Parallel interconnected kinetic asymmetric transformation (PIKAT) with an immobilized ω-transaminase in neat organic solvent

Article abstract of DOI:10.3390/molecules25092140

Comprising approximately 40% of the commercially available optically active drugs, α-chiral amines are pivotal for pharmaceutical manufacture. In this context, the enzymatic asymmetric amination of ketones represents a more sustainable alternative than traditional chemical procedures for chiral amine synthesis. Notable advantages are higher atom-economy and selectivity, shorter synthesis routes, milder reaction conditions and the elimination of toxic catalysts. A parallel interconnected kinetic asymmetric transformation (PIKAT) is a cascade in which one or two enzymes use the same cofactor to convert two reagents into more useful products. Herein, we describe a PIKAT catalyzed by an immobilized ω-transaminase (ωTA) in neat toluene, which concurrently combines an asymmetric transamination of a ketone with an anti-parallel kinetic resolution of an amine racemate. The applicability of the PIKAT was tested on a set of prochiral ketones and racemic α-chiral amines in a 1:2 molar ratio, which yielded elevated conversions (up to >99%) and enantiomeric excess (ee, up to >99%) for the desired products. The progress of the conversion and ee was also monitored in a selected case. This is the first report of a PIKAT using an immobilized ωTA in a non-aqueous environment.

Full text of DOI:10.3390/molecules25092140

molecules
Article
Parallel Interconnected Kinetic Asymmetric
Transformation (PIKAT) with an Immobilized
ω-Transaminase in Neat Organic Solvent
Wesley Böhmer, Lucien Koenekoop, Timothée Simon and Francesco G. Mutti *
Van ‘t Hoff Institute for Molecular Sciences, HIMS Biocat, University of Amsterdam,
1098 XH Amsterdam, the Netherlands; wbohmer91@gmail.com (W.B.); lucien.koenekoop@gmail.com (L.K.);
timothee.simon@outlook.com (T.S.)
* Correspondence: f.mutti@uva.nl; Tel.: +31-205-258-264
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editors: Antimo Gioiello and Bruno Cerra
Received: 6 April 2020; Accepted: 30 April 2020; Published: 3 May 2020
Abstract: Comprising approximately 40% of the commercially available optically active drugs, α-chiral
amines are pivotal for pharmaceutical manufacture. In this context, the enzymatic asymmetric amination
of ketones represents a more sustainable alternative than traditional chemical procedures for chiral amine
synthesis. Notable advantages are higher atom-economy and selectivity, shorter synthesis routes, milder
reaction conditions and the elimination of toxic catalysts. A parallel interconnected kinetic asymmetric
transformation (PIKAT) is a cascade in which one or two enzymes use the same cofactor to convert
two reagents into more useful products. Herein, we describe a PIKAT catalyzed by an immobilized
ω
-transaminase (
a ketone with an anti-parallel kinetic resolution of an amine racemate. The applicability of the PIKAT
was tested on a set of prochiral ketones and racemic -chiral amines in a 1:2 molar ratio, which yielded
ωTA) in neat toluene, which concurrently combines an asymmetric transamination of
α
elevated conversions (up to >99%) and enantiomeric excess (ee, up to >99%) for the desired products.
The progress of the conversion and ee was also monitored in a selected case. This is the first report of a
PIKAT using an immobilized ωTA in a non-aqueous environment.
Keywords: biocatalysis; asymmetric catalysis; kinetic resolution; α-chiral amines; enzyme immobilization;
EziG; cascades; PIKAT; transaminases; aminotransferases
1. Introduction
ω
-Transaminase enzymes (
stereo-selectivity under mild reaction conditions and are frequently applied as catalysts for the
synthesis of -chiral amines [ ]. Among the many enzymes that have been applied for the synthesis
of optically active compounds, -TAs can enable both asymmetric synthesis from prochiral ketones
and kinetic resolution of racemic amines [ ]. The enzymatic transamination reaction proceeds via
the pyridoxal 50-phosphate cofactor (PLP), which performs the reversible transfer of an amine group
from an amine donor to a carbonyl compound acceptor. Whereas - and -transaminases are used for
the synthesis of - and -amino acids, respectively, -TAs are distinguished from other members of
ω-TAs) display high reactivity and exquisite chemo-, regio- and
α
1–8
ω
1–8
α
β
α
β
ω
the transaminase sub-class by their ability to convert substrates that are devoid of a carboxylate group,
thereby making them attractive catalysts in chemical synthesis [7].
During the past decade, the applicability of
ω-TAs in organic synthesis has been tremendously
enhanced in terms of substrate acceptance and optimization of the reaction conditions to solve
(or at least alleviate) challenging issues such as enzymes’ substrate/(co)product inhibition as well as
unfavorable reaction equilibrium (i.e., for asymmetric amination of ketones) [
3,6]. This improvement
has been achieved through protein engineering (i.e., structurally-guided or directed evolution) of
Molecules 2020, 25, 2140; doi:10.3390/molecules25092140
www.mdpi.com/journal/molecules

