Introduction

Chiral amines are valuable building blocks for the synthesis of a vast selection of active pharmaceutical ingredients (APIs), agrochemicals and fine chemicals . These compounds can be obtained by chemical and biotechnological approaches, whereas biocatalytic applications frequently enable the highest stereo-, regio- and chemoselective conversion from the respective starting materials being a significant process advantage over conventional chemical approaches . In addition, in the past decades the scientific advances in protein engineering techniques have provided a better access to specifically improved enzymes for tailor-made bioprocesses, also in the chiral amine synthesis . Especially amine transaminases , amine dehydrogenases , and imine reductases (IREDs) have recently attracted a lot of attention for synthesizing a wide selection of chiral amines. A special case are IREDs, which allow a direct synthesis of enantiopure secondary amines from prochiral imines . Within this relatively young group of enzymes, Mitsukura et al. first reported the purification and characterization of the (R )-IRED from Streptomyces sp. GF3587 reducing 2-methyl-1-pyrroline (2-MP) to (R )‑2‑methylpyrrolidine ((R )-2-MPN) with an enantioselectivity of 99%. The same reduction was performed using purified (S )-IRED from Streptomyces sp. GF3546 synthesizing (S )‑2‑methylpyrrolidine ((S )-2-MPN) . Since then, this beneficially conversion yielding optically active secondary and tertiary amines by using nicotinamide adenine dinucleotide [phosphate] (NAD[P]H) as the cofactor has been broadened to a wide substrate scope from acyclic imines up to five and six membered heterocycles . Also, the IRED-catalyzed asymmetric reductive amination of ketones has been reported recently , boosting this asymmetric biosynthesis in the research field.
A significant amount of IRED-based reports focus on the optimization of the enzymatic reaction, either by process or reaction engineering while optimizing the relevant environmental conditions (pH, temperature, etc.), or the biocatalyst itself via enzyme engineering (K M, k cat, substrate scope, etc.). Unfortunately, the downstream-processing (DSP) within such biocatalytic processes is often not investigated in detail and typically limited to an initial pH-shift of the aqueous reaction medium, followed by single or even multiple extractions and final distillation or column purification steps. Alternative non-conventional reaction media such as ionic liquids , deep eutectic solvents and other could be used to simplify the DSP but may suffer from major drawbacks like activity losses of the biocatalyst and a high complexity associated with a cost-intensive system. In addition, in situ -product removal (ISPR) techniques such as crystallization or membranes would be also a powerful addition, but are currently not widely applicable for IRED-catalyzed reaction systems .
This study aims at presenting the utilization of ion exchange resins as an easy option to perform the DSP of IRED-catalyzed reaction systems, including its potential usage on a preparative scale. A small selection of ion exchange materials was mentioned in the past within biocatalytic processes, but their applications are still somehow limited with a few noticeable exceptions . In this study, the ion exchange resins are applied to the IRED-reaction system to selectively remove the reactants from the reaction solution without any significant changes to the aqueous reaction medium, which also minimizes the processing steps during the DSP. In addition, this strategy opens the possibility of simply reusing the biocatalyst without any potentially harmful biocatalyst immobilization steps. In contrast to classical adsorbers, ion exchange resins offer a more specific interaction with charged reactants such as ammonium- and iminium-ions. This concept was also recently reported for a decarboxylase-reaction system for the synthesis of a benzoic acid derivate using the commercially available anion exchange resin Dowex 1x2 (Cl) .
The presented ion exchange resin-based DSP-concept involves a simple addition of the ion exchange resin to the reaction medium for selectively capturing the reactants. This is followed by a filtration step to remove the resin particles and a final release of the product from the resin into ether (Figure 1). Due to the ubiquitous use of 2-methyl-1-pyrroline as a model compound in IRED-catalyzed reactions it was specifically chosen for this study and used for the preparative synthesis and isolation of the corresponding enantiopure product (S )-2-methylprrolidine. The highly (S )-selective IRED fromPaenibacillus elgii B69 was applied in whole cells as a model biocatalyst.