Continuous Flow Bioamination of Ketones in Organic Solvents at Controlled Water Activity using Immobilized ω-Transaminases

Article abstract of DOI:10.1002/adsc.201901274

Compared with biocatalysis in aqueous media, the use of enzymes in neat organic solvents enables increased solubility of hydrophobic substrates and can lead to more favorable thermodynamic equilibria, avoidance of possible hydrolytic side reactions and easier product recovery. ω-Transaminases from Arthrobacter sp. (AsR?ωTA) and Chromobacterium violaceum (Cv?ωTA) were immobilized on controlled porosity glass metal-ion affinity beads (EziG) and applied in neat organic solvents for the amination of 1-phenoxypropan-2-one with 2-propylamine. The reaction system was investigated in terms of type of carrier material, organic solvents and reaction temperature. Optimal conditions were found with more hydrophobic carrier materials and toluene as reaction solvent. The system's water activity (a_w) was controlled via salt hydrate pairs during both the biocatalyst immobilization step and the progress of the reaction in different non-polar solvents. Notably, the two immobilized ωTAs displayed different optimal values of a_w, namely 0.7 for EziG³?AsR?ωTA and 0.2 for EziG³?Cv?ωTA. In general, high catalytic activity was observed in various organic solvents even when a high substrate concentration (450–550 mM) and only one equivalent of 2-propylamine were applied. Under batch conditions, a chemical turnover (TTN) above 13000 was obtained over four subsequent reaction cycles with the same batch of EziG-immobilized ωTA. Finally, the applicability of the immobilized biocatalyst in neat organic solvents was further demonstrated in a continuous flow packed-bed reactor. The flow reactor showed excellent performance without observable loss of enzymatic catalytic activity over several days of operation. In general, ca. 70% conversion was obtained in 72 hours using a 1.82 mL flow reactor and toluene as flow solvent, thus affording a space-time yield of 1.99 g L^?1 h^?1. Conversion reached above 90% when the reaction was run up to 120 hours. (Figure presented.).

Full text of DOI:10.1002/adsc.201901274

Title: Continuous Flow Bioamination of Ketones in Organic Solvents at
Controlled Water Activity using Immobilized ω-Transaminases
Authors: Wesley Böhmer, Alexey Volkov, Karima Engelmark
Cassimjee, and Francesco Mutti
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10.1002/adsc.201901274
Advanced Synthesis & Catalysis
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DOI: 10.1002/adsc.201901274
Continuous Flow Bioamination of Ketones in Organic Solvents
at Controlled Water Activity using Immobilized ω-
Transaminases
Wesley Böhmer,a Alexey Volkov,b Karim Engelmark Cassimjee,b and Francesco G.
Muttia,*
a
Van ’t Hoff Institute for Molecular Sciences, HIMS-Biocat, University of Amsterdam, Science Park 904, 1098 XH,
The Netherlands.
EnginZyme AB, Tomtebodavägen 6, 171 65 Solna, Sweden
b
* Corresponding author. E-mail address: f.mutti@uva.nl
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.
Abstract. Compared with biocatalysis in aqueous media, the In general, high catalytic activity was observed in various
use of enzymes in neat organic solvents enables increased organic solvents even when a high substrate concentration
solubility of hydrophobic substrates and can lead to more (450-550 mM) and only one equivalent of 2-propylamine
favorable thermodynamic equilibria, avoidance of possible were applied. Under batch conditions, a chemical turnover
hydrolytic side reactions and easier product recovery. ω- (TTN) above 13000 was obtained over four subsequent
Transaminases from Arthrobacter sp. (AsR-ωTA) and reaction cycles with the same batch of EziG-immobilized
Chromobacterium violaceum (Cv-ωTA) were immobilized on ωTA. Finally, the applicability of the immobilized
controlled porosity glass metal-ion affinity beads (EziG) and biocatalyst in neat organic solvents was further demonstrated
applied in neat organic solvents for the amination of 1- in a continuous flow packed-bed reactor. The flow reactor
phenoxypropan-2-one with 2-propylamine. The reaction showed excellent performance without observable loss of
system was investigated in terms of type of carrier material, enzymatic catalytic activity over several days of operation.
organic solvents and reaction temperature. Optimal conditions In general, ca. 70% conversion was obtained in 72 hours
were found with more hydrophobic carrier materials and using a 1.82 mL flow reactor and toluene as flow solvent,
toluene as reaction solvent. The system’s water activity (aw) thus affording a space-time yield of 1.99 g L-1 h-1.
was controlled via salt hydrate pairs during both the Conversion reached above 90% when the reaction was run
biocatalyst immobilization step and the progress of the up to 120 hours.