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novel
ω
-TAs possessing a broader substrate scope [
9,
10], as well as the development of displacement
strategies for the shift of reaction equilibria [11
–
15]. Therefore, ω-TA-catalyzed reactions have been
implemented in industry, and current prominent applications entail (chemo)enzymatic cascades and
processes with immobilized biocatalysts in batch or flow reactors [3,6,9,16–24].
As mentioned above, recurrent challenges in
alanine (or another -amino acid) as the sacrificial amine donor in an aqueous environment are
unfavorable reaction equilibrium and frequent enzyme inhibition by the amine product and pyruvate
co-product [11 25 32]. However, our group and others have demonstrated that these limitations can be
addressed (or at least mitigated) by performing the transamination reaction in a neat organic solvent
at controlled water activity (aw) and using inexpensive 2-propylamine as the amine donor [33 35].
Biocatalyst recyclability and long-time stability under process conditions are also critical factors for
the successful implementation of enzymes such as -TAs in industry, particularly in continuous
ωTA-catalyzed reactions using the “classical”
α
,
–
–
ω
flow processes [36]. In this context, the development and application of immobilization strategies
has enabled enzyme recyclability, thereby enhancing biocatalysts’ performance in both batch and
flow reactors [37
namely entrapment, cross-linking and support binding [17–23,42–50]. Our group recently reported a
highly efficient kinetic resolution of a racemic -chiral amine in both batch and continuous flow using
–41]. A number of these immobilization techniques have been applied on ωTAs,
α
ω
-TAs immobilized on polymer-coated controlled porosity glass beads (EziG) [51]. Notably, the same
immobilized EziG3-
ωTA biocatalyst also led to excellent activities for the asymmetric synthesis of
α
-chiral amines from prochiral ketones in neat organic solvents when the system’s water activity (aw)
was controlled with hydrate salt pairs [35]. In particular, we obtained conversions of 80% or up to 96%
for the asymmetric synthesis of (R)-phenoxy-2-propylamine from phenoxyacetone by using only one
or two equivalents of 2-propylamine as the amine donor, respectively. Thus, we could increase the
substrate concentration from 50 mM to 400 mM without any apparent loss in catalytic performance.
The use of the immobilized EziG3-
ωTA in an organic solvent as a reaction medium did not negatively
affect—and possibly even increased—biocatalyst stability, as the reaction could be conducted in a
continuous flow reactor for several days.
Nature benefits from a large number of chemically interconnected processes that simultaneously
occur in the same living systems. Concurrent artificial catalytic processes inspired by such systems have
been developed for the synthesis of enantioenriched compounds [52–56]. Some of these cascades entail
strictly parallel biocatalytic reactions; an interesting example is parallel interconnected asymmetric
kinetic transformation (PIKAT), in which two redox reactions are generally combined in one-pot in an
anti-parallel fashion. Thus, one enzyme—or two enzymes operating in tandem—convert two reagents
into two or more useful products via internal cofactor recycling [52
for the asymmetric synthesis of vicinal diols or -haloalcohols combined with an anti-parallel kinetic
resolution of racemic secondary alcohols; the process was catalyzed by an alcohol dehydrogenase
(ADH) that required nicotinamide adenine dinucleotide as cofactor (NAD(P)+/NAD(P)H) [57
58].
An extension of the approach combined either an asymmetric sulfoxidation or a Baeyer-Villiger
oxidation with an anti-parallel kinetic resolution of secondary alcohols [59 60]; in this case, two
enzymes were required, namely a Baeyer-Villiger monooxygenase (BVMO) and ADH, which internally
recycled the NADP cofactor. One PIKAT example was also reported with an TA via internal recycling
,56]. PIKATs were first reported
β
,
,
ω
of the PLP cofactor; in this case, the PIKAT was incorporated into a slightly longer cascade whereby
L-threonine was converted to L-homoalanine in an anti-parallel combination with the kinetic resolution
of racemic α-chiral primary amines [61].
In this work, we describe a novel PIKAT using an EziG3-immobilized and (R)-selective
from Arthrobacter sp. (EziG3-AsR-
ωTA) in a neat organic solvent. The immobilization of AsR-
ω
TA
TA
ω
on EziG controlled porosity glass beads was previously investigated and exhibited high retention of
enzyme activity and excellent stability in both aqueous and organic media [35 51]. Herein, the same
,
biocatalyst preparation was employed in the organic solvent for the one-pot asymmetric formal
reductive amination of a prochiral ketone combined with an anti-parallel kinetic resolution of a racemic

Molecules 2020, 25, 2140
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α
-chiral primary amine (Scheme 1). Due to the more favorable thermodynamic equilibrium for such a
PIKAT in the non-aqueous medium, ketone substrates could be combined with racemic amine donors
in a 1:2 molar ratio. In a previous study using EziG3-AsR-
TA, toluene was found to be the best solvent
ω
when the system’s water activity (aw) was fixed and controlled at 0.7 using salt hydrate pairs [35].
Therefore, the same reaction conditions were applied in this study. Over time, the racemic amine
(rac-4–6b) undergoes resolution to yield the ketone by-product with a theoretical maximum conversion
of 50% and, in principle, leaving the (S)-configured amine enantiomer untouched; in the same pot
and anti-parallel direction, the prochiral ketone substrate (1–3a) gets converted to an enantiopure
(R)-configured amine product.
Scheme 1.
EziG3-immobilized (R)-selective
controlled water activity (aw). Hydrate salt pair Na2HPO3
control aw at 0.7. The reaction involves the formal reductive amination of a prochiral ketone (1 eq.)
combined with the kinetic resolution of a racemic amine (2 eq.) in one pot and anti-parallel direction.
PLP represents the pyridoxal-50-phosphate cofactor that becomes pyridoxamine-50-phosphate in the
catalytic cycle.
Parallel interconnected kinetic asymmetric transformation (PIKAT) using an
TA from Arthrobacter sp. (EziG3-AsR-
TA) in neat toluene and at
5H2O/Na2HPO4 7H2O was used to fix and
ω
ω
•
•
2. Results
2.1. Biocatalyst Preparation
EziG3 (Fe Amber) beads were used as immobilization carrier material. These beads are made
of high porosity glass with pores of well-defined size and geometry (pore diameter: 30
±
5 nm; pore
volume: 1.8 mL g−1). The beads’ glass surface (internal and external) is coated with a semi-hydrophobic
polymer (total bead’s bulk density: 0.21–0.25 g mL−1), which is further derivatized to chelate Fe(III)
cations. These Fe(III) centers enable the chemical coordination binding between the enzyme and the
carrier [45]. Enzyme loading capacity as high as 30% w w−1 was reported with this type of carrier

