Date: 16 May 2025 | Category: News
Ana I. Benitez-Mateos, David Lim, David Roura Padrosa, Valentina Marchini, Hao Wu, Frederic Buono, and Francesca Paradisi*
The Paradisi Group from the University of Bern in Switzerland have used a bienzymatic system within a continuous flow reactor to reduce heterocyclic imines at concentrations of up to 100 mM with excellent stereoselectivity, while maintaining operational stability of the enzymes for up to 35 hours. In this case, the Vapourtec R-Series system was used in combination with a short glass column containing the immobilised enzymes, which acted as a packed-bed reactor (PBR).
Heterocyclic chiral amines within pharmaceuticals
Heterocyclic chiral amines are privileged scaffolds in nature and within pharmaceuticals and drug development, however a general synthetic strategy to prepare this broad class is still lacking. Organo- and metallocatalysis to effect reduction of imines are both viable approaches, however both require high catalytic loading or harsh reaction conditions, leading to functional group incompatibility, as well as issues with stereoselectivity [2]. The use of enzymes as reducing agents, however, offers access to high substrate selectivity, milder reaction conditions, a greener approach and an improved reaction safety profile. While the preparation of chiral piperidine scaffolds through biocatalytic means is well-elucidated, there are still challenges with preparation of chiral heterocyclic amines such as morpholines, piperazines and thiomorpholines.
Imine reductases (IREDs) were discovered 15 years ago by Nagasawa and co-workers [3] and are a promising approach towards asymmetric reduction of prochiral imines to the corresponding amine. These enzymes are NADPH-dependent, however IRED systems can be combined with co-factor recycling systems to regenerate NADP+ in situ. While the use of immobilising technology provides an opportunity for enzyme stabilisation, the protein rigidification that occurs upon tethering to a solid support can cause IREDs to completely lose their bioactivity – very few successful immobilisations of IREDs have been reported to date. The first successful covalent immobilization of IREDs was reported in 2024 by Gaun et al., demonstrating the utility of an immobilised IRED to produce (S)-nornicotine with >99% chiral purity on a multi-gram scale [4]. This demonstrates that if enzymatic activity can be maintained with immobilisation of the IRED, the use of solid supports in combination with flow chemistry offers the possibility for rapid scale-up of a green and sustainable solution to the heterocyclic imine reduction conundrum.
The Paradisi group’s approach
After confirming activity of several IREDs across a range of pharmaceutically relevant imines, the group successfully immobilised several enantiocomplementary IRED enzymes onto a solid support. Tailored approaches were required for individual IREDs to produce immobilized enzymes that produced both good activity and reusability. Use of catalytic quantities of the immobilised co-factor glucose dehydrogenase (GDH) from Bacillus megaterium (BmGDH) allowed costs to be minimised as use of stoichiometric cofactor would be costly. The use of the dual enzyme system also allowed NADP+ to be used as a substrate, rather than NADPH, representing a further significant reduction in cost. In batch mode, 90% conversion was maintained for at least 10 reaction cycles using the same biocatalysts, and enzyme leaching was minimised through use of irreversible covalent immobilisation.
When the reaction was translated into flow mode, optimal ratios of immobilized IRED-4/IRED-5 and BmGDH produced high yields and high enantioselectivities, Scheme 1. At 10 mM substrate concentration, IRED-4 produced (S)-3-methylthiomorpholine with 98% yield and IRED-5 produced (R)-3-methylthiomorpholine with 91% yield, both with ee >99%. Concentrations up to 100 mM are feasible, and the immobilized enzymes demonstrated high operational stability for over 35h.
Immobilised enzymes: the future of catalysis?
The use of a short glass column containing two different immobilised enzymes as a packed-bed reactor (PBR) has facilitated the bienzymatic reduction of imines to the corresponding amine with excellent yields, high enantioselectivity and reproducibility. This example demonstrates the significant impact flow chemistry can have, and provides a powerful example of low-cost, scalable, and efficient flow chemistry systems with excellent green credentials.
Talk to Vapourtec today to find out how the use of continuous flow chemistry can impact your research endeavours.
References:
[1] Biocatalytic Reduction of Heterocyclic Imines in Continuous Flow with Immobilized Enzymes. A. I. Benítez-Mateos, D. Lim, D. Roura Padrosa, V. Marchini, H. Wu, F. Buono, F. Paradisi, ACS Sustainable Chem. Eng. 2025, 13, 13 500−5018). https://doi.org/10.1021/acssuschemeng.4c09676
[2] (a) Development of heterogeneous catalyst systems for the continuous synthesis of chiral amines via asymmetric hydrogenation. (T. Yasukawa, R. Masuda, S. Kobayashi, Nature Catalysis 2019, 2 (12), 1088−1092). https://doi.org/10.1038/s41929-019-0371-y (b) Chiral Bronsted Acid-Catalyzed Metal-free Asymmetric Direct Reductive Amination Using 1-Hydrosilatrane. (V. Skrypai, S. E. Varjosaari, F. Azam, T. M. Gilbert, M. J. Adler, J. Org. Chem. 2019, 84, (9), 5021−5026). https://doi.org/10.1021/acs.joc.8b03073
[3] Process for production of optically active amine derivative. (T. Nagasawa, T. Yoshida, K. Ishida, H. Yamamoto, N. Kimoto, US20110262977A1, 2011). https://patents.google.com/patent/US20110262977
[4] Efficient Synthesis of (S)-Nornicotine using Co-Immobilized IRED and GDH in Batch and Continuous Flow Reaction Systems. (s. Guan, W. Zhou, Y. Yue, S. Wang. B. Chen, H. Yang, Org. Process Res. Dev., 2024, 28 (5), 2050−2060). https://doi.org/10.1021/acs.oprd.4c00130