reaction in different non-polar solvents. Notably, the two
immobilized ωTAs displayed different optimal values of aw,
Keywords: asymmetric synthesis, biocatalysis, α-chiral
amines, flow chemistry, transaminases.
namely 0.7 for EziG3-AsR-ωTA and 0.2 for EziG3-Cv-ωTA.
an aqueous buffer, such as the low solubility of
hydrophobic substrates and the possible occurrence of
unwanted hydrolytic side reactions; in certain cases,
thermodynamic equilibria can become more favorable
in a non-aqueous medium and product recovery can
also be facilitated.[5] In this context, enzyme
immobilization and microenvironment optimization
have proven to enhance stability and catalytic
performance in neat organic solvents.[6] However, it is
important to remark that dynamic and catalytic
properties of enzymes in non-aqueous media are
dependent on the presence of a limited but critical
amount of water.[7] On the one hand, less than a
monolayer of water is required for an isolated enzyme
molecule to show activity in an organic solvent. On the
other hand, the inherent conformational rigidity of an
Introduction
The use of enzymes in laboratory and industrial scale
organic synthesis has predominantly been restricted to
either monophasic aqueous reaction media in presence
of an optional water-miscible cosolvent or bi-phasic
aqueous-organic media or reverse-micelles systems in
which the enzyme is still dissolved in a significant
amount of water.[1] However, many different enzyme
families have been applied in neat organic solvents or
supercritical gases over the last few decades,[2]
whereas more recent contributions entail the use of
ionic liquids [3] and deep eutectic solvents.[4] Notably,
the use of enzymes in non-aqueous media enables the
avoidance of some drawbacks of biotransformations in
1
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10.1002/adsc.201901274
Advanced Synthesis & Catalysis
enzyme in an organic medium with minimal water
content preserves its native folding state.[8] However,
the impact of these factors significantly depends on the
biocatalyst form, which can be either an isolated
enzyme as lyophilized powder, or immobilized on a
carrier material, or entrapped in a polymeric matrix, or
even a whole cell biocatalyst.
critical in non-aqueous biocatalysis, and this water
partitions between all components in the system,
including the solvent, enzyme, support matrix and the
headspace of the reaction vessel. The water activity
(aw) is a more reliable parameter than the water content
(cw) for analysis and comparison of enzyme
performance in different solvents because the
hydration state of an enzyme is always fixed by the aw
value independently of the solvent of choice.[27]
Optimization of the reaction system was performed in
terms of carrier material, solvent and reaction
temperature. Under optimal reaction conditions, the
immobilized (R)-selective transaminase was employed
in a packed-bed flow reactor in a neat organic solvent
showing high catalytic performance.
Hydrolases were the first and remain the most
frequently applied enzymes in neat non-aqueous
environments.[2b, 9] Oxidoreductases, including alcohol
dehydrogenases[10] and peroxidases[11] as well as
alcohol and phenol oxidases[12] were found to be active
in neat organic solvents in the 1980s, and further
studies on oxidoreductases were conducted during this
decade.[13] Examples with lyases comprise
hydroxynitrile
lyases,[14]
cyclases,[15]
decarboxylases,[13d, 13e, 16] hydroperoxide lyases[17] and
benzaldehyde lyases.[18]
Results and Discussion
In contrast, transferases such as ω-transaminases
(ωTAs) have only sporadically been applied in neat
organic solvents. Transaminases are pyridoxal
phosphate-dependent (PLP) enzymes that enable the
transfer of an amino group from a simple donor
molecule to a prochiral carbonyl compound, thereby
generating a wide range of high-value chiral α-amine
containing compounds.[19] Transaminases possess
significant potential for industrial application due to
high turnover rates, excellent enantioselectivity and
economical cofactor (PLP) regeneration.[20] An
engineered and immobilized ωTA was applied for the
production of Sitagliptin in a neat organic solvent.[21]
In a concomitant publication, we reported that wild-
type ωTAs retain elevated catalytic activity for the
amination of prochiral ketones in neat organic solvents
when applied as lyophilized cell extracts.[22] The
activity of the ωTA’s lyophilized cell extract was
retained by the addition of a small amount of water
below the saturation value in an organic medium. The
system exhibited a more favorable thermodynamic
equilibrium for the amination compared to the same
reaction in aqueous buffer as well as an apparent lack
of substrate inhibition, and it enabled simple product
recovery along with biocatalyst recycling. In later
studies, the same strategy was exploited for the
synthesis of enantiomerically pure 3-substituted
cyclohexylamines and both enantiomers of valinol.[23]
E. coli whole cells expressing ωTAs were then applied
in a flow reactor.[24] Finally, ωTAs have recently been
implemented in neat MTBE as enzyme particles
coated with ionic liquid.[25]
Two stereocomplementary ωTAs, namely the (R)-
[28]
selective ωTA from Arthrobacter sp. (AsR-ωTA)
and the (S)-selective ωTA from Chromobacterium
[29]
violaceum (Cv-ωTA)
were chosen for application
in neat organic media. In this study, we decided to use
purified ωTAs in order to be able to quantify the
amount of immobilized enzyme loaded on the carrier
material with high precision and accuracy. However,
the efficiency of the selective immobilization of ωTA
from crude lysate on EziG3 has already been
demonstrated.[26] Therefore, we applied a typical
enzyme loading of 10%
w
w-1 ωTA. The
immobilization of ωTAs was performed on three types
of EziG polymer-coated controlled porosity glass
carrier materials possessing distinct surface properties.