Molecules 2020, 25, 2140
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material, due to the high surface area of the porous glass per unit of volume [62]. The advantages of
metal-ion affinity binding compared with other methods for enzyme immobilization onto a support
are: (1) higher enzyme loading capacity; (2) enhanced retention of catalytic activity and stability due to
a more oriented binding; (3) possibility to detach the deactivated enzyme, thereby enabling carrier
recycling. The other immobilization methods onto a support might have the advantage of requiring
less expensive carrier materials (e.g., polymeric materials).
We previously reported the immobilization of an (R)-selective
EziG3 (forming EziG3-AsR-
TA), through the Fe(III)-ion affinity binding between the enzyme and
the carrier material. In practice, a genetically fused and N-terminal poly-histidine (His-6) tag of the
TA interacts with high affinity with the Fe(III) ions onto the polymeric surface of the EziG beads.
We previously employed EziG3-AsR- TA with an enzyme loading of 10% w w−1 (ratio of enzyme to
bead), and quantitative enzyme immobilization was achieved within 2 h incubation time [51]. In this
ωTA from Arthrobacter sp. on
ω
ω
ω
work, we applied the same immobilization conditions, namely AsR-ωTA (2 mg, purified enzyme),
EziG3 (Fe Amber) beads (20 mg) in KPi buffer (1 mL, 100 mM, pH 8.0) supplemented with PLP
(0.1 mM). Under the optimized aqueous reaction conditions (HEPES buffer, pH 7.0, 250 mM; PLP
25
µ
M), the specific catalytic activity was determined for the transamination of rac-methylbenzylamine
◦
6b (100 mM) with sodium pyruvate (50 mM) at 30 C [51]. Purified free AsR-
activity of approximately 800 U
an apparent activity of approximately 380 U
ω
TA exhibited an
TA exhibited
Mol−1enzyme. The lower apparent activity of the
µ
Mol−1enzyme, whereas immobilized EziG3-AsR-
ω
µ
immobilized biocatalyst must be, at least in part, attributed to additional mass transfer limitations in
heterogeneous catalysis (i.e., solid supported enzyme in a liquid medium); this is particularly valid for
the kinetic resolution of rac-methylbenzylamine that has an elevated reaction rate in a homogeneous
aqueous system.
2.2. PIKAT Substrate Selection
For screening of the PIKAT with the immobilized AsR-
and three racemic -chiral primary amines comprising aliphatic and aromatic substrates (Scheme 1).
Thus, prochiral ketone substrates 3a were combined with racemic amine donors 6b in a 1:2
molar ratio. Toluene was used as the reaction solvent, and the water activity (aw) was controlled using
a hydrate salt pair (i.e., Na2HPO3 5H2O/Na2HPO4
7H2O, 1:1 w w−1), as reported in our previous
study [35]. AsR- TA—a recombinant (R)-selective enzyme originated from Arthrobacter sp.—was
ωTA, we selected three prochiral ketones
α
1
–
4–
•
•
ω
immobilized on EziG3 (Fe Amber) controlled porosity glass beads and prepared for reactions in organic
solvent, as previously reported [35,51]. The reactions were performed in dark glass vials (2 mL) with
screw caps and shaken in a thermomixer at 25 ◦C.
Initial experiments indicated the formation of imine side products in the final reaction mixture;
these imines arose from the reaction between ketones and amines that were present as substrates and
products in the reaction mixture. In fact, the (reversible) imine formation between ketones and amines
is more favored in an organic solvent than in an aqueous buffer. Due to the complexity of the obtained
reaction mixture, quantification of conversion and enantiomeric excess (%ee) of the final products
proved difficult. Therefore, we decided to modify the established work-up procedure for enzymatic
transamination reactions such that ketones and amines could be separately extracted from the mixture
and analyzed in two discrete steps. Although this work-up procedure would require improvements
for a preparative or industrial scale application, possibly in a continuous process, separation of the
products allowed us to quantify conversions and ee by adding an internal standard (IS). Conversions
were determined according to the gas chromatography (GC) peak area ratios between the products
and IS in the different extraction phases. ee values were obtained after derivatization to acetoamido
and analyzed using a chiral GC column.

