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.