The first type of material, henceforth called EziG1 (Fe
Opal), is covered with a hydrophilic polymeric surface.
EziG2 (Fe Coral) has a hydrophobic surface polymer
and EziG3 (Fe Amber) is covered with a semi-
hydrophobic polymer surface (Table S3). The
immobilization of ωTAs was performed by incubating
the desired amount of enzyme in buffer supplied with
EziG carrier material according to our previously
reported protocol.[26] The progress of the
immobilization was determined by measuring the
amount of enzyme remaining in the immobilization
buffer with a Bradford assay (see SI – section 2.2).
Quantitative immobilization of AsR-ωTA on EziG
carrier materials was obtained within three hours of
incubation.[26]
A practical method for aw control involves the use
of insoluble salts possessing a varied stoichiometry of
co-crystallized water molecules. By incubating the
organic reaction medium with a specific pair of salts,
it is possible to dynamically “buffer” the water content
during the reaction and thus fix the aw. A selection of
salt hydrate pairs for setting aw is available from the
literature.[30]
Hydrophilic carrier materials tend to retain a
significant amount of water. Therefore, in the present
study, the water content (cw) of EziG-immobilized
ωTA had to be lowered after the immobilization step
and controlled during the reaction in organic solvent in
To the best of our knowledge, there are no reports
about the application of immobilized ωTAs (as
isolated enzymes) in neat organic solvents and
continuous flow reactors. In a previous study, we
optimized the immobilization of transaminases on
controlled porosity glass carrier material (EziG) for
their application in an aqueous environment.[26] Herein,
we report the application of both an (R)-selective and
an (S)-selective transaminase as immobilized enzymes
in neat organic solvents at controlled aw (water
activity). In fact, the residual amount of water in a
given solvent (i.e., below the water saturation level) is
2
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10.1002/adsc.201901274
Advanced Synthesis & Catalysis
order to obtain catalytic activity. Our previously
reported methodology based on the lyophilization of
ωTAs as cell extracts was not applicable for EziG-
immobilized enzymes and resulted in the complete
loss of catalytic activity.[22] Other attempts aimed at
reducing the water content of the immobilized ωTA by
the gentle flowing of a stream of air or dinitrogen were
also unsuccessful. Optimizing cw (and consequently
aw) in the immobilized ωTA using salt hydrate pairs
proved to be successful, particularly in combination
with a hydrophilic solvent. The reason for using a
hydrophilic solvent is to gently and gradually remove
the excess of water from the enzyme-carrier complex
while preserving enzyme activity. Optimal cw could be
set in the system via simple washing of the wet
immobilized ωTA with hydrophilic organic solvent in
the presence of salt hydrate pairs (i.e.,
The transamination of 1-phenoxypropan-2-one (1a,
50 mM) using 2-propylamine 2b (150 mM) as the
amine donor was used as a model reaction (Scheme 1).
In particular, the amination of these types of substrates
is thermodynamically favored in non-aqueous
environment as demonstrated in the previous study
that was conducted using lyophilized crude enzyme in
batch system.[22] Among the others, the amine product
1b is an analogue of the drug Mexiletine.[29b]
Na2HPO4•2H2O/Na2HPO4•7H2O
Na2HPO4•7H2O/Na2HPO4•12H2O).
or
Then,
the
Scheme 1. Reductive amination of 1-phenoxypropan-2-one
(1a) with 2-propylamine (2b) catalyzed by EziG3-AsR-ωTA
in neat organic solvents at controlled aw.
hydrophilic solvent was removed and hydrophobic
solvents (also pre-equilibrated at the set aw using the
same type of salt hydrate pairs) were instead applied
during the reaction to prevent the further stripping of
enzyme-bound water, thereby ensuring high catalytic
activity of the immobilized ωTA.