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2.3. PIKAT Screening of Prochiral Ketones and Racemic Amines
Prochiral ketones
1
–
3a were successfully employed in the PIKAT using rac-
4
–
6b as amine donors.
The reactions were performed at 25 ◦C in toluene at aw of 0.7 (Scheme 1). The work-up procedure
entailed extraction with an HCl aqueous solution followed by treatment with a basic aqueous solution
of KOH.
In contrast, for PIKATs with ketone 2a, the work-up had to be performed using K2CO3 instead of
KOH in the basic extraction step to avoid ester hydrolysis of the final amine product 2b. The results
are shown in Figure 1 (see details in Appendix A, Table A1), wherein each data is the average value
obtained from two independent reactions.
The PIKAT of ketone 1a led to variable conversion levels for the formal reductive amination step
(from 48% to 90%) depending on the amine donor (Figure 1a); however, the amine product (R)-1b was
always obtained with >99% ee. The conversion of 1a into (R)-1b and the outcome of the anti-parallel
kinetic resolution of rac-4–6b were obviously mutually influenced; in particular, kinetic resolution
theoretically could not exceed the 50% value, as the prochiral ketone and the racemic amine were
always applied in a 1:2 molar ratio. In contrast, the kinetic resolution of rac-6b afforded an apparent
conversion of 51
±
1%, although the ketone 1a was converted into (R)-1b with 90
±
7% conversion. This
apparent discrepancy must be attributed to the probably higher volatility of rac-6b, thereby resulting
in an apparent slightly higher formation of acetophenone 6a. Furthermore, the unreacted amine donor
6b resulted in 80% ee for the (S)-configured enantiomer, thus indicating that both enantiomers can be
accepted by the AsR-ωTA during the kinetic resolution. The imperfect enantioselectivity of the kinetic
resolution step was a general trend, as amines 4b and 5b were obtained in average values of 40% and
2% ee, respectively.
The PIKAT of ketone 2a yielded higher conversion for the asymmetric formal reductive amination
step such that (R)-2b was obtained with conversion between 77% and 91% (Figure 1b). Notably,
the amine product for the amination of this ketone substrate was also always obtained in >99% ee.
Analysis of the composition of the reaction mixtures revealed that the kinetic resolutions of the amine
donors rac-4–6b additionally yielded enantioenriched mixtures of the (S)-configured enantiomers at
the end of the reaction (between 64 ± 1% and 80 ± 1% ee).
The best performing PIKAT was observed with ketone 3a; the asymmetric formal reductive
amination step afforded quantitative conversion for all of the three investigated cases (Figure 1c). As
previously observed for ketones 1a and 2a, the amination yielded the amine product (R)-3b in optically
pure form (ee >99%). As observed in the previous cases, AsR-ωTA accepted both enantiomers of the
racemic amine donors with a preference for the (R)-configured ones, thereby leading to enantioenriched
mixture of (S)-4–6b at the end of the process (between 52 ± 2% and 59 ± 9% ee).
2.4. Time Studies
In order to examine the progress of the PIKAT reaction, we performed a time study with ketone
1a and racemic amine donor 6b. The progress of the conversion (Figure 2a) and enantiomeric excess
(ee, Figure 2b) were monitored throughout the course of the reaction (see Appendix A, Table A2 for
details). The asymmetric formal reductive amination step of 1a gradually proceeded until 90
±
7%
conversion; concomitantly, rac-6b was converted into acetophenone (6a) until 50 ± 1% conversion.
Figure 2b shows that the enantioselectivity of the formal reductive amination step remained
perfect throughout the course of the reaction (>99% ee), whereas the ee of the amine donor mixture
gradually increased from 0% to ca. 80% (S) at the end of the reaction. It is evident that the ee of
(S)-6b increased more rapidly at the beginning of the PIKAT, as (R)-6b is virtually the only accepted
enantiomer by the AsR-ωTA at this stage; however, the ee curve flattens after 20 h reaction time,
thus indicating that a considerable portion (S)-6b eventually starts to be accepted by AsR-ωTA. This
behavior results in the imperfect selectivity of the anti-parallel kinetic resolution step.

Molecules 2020, 25, 2140
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Figure 1. Screening of prochiral ketone substrates and racemic amine donors for the PIKAT. The y-axis
represents either the percentage of conversion to product or the ee of the amines for the anti-parallel
asymmetric formal reductive amination and kinetic resolution steps. Prochiral ketone substrates in the
PIKAT reaction: (
Reaction conditions: toluene (aw = 0.7, reaction volume: 1 mL),
immobilized EziG3-AsR-
TA (total mass of enzyme plus carrier material: ca. 22 mg; enzyme loading
on carrier material: 10% w w−1), hydrate salt pair (40 mg; ratio Na2HPO3
5H2O/Na2HPO4 7H2O, 1:1,
w w−1) in a dark 2 mL glass vial; agitation in a thermomixer at 900 rpm, 25 ◦C, for 72 h.
a)
1a, (b)
2a, and (
c)
3a; these ketones were tested with racemic amine donors
4–6b.
1
–
3a (50 mM), rac- 6b (100 mM),
4–
ω
•
•

Molecules 2020, 25, 2140
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Figure 2. Time study of the PIKAT combining prochiral ketone 1a with racemic amine 6b: (
a) progress
of conversion and (
b
) ee progress over the time. Reaction conditions: toluene (aw = 0.7, reaction volume:
1 mL), 1a (50 mM), rac-6b (100 mM), immobilized EziG3-AsR-ωTA (total mass of enzyme plus carrier
material: ca. 22 mg; enzyme loading on carrier material: 10% w w−1), hydrate salt pair (40 mg; ratio
Na2HPO3
•
5H2O/Na2HPO4
7H2O, 1:1, w w−1) in a dark 2 mL glass vial; agitation in a thermomixer at
•
900 rpm, 25 ◦C, for 72 h.
2.5. Further Studies
Since the PIKAT is based on a reversible amine transfer, we decided to study the influence of
the molar ratio between prochiral ketone and a racemic amine donor on the overall thermodynamic
equilibrium of the process. Therefore, we decided to increase the prochiral ketone substrate
concentration from 50 mM up to 100 mM while keeping the racemic amine concentration constant at
100 mM. Thus, we could check whether an excess of ketone (i.e., from >1 up to 2 eq. In this study)
could push the anti-parallel kinetic resolution of the racemic amine to completion at a given ketone
substrate concentration. Crucially, high ee values could also be accessible for the kinetic resolution of
the racemic amine donor, while the formal reductive amination reaction would always provide >99%
ee of the desired amine product. Ketone 1a and amine donor rac-6b were chosen as model substrates,
and the results are reported in Table A3 of Appendix A. These data clearly show that altering the molar
ratios between prochiral ketone and racemic amine for the PIKAT did not influence the outcome of
the kinetic resolution step, as (S)-6b was always obtained with an ee around 70% when 1a was added
from 50 mM to 80 mM concentration. Moreover, increasing the concentration of 1a up to 90 mM and
100 mM was detrimental for the selectivity of the kinetic resolution, as the ee of (S)-6b decreased to
average values of 48% and 28%, respectively. In contrast, as observed in all this study’s experiments,

