The production of active pharmaceutical ingredients (APIs) and fine chemicals is accelerating due to the advent of novel microreactors and new materials for immobilizing customized biocatalysts that permit long-term use in continuous-flow reactors. This work studied the scalability of a tunable U-shape magnetic nanoparticles (MNPs)-based microreactor. The reactor consisted of a polytetrafluoroethylene tube (PTFE) of various inner diameters (ID = 0.75 mm, 1.50 mm, or 2.15 mm) and six movable permanent magnets positioned under the tube to create reaction chambers allowing the fluid reaction mixture to flow through and above the enzyme-loaded MNPs anchored by permanent magnets. The microreactors with various tube sizes and MNP capacities were tested with the preparative scale kinetic resolution of the drug-like alcohols 4-(3,4-dihydroisoquinolin-2(1H)-yl)butan-2-ol (±)-1a and 4-(3,4-dihydroquinolin-1(2H)-yl)butan-2-ol (±)-1b, utilizing Lipase B from Candida antarctica immobilized covalently onto MNPs, leading to highly enantioenriched products [(R)-2a,b and (S)-1a,b]. The results in the U-shape MNP flow reactor were compared with reactions in the batch mode with CaLB-MNPs using similar conditions. Of the three different systems, the one with ID = 1.50 mm showed the best balance between the maximum loading capacity of biocatalysts in the reactor and the most effective cross-section area. The results showed that this U-shaped tubular microreactor might be a simple and flexible instrument for many processes in biocatalysis, providing an easy-to-set-up alternative to existing techniques.
This study implements a convenient microreactor for biocatalysis with enzymes immobilized on magnetic nanoparticles (MNPs). The enzyme immobilized onto MNPs by adsorption or by covalent bonds was lipase B from Candida antarctica (CaLB). The MNPs for adsorption were obtained by covering the magnetite core with a silica shell and later with hexadecyltrimethoxysilane, while for covalent immobilization, the silica-covered MNPs were functionalized by a layer forming from mixtures of hexadecyl- and 3-(2-aminoethylamino)propyldimethoxymethylsilanes in 16:1 molar ratio, which was further activated with neopentyl glycol diglycidyl ether (NGDE). The resulting CaLB-MNPs were tested in a convenient continuous flow system, created by 3D printing to hold six adjustable permanent magnets beneath a polytetrafluoroethylene tube (PTFE) to anchor the MNP biocatalyst inside the tube reactor. The anchored CaLB-MNPs formed reaction chambers in the tube for passing the fluid through and above the MNP biocatalysts, thus increasing the mixing during the fluid flow and resulting in enhanced activity of CaLB on MNPs. The enantiomer selective acylation of 4-(morpholin-4-yl)butan-2-ol (±)-1, being the chiral alcohol constituent of the mucolytic drug Fedrilate, was carried out by CaLB-MNPs in the U-shape reactor. The CaLB-MNPs in the U-shape reactor were compared in batch reactions to the lyophilized CaLB and to the CaLB-MNPs using the same reaction composition, and the same amounts of CaLB showed similar or higher activity in flow mode and superior activity as compared to the lyophilized powder form. The U-shape permanent magnet design represents a general and easy-to-access implementation of MNP-based flow microreactors, being useful for many biotransformations and reducing costly and time-consuming downstream processes.
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