster and greener procedures.3
Here below, I summarize three different applications exploited during my PhD thesis that clearly demonstrate the potential of performing biotransformations in a continuous flow fashion:
1. Chemo-enzymatic continuous flow synthesis of Captopril
A chemo-enzymatic route for the synthesis of the antihypertensive drug Captopril [i.e., (S)-1-((S)-3- mercapto-2-methylpropanoyl) pyrrolidine-2-carboxylic acid] was developed in a continuous flow reactor. The process consists of four steps: the first one was a biocatalyzed regio- and stereo-selective oxidation (using Ca-alginate-immobilized cells of A. aceti) of a cheap commercially available prochiral diol (i.e., 2- methyl-1,3-propandiol, 1, Scheme 1). This reaction was performed with a segmented air-liquid flow stream. The air was necessary to guarantee the oxygen required for the reaction.
After isolation of the obtained carboxylic acid 2, the first step was followed by three chemical reactions (i.e., chlorination, amide coupling and nucleophilic substitution).
The continuous flow process leads to different advantages compared to the batch one: first, the overall reaction time was dramatically reduced from 3 days (batch) to 100 minutes (flow), increasing the overall yield from batch (45%) to flow (65%). In addition, it was possible to perform in-line purification procedures as in-line quenching and liquid/liquid separations, avoiding in this way the traditional, time-consuming work-ups and purification of intermediates. Only one column-chromatography purification was performed at the end of the process (Scheme 2).
2. Stereoselective reduction of di-ketones using an immobilized ketoreductase/glucose dehydrogenase mixed bed reactor
The stereoselective biocatalyzed reduction of di-ketones was optimized in flow to obtain the mono-alcohol products, some of them key intermediates for the synthesis of hormonal contraceptives (e.g., compound 6c, Scheme 3). The reactions were performed in a reactor packed with two immobilized enzymes, a ketoreductase (KRED1) from Pichia glucozyma and a glucose dehydrogenase (GDH) from Bacillus megaterium. The KRED1 performs the reductive reaction, while the glucose dehydrogenase was necessary to regenerate the cofactor used, that is NADP+. The BmGDH was able to do so by oxidizing glucose to gluconic acid.
Reaction parameters (i.e., stoichiometry, concentration, temperature, pressure, residence time) were optimized and a complete conversion was observed with residence times between 7 minutes and 3 hours to form the enantiopure mono-alcohol. For all the substrates, the reaction was carried out in continuously for 15 days with no significant change of the chemical composition of the outflow solution. Noteworthy, after 6 months of operation, the flow reactor only lost 30-32% of the original activity
3. Oxidation of amines to aldehydes using an immobilized form of pure transaminase from Halomonas elongata
Benzylamine-derivatives were oxidized into the corresponding aldehyde-compounds using an immobilized transaminase from Halomonas elongata in a flow reactor (Scheme 4). This class of molecules is commonly employed as flavour and fragrance component in food, beverage, cosmetics and pharmaceuticals.
For all the substrates, conversions ≥ 90% were reached within a residence time between 3 and 10 minutes. The products were directly purified in-line, with acidification and extraction with ethyl acetate. In some cases, the aldehyde product remained attached to the support used for the immobilization of the enzyme and an adjustment in the flow configuration was necessary. In these cases, a liquid/liquid biphasic system with toluene and buffer was used and an extraction with toluene was performed. The bioreactor was stable after several weeks of continuous work.
Flow chemistry and biocatalysis offer a broad spectrum of new and interesting possibilities that can change the idea of organic chemistry. During my PhD, I developed new chemo-enzymatic routes for the synthesis of pharmaceutically interesting intermediates, APIs and fragrances that can be considerable alternatives to the traditional chemical pathways. In all cases, the reactions were completely stereoselective, conversions were higher and productivities were increased compared to batch procedures, thus reducing the waste by reusing the biocatalysts for several cycles.
1) Ley, S. V. On being green: Can flow chemistry help? Chem. Rec. 2012, 12, 378–390
2) Tsubogo, T.; Oyamada, H.; Kobayashi Multistep continuous-flow synthesis of (R)- and (S)-rolipram using heterogeneous catalysts S. Nature 2015, 520, 329–332;
3) Bryan M. C., Dillon B., Hamann L. G., Hughes G. J., Kopach M. E., Peterson E. A., Pourashraf M., Raheem I., Richardson P., Richter D., Sneddon H. F., Sustainable Practices in Medicinal Chemistry: Current State and Future Directions, J. Med. Chem, 2013, 56, 6007-6012
PhD in Chemistry at the University of Milan.
Department of Pharmaceutical Sciences (DISFARM), University of Milan, via Mangiagalli 25, 20133, Milan, Italy. E-mail: firstname.lastname@example.org