Molecules 2020, 25, 2140
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the asymmetric formal reductive amination step proceeded with excellent enantioselectivity such that
(R)-1b was always obtained in >99% ee.
In another set of reactions, we either doubled the enzyme concentration in the reaction or added
another portion of fresh enzyme after 24 h reaction time. No improvements were observed (data not
shown).
3. Discussion
Prochiral ketone substrates
in a parallel interconnected kinetic asymmetric transformation (PIKAT). The asymmetric synthesis
step yielded (R)-configure amines 3b with average conversions from 48% to >99% and virtually
1–3a were successfully employed with racemic amine donors 4–6b
1–
perfect enantioselectivity (>99% ee). Notably, ketone 3a was quantitatively converted in the three
investigated cases and amination of ketone 2a also led to elevated conversions (77% or higher), whereas
ketone 1a was converted with slightly lower conversions (average values from 48 to 90%) (Figure 1;
see Appendix A, Table A1 for details).
Overall, the asymmetric formal reductive amination step appeared to be only minorly affected by
the type of racemic amine donor that was applied. The anti-parallel kinetic resolution step showed a
consistent trend depending on the applied amine donor; for instance, rac-6b was always converted to
higher conversions compared with rac-4b and rac-5b when combined with the same type of ketone in
the amination step. In the combination between 1a and rac-5b, the kinetic resolution showed a low
conversion (19
±
3%) and nearly racemic amine was obtained (ee 2%); accordingly, the concurrent
asymmetric formal reductive amination step led to moderate conversion, albeit with an excellent ee of
>99% (Appendix A, Table A1, entry 2). As the conversion in the kinetic resolution step varied from
moderate to high and the enantiomeric excess values were lower than expected, we postulated that the
AsR-ωTA is less enantioselective for the kinetic resolution step, and thus, over time, both enantiomers
got transformed. The time study of 1a with rac-6b (Figure 2a) showed that both asymmetric synthesis
and kinetic resolution went towards completion. Compound 1a was converted to (R)-1b with 90
±
7% conversion after 72 h of reaction time, whereas the rac-6b concentration gradually decreased
to reach 50 1% after 72 h. The ee percentage values displayed in Figure 2b demonstrate excellent
±
selectivity for the asymmetric formal reductive amination, such that exclusively (R)-1b was formed.
Furthermore, the originally racemic 6b became an enantioenriched mixture of the (S)-enantiomer over
time, as mainly the (R)-enantiomer was converted to 6a. After 72 h, the (S)-enantiomer comprised
approximately 90% of 6b.
In a subsequent set of experiments, we attempted to increase the conversion and thus the ee
in the kinetic resolution of rac-6b by adding more of the prochiral ketone 1a to the reaction. In this
manner, we would sacrifice some conversion in the asymmetric synthesis of (R)-1b while aiming to
gain a higher enantioenrichment of (S)-6b. Unfortunately, the use of an excess of 1a (up to equimolar
with rac-6b) did not provide any evident improvement of the reaction outcome (see Appendix A,
Table A3 for details). As expected, conversion of 1a to (R)-1b was slightly lower than in the previous
experiments (Appendix A, Table A1, entry 3) and further decreased while increasing the concentration
of 1a. With the exception of Table A3, entries 5 and 6, all of the reactions exhibited ca. 36-40 mM
product formation of (R)-1b from 1a (Appendix A, Table A3). Higher concentration of 1a (90 and 100
mM; Appendix A, Table A3, entries 5 and 6) hampered the PIKAT such that the average productivity
of (R)-1b was 31 mM and 20 mM, respectively. We attribute this effect to the known
inhibition at higher concentrations of some ketone substrates [11 26 28 32]. On the other hand, kinetic
resolution of rac-6b was not improved by increasing the concentration of 1a, as the ee values never
exceeded 78 2% (Appendix A, Table A3) and never improved compared with the value of 80 0%
ωTA enzyme’s
,
,
–
±
±
reported in Appendix A, Table A1 entry 3. Finally, doubling the amount of immobilized enzyme or
adding fresh enzyme after 24 h of reaction time did not benefit the reaction outcome.

Molecules 2020, 25, 2140
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4. Materials and Methods
4.1. Chemicals and Carrier Materials
Ketones
1–
3a, racemic amines
4
–
6b and pyridoxal-50-phosphate (PLP) were purchased from
Sigma-Aldrich (Steinheim, Germany). EziG3 (Fe Amber) enzyme carrier material was provided by
EnginZyme AB (Solna, Sweden). Further details for equipment and analytical determination are
presented in Section 4.6. All reaction solvents were degassed before use. All of the water-equilibrated
solvents were prepared by shaking hydrate salt pairs in the organic solvent for 1 h at RT.
4.2. Expression and Purification of ωTAs
C-terminal His-tagged (R)-selective
ωTA from Arthrobacter sp. (AsR-ωTA) [63,64] was expressed
and purified as previously reported [51].
4.3. Bradford Assay
Protein assay dye reagent concentrate (BioRad Laboratories, Veenendaal, the Netherlands)
was diluted 5 times with MilliQ water and filtered through a paper filter. This diluted stock
◦
solution was freshly prepared before use and kept in the dark at 4 C. The albumin calibration
line was determined in the standard range of 200–1000
to the supplier-provided protocol. A low-concentration assay was used in cases of lower protein
concentrations (<25 L diluted stock solution
g mL−1). Samples were prepared by mixing 980
and 20 L protein sample (for low-concentration assay: 800 L diluted stock solution and 200
protein sample) followed by incubation for 5–10 min at room temperature. Absorption at 595 nm
was measured and plotted against the protein concentration. Diluted TA enzyme samples were
µ
g mL−1 protein concentration according
µ
µ
µ
µ
µL
ω
then prepared and measured in the same fashion in order to determine their concentration using the
albumin calibration line.
4.4. Immobilization of AsR-ωTAs on EziG3 Carrier Materials
EziG3 carrier material (20
immobilization buffer (KPi, 1 mL, 100 mM, pH 8.0) supplemented with PLP (0.1 mM). Purified
-TA (2 mg, equal to 10% w w−1; enzyme loading to support material) was added to the suspension.
The mixture was shaken on an orbital shaker (120 rpm) for 3 h at 4 ◦C. Small aliquots from the aqueous
phase (20 L) were taken before and after the immobilization procedure, and their concentrations
±
0.2 mg) was cooled down in an ice bath and suspended in an
ω
µ
were determined using the Bradford assay (see Section 4.3 for details). Once full immobilization was
obtained, the immobilized enzyme was let to sediment and the buffer was removed by pipetting.
We recovered the immobilized enzyme by sedimentation (1 min), since the use of other separation
techniques such as centrifugation or vacuum filtration can lead to partial enzyme deactivation.
4.5. PIKAT in Organic Solvents with Immobilized ωTAs
In a dark glass vial (2 mL), immobilized EziG3-AsR-
loading to support material) and hydrate salt pair (Na2HPO3
40 mg, 1:1, w w−1) were suspended in EtOAc (1 mL, at fixed aw of 0.7) and shaken for 15 min (900 rpm,
thermomixer). The EziG3-AsR-
TA was allowed to sediment and the solvent was removed by
pipetting. The EziG3-AsR-
TA with hydrate salt pair was resuspended in another aliquot of EtOAc,
and the above-described process was repeated twice. Next, the EziG3-AsR-
TA with hydrate salt pair
ω
TA (total mass 22 mg, 10% w w−1 enzyme
•
5H2O/Na2HPO4 7H2O, total mass
•
ω
ω
ω
was washed with toluene (1 mL, at fixed aw of 0.7) and then allowed to sediment, after which the
solvent was removed by pipetting. Finally, toluene (1 mL total reaction volume, at fixed aw of 0.7) was
added as a reaction solvent. The ketone substrate
donor 6b (final concentration: 100 mM) were added, and the reaction vials were shaken in an upright
position (900 rpm, thermomixer) for 72 h at 25 ◦C. Work-up was performed by separating ketone and
1–3a (final concentration: 50 mM) and racemic amine
4–

Molecules 2020, 25, 2140
10 of 16
amine compounds from each other by extraction. First, the EziG3-AsR-
ωTA was left to sediment and
the organic reaction mixture was separated from the biocatalyst by pipetting. Then, naphthalene was
added as an internal standard to the organic reaction mixture from a concentrated stock solution (50
from 1 M stock in toluene, final concentration 50 mM). The amine compounds were selectively extracted
µL
from the toluene reaction phase using an aqueous 2 M HCl solution (400
the possible extraction of ketones. Following mixing and centrifugation (14 krpm, 5 min, 4 C),
the two layers were separated and the organic layer was kept separate. The acidic aqueous layer was
µ
L), thereby minimizing
◦
re-extracted with toluene (2
were combined and dried over MgSO4 prior to analysis. Then, the acidic aqueous layer—containing
the protonated amine products—was basified using an aqueous solution of either KOH (200 L of a 10
M stock solution) or K2CO3 (for reactions with substrate 2a), and extracted with EtOAc containing
50 mM naphthalene as an internal standard (2 500 L). Drying over MgSO4 was performed prior
×
500 µL) to selectively remove potential traces of ketone. The organic layers
µ
×
µ
to analysis. Analysis was performed by separately injecting ketone- and amine-containing solutions
on GC equipped with an achiral column (see Section 4.6 for details on analytical equipment and
determination). For the determination of enantiomeric excess, the samples containing amines were
derivatized by incubation with 4-dimethylaminopyridine in acetic anhydride (final concentration:
5 mg mL-1) for 30 min (170 rpm, RT). Samples were quenched by the addition of water (500
µL) and
shaken for 30 min (170 rpm, RT). Following centrifugation, the organic layer was dried over MgSO4
and analyzed by GC equipped with a chiral column (see Section 4.6 for details on analytical equipment
and determination).
4.6. Analytical Equipment and Determination
The biotransformation conversions were determined using a 7890A GC system (Agilent
Technologies, Santa Clara, CA, USA) equipped with a FID detector with H2 as carrier gas and
a DB-1701 column from Agilent (30 m, 250
0.25 M). Chiral samples were measured using a CP-DEX column from Agilent (25 m, 320
or a HydroDex-β-TBDAc column (50 m, 250 µM) from Macherey-Nagel (Dueren, Germany):
µ
m, 0.25
µ
M), or a HP-5 column from Agilent (30 m, 320
µ
m,
µ
µm, 0.25 µ
M)
•
DB1701-30 m-method-A: constant pressure 6.9 psi, T injector 250 ◦C, split ratio 50:1, T initial 80 ◦C,
hold 6.5 min; gradient 10 ◦C/min up to 160 ◦C, hold 5 min; gradient 20 ◦C/min up to 200 ◦C, hold
2 min; gradient 20 ◦C/min up to 280 ◦C, hold 1 min.
•
DB1701-30 m-method-B: constant pressure 6.9 psi, T injector 250 ◦C, split ratio 50:1, T initial 60 ◦C,
hold 6.5 min; gradient 20 ◦C/min up to 100 ◦C, hold 1 min; gradient 20 ◦C/min up to 280 ◦C, hold
1 min.
•
•
•
HP-5-method-A: constant pressure 4 psi, T injector 250 ◦C, split ratio 30:1, T initial 60 ◦C; gradient
5 ◦C/min up to 150 ◦C, hold 1 min; gradient 10 ◦C/min up to 250 ◦C, hold 1 min.
HP-5-method-B: constant pressure 4 psi, T injector 250 ◦C, split ratio 30:1, T initial 40 ◦C, hold 2
min; gradient 5 ◦C/min up to 80 ◦C, hold 2 min; gradient 20 ◦C/min up to 250 ◦C.
CP-DEX-method-A: constant flow 1.4 mL/min, T injector 200 ◦C, split ratio 40:1, T initial 100 ◦C,
hold 2 min; gradient 1 ◦C/min up to 130 ◦C, hold 5 min; gradient 10 ◦C/min up to 170 ◦C, hold
10 min.; gradient 10 ◦C/min up to 180 ◦C, hold 1 min.
•
•
•
CP-DEX-method-B: constant flow 1.4 mL/min, T injector 200 ◦C, split ratio 40:1, T initial 100 ◦C,
hold 2 min; gradient 1 ◦C/min up to 118 ◦C, hold 5 min; gradient 10 ◦C/min up to 170 ◦C, hold
10 min.; gradient 10 ◦C/min up to 180 ◦C, hold 1 min.
CP-DEX-method-C: constant flow 1.5 mL/min, T injector 200 ◦C, split ratio 20:1, T initial 60 ◦C,
hold 2 min; gradient 5 ◦C/min up to 100 ◦C, hold 2 min; gradient 10 ◦C/min up to 180 ◦C, hold
1 min.
HydroDex-β
-TBDAc-method: constant flow 1 mL/min, T injector 220 ◦C, split ratio 20:1, T initial
100 ◦C, hold 2 min; gradient 1 ◦C/min up to 150 ◦C, hold 8 min; gradient 10 ◦C/min up to 170 ◦C,
hold 1 min.

Molecules 2020, 25, 2140
11 of 16
5. Conclusions
In this work, we report the successful development of the first example of parallel interconnected
kinetic asymmetric transformation (PIKAT) using an immobilized -transaminase in a neat organic
solvent. PIKATs have been achieved almost exclusively using oxidoreductase enzymes, and other
enzyme classes such as transferases have rarely been considered. The PIKAT with TA entails an
ω
ω
asymmetric formal reductive amination of a prochiral ketone combined with a concomitant and
anti-parallel kinetic resolution of a racemic amine. The use of a neat organic solvent is critical for
the successful application of the PIKAT concept with
ωTA, as it enables the appropriate tuning of
the thermodynamic equilibrium between two anti-parallel reactions, also using structurally diverse
substrates. The formal reductive amination step always proceeded with excellent selectivity, thus
yielding the (R)-configured amine product in >99% ee. Conversely, the immobilized
Arthrobacter sp. displayed only moderate selectivity in the kinetic resolution step, thereby resulting
in a maximum of 80 1% ee value for the less converted (S)-configured amine donor. Time studies
ωTA from
±
showed that the kinetic resolution step proceeded with main (or even sole) consumption of the
(R)-configured amine until a conversion of ca. 35% was obtained, at which stage it appeared that the
other enantiomer started to be accepted as well, ultimately leading to a 51
In contrast, the anti-parallel formal reductive amination proceeded smoothly until 90
and >99% ee of the final amine product was obtained. The enantioselectivity of the kinetic resolution
step can potentially be enhanced by testing other available TAs and/or engineering new and more
±
1% conversion and 80% ee.
±
7% conversion,
ω
selective variants that are customized for this process. Admittedly, one limitation of the current
process is the elaborated work-up procedure entailed in the selective extraction of the amine products
(and ketone by-product) from the reaction mixture. Therefore, further work is required to develop
more efficient procedures for the isolation of pure products, such as using fractional distillation and/or
selective ion-exchange or hydrophobic resins [27]. In this context, strategies for selective product
removal could be implemented along with PIKAT running in continuous flow reactors. In summary,
this work provides a proof-of-concept for such a PIKAT process, which was notably applied with a
selected range of structurally diverse ketones and racemic amines that comprise aliphatic, aromatic
and arylaliphatic substrates.
Author Contributions: All of the authors contributed to the design of the experiments. Studying and optimizing
the procedure for enzyme immobilization, W.B.; performing and analyzing the screening of prochiral ketones with
racemic amines, analyzing the data and performing the time studies, W.B. and L.K.; performing and analyzing
initial reactions that lead to a better understanding of the process and reactivity, T.S.; conceiving and directing the
project and receiving the funding, F.G.M.; writing the manuscript, W.B. and F.G.M. All authors have read and
agree to the published version of the manuscript.
Funding: This project has received funding from the European Research Council (ERC) under the European
Union’s Horizon 2020 Research and Innovation programme (ERC-StG, Grant Agreement N◦ 638271, BioSusAmin).
Dutch funding from the NWO Sector Plan for Physics and Chemistry is also acknowledged.
Acknowledgments: We kindly thank K. E. Cassimjee and Alexey Volkov (EnginZyme AB) for providing EziG3
Fe-Amber beads.
Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the study design;
data collection, analyses, or interpretation; manuscript writing; or the decision to publish the results.
Appendix A
This section contains experimental data (Tables A1–A3), and GC retention times and analytical
methods (Table A4).

Molecules 2020, 25, 2140
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Table A1. Screening of substrate ketones (1–3a) and amine donors (rac-4–6b) in the PIKAT.
Formal Reductive Amination Step
Kinetic Resolution Step
Conversion
1–3a to 1–3b [%]
ee
Conversion
4–6b to 4–6a [%]
ee
Entry
Ketone Substrate
Amine Donor
1–3b [%]
4–6b [%]
1
2
3
4
5
6
7
8
9
1a
2a
3a
4b
5b
6b
4b
5b
6b
4b
5b
6b
62 ± 10
48 ± 15
90 ± 7
91 ± 0
87 ± 1
77 ± 0
>99
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99(R)
33 ± 6
19 ± 3
51 ± 1
47 ± 3
39 ± 0
54 ± 1
37 ± 1
35 ± 1
44 ± 4
40 ± 10 (S)
2 ± 1 (S)
80 ± 0 (S)
80 ± 1 (S)
64 ± 1 (S)
75 ± 1 (S)
52 ± 2 (S)
48 ± 2 (S)
59 ± 9 (S)
>99
>99
Reaction conditions: toluene (aw = 0.7, reaction volume: 1 mL),
EziG3-AsR-
10% w w−1), hydrate salt pair (40 mg; ratio Na2HPO3
agitation in a thermomixer at 900 rpm, 25 ◦C, for 72 h.
1
–
3a (50 mM), rac-4–6b (100 mM), immobilized
ω
TA (total mass of enzyme plus carrier material: ca. 22 mg; enzyme loading on carrier material:
•
5H2O/Na2HPO4
•
7H2O, 1:1, w w−1) in a dark 2 mL glass vial;
Table A2. Time study using ketone 1a and amine donor rac-6b.
Formal Reductive Amination Step Kinetic Resolution Step
Reaction
Time [h]
Conv.
1a to 1b [%]
ee
1b [%]
Conv.
6b to 6a [%]
ee *
6b [%]
Entry
1
2
3
4
5
6
7
8
1
2
3
5
8
24
48
72
8 ± 4
15 ± 8
24 ± 2
39 ± 4
55 ± 7
84 ± 1
90 ± 3
90 ± 7
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
5 ± 2
9 ± 4
6 (S)
9 (S)
13 ± 1
17 ± 8
29 ± 3
42 ± 1
47 ± 2
51 ± 1
14 (S)
25 (S)
40 (S)
63 (S)
69 (S)
80 (S)
* In this study, the ee was measured for one sample at each time point. Reaction conditions: toluene (aw = 0.7,
reaction volume: 1 mL), 1a (50 mM), rac-6b (100 mM), immobilized EziG3-AsR-
TA (total mass of enzyme plus
carrier material: ca. 22 mg; enzyme loading on carrier material: 10% w w−1), hydrate salt pair (40 mg; ratio
Na2HPO3 5H2O/Na2HPO4
7H2O, 1:1, w w−1), in a dark 2 mL glass vial; agitation in a thermomixer at 900 rpm,
25 ◦C, for 72 h.
ω
•
•
Table A3. PIKAT of 1a with rac-6b using higher concentrations of 1a (50–100 mM) in order to possibly
thermodynamically push the kinetic resolution of rac-6b to completion.
Formal Reductive Amination Step
Kinetic Resolution Step
Ketone 1a Conc.
[mM]
Conv.
1a to 1b [%]
Productivity
1a to 1b [mM]
ee
1b [%]
Conv.
6b to 6a [%]
ee
6b [%]
Entry
1
2
3
4
5
6
50
60
70
80
90
79 ± 1
66 ± 3
51 ± 2
49 ± 4
34 ± 7
20 ± 6
40
40
36
39
31
20
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
49 ± 1
49 ± 3
44 ± 2
47 ± 4
38 ± 7
28 ± 6
77 ± 2 (S)
78 ± 2 (S)
63 ± 2 (S)
70 ± 10 (S)
48 ± 15 (S)
27 ± 11 (S)
100
Reaction conditions: toluene (aw = 0.7, reaction volume: 1 mL), 1a (50–100 mM), rac-6b (100 mM), immobilized
EziG3-AsR-
TA (total mass of enzyme plus carrier material: ca. 22 mg; enzyme loading on carrier material:
10% w w−1), hydrate salt pair (40 mg; ratio Na2HPO3 7H2O, 1:1, w w−1), in a dark 2 mL glass vial;
5H2O/Na2HPO4
agitation in a thermomixer at 900 rpm, 25 ◦C, for 72 h.
ω
•
•

Molecules 2020, 25, 2140
13 of 16
Table A4. GC retention times and methods.
Compound
Retention Time
GC Method
1a
1b
(R)-1b
(S)-1b
2a
13.6
13.7
43.7
43.3
6.5
HP-5-method-A
HP-5-method-A
CP-DEX-method-A
CP-DEX-method-A
HP-5-method-A
HP-5-method-A
2b
6.9
(R)-2b
(S)-2b
3a
14.6
14.4
2.8
HydroDex-β-TBDAc-method
HydroDex-β-TBDAc-method
DB1701-30m-method-B
DB1701-30m-method-B
CP-DEX-method-C
3b
2.7
(R)-3b
(S)-3b
4a
15.1
14.4
3.7
CP-DEX-method-C
DB1701-30m-method-A
DB1701-30m-method-A
CP-DEX-method-A
4b
4.5
(R)-4b
(S)-4b
5a
13.3
12.6
14.2
13.5
41.0
40.9
10.3
9.0
CP-DEX-method-A
DB1701-30m-method-A
DB1701-30m-method-A
CP-DEX-method-A
5b
(R)-5b
(S)-5b
6a
6b
(R)-6b
(S)-6b
CP-DEX-method-A
DB1701-30m-method-A
DB1701-30m-method-A
CP-DEX-method-A
27.9
27.5
CP-DEX-method-A
naphthalene (internal standard)
12.1
12.8
12.4
DB1701-30m-method-A
DB1701-30m-method-B
HP-5-method-A
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