Table 1. Optimization of conditions for the transamination of 1a (50 mM) with 2b (150 mM) catalyzed by EziG3-AsR-ωTA
(22 mg, enzyme loading: 10% w w-1) in neat organic solvents (1 mL) at controlled water activity (aw) as specified, at 25 °C
and shaking on an orbital shaker at 900 rpm for 72 h. 900 rpm agitation was selected because the immobilized enzyme
appeared to have the best contact with the solvent at this rate (i.e., all the beads were evenly distributed in the heterogeneous
system and not prevalently at the bottom of the reaction mixture).
Entry Immobilization buffer
aw equilibration
solvent
Reaction
solvent
Salt hydrate pairs
Conv.
[%][3]
1
2
3
4
KPi (100 mM, pH 8.0)
KPi (100 mM, pH 8.0)
KPi (100 mM, pH 8.0)
KPi (100 mM, pH 8.0)
MTBE[1]
MTBE[1]
EtOAc[1]
EtOAc[1]
MTBE[1]
none
none
none
4±0
8±1
28
Toluene[1]
Toluene[1]
Toluene[1]
Na2HPO4•2H2O/
Na2HPO4•7H2O
Na2HPO3•5H2O/
Na2HPO4•7H2O
Na2HPO3•5H2O/
Na2HPO4•7H2O
Na2HPO3•5H2O/
Na2HPO4•7H2O
Na2HPO3•5H2O/
Na2HPO4•7H2O
Na2HPO3•5H2O/
Na2HPO4•7H2O
Na2HPO4•2H2O/
Na2HPO4•7H2O
Na2HPO3•5H2O/
Na2HPO4•7H2O
Na2HPO3•5H2O/
Na2HPO4•7H2O
87
5
KPi (100 mM, pH 6.0)
KPi (100 mM, pH 6.5)
KPi (100 mM, pH 7.0)
KPi (100 mM, pH 7.5)
KPi (100 mM, pH 8.0)
MOPS (100 mM, pH 7.5)
HEPES (100 mM, pH 7.5)
Tris (100 mM, pH 7.5)
EtOAc[2]
EtOAc[2]
EtOAc[2]
EtOAc[2]
EtOAc[2]
EtOAc[1]
EtOAc[2]
EtOAc[2]
Toluene[2]
Toluene[2]
Toluene[2]
Toluene[2]
Toluene[2]
Toluene[1]
Toluene[2]
Toluene[2]
88±6
81±14
92±4
85±7
96±1
21±2
75±5
91±2
6
7
8
9
10
11
12
[1] aw = 0.6. [2] aw = 0.7. [3] Conversion to amine product in 72 h reaction time. Values are depicted with standard deviations
over three independent experiments. All reactions afforded >99% enantiomeric excess of the amine product.
Application of a lyophilized crude cell extract of AsR-
ωTA in MTBE was shown to best operate at a water
activity of aw = 0.6.[22] However, setting the hydration
state of immobilized EziG3-AsR-ωTA with MTBE (aw
= 0.6), followed by transamination either in toluene (aw
= 0.6) or MTBE (aw = 0.6) resulted in only 4% and 8%
conversions, respectively (Table 1, entries 1–2). A
significant increase in conversion (28%) was observed
3
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10.1002/adsc.201901274
Advanced Synthesis & Catalysis
water content. Conversely, hydrophobic EziG2-AsR-
ωTA exhibited a dramatic drop in conversion at low
aw. EziG3 (Fe Amber) was chosen as the carrier
material for further studies due to its superior
performance in the range of 0.4 ≤ aw ≤ 0.7. Notably,
the aw values that are reported in this work with one
significant digit must be considered as accurate
because the original literature values are reported with
an error on the second significant digit.[30]
when EtOAc (aw = 0.6) was used to set the hydration
state of EziG3-AsR-ωTA and the immobilized enzyme
was then applied in toluene (aw = 0.6) (Table 1, entry
3). Although immobilized EziG3-AsR-ωTA was
obtained in a sufficiently active form using EtOAc at
aw of 0.6 for equilibration, we postulated that some
additional water molecules could be adsorbed by the
immobilized ωTA over time, thus reducing its
catalytic activity during the progress of the reaction.
Indeed, when a pair of disodium hydrogen phosphate
di- and hepta-hydrate salts was added to the reaction
mixture (1:1, w w-1 ratio salt hydrate pair to
immobilized ωTA), we observed a remarkable
increase in conversion (87%, Table 1, entry 4). The
addition of the salt hydrate pairs to the reaction
mixture also significantly increased the system’s
reproducibility; therefore, all further experiments were
performed with salt hydrate pairs in the reaction.
Enzyme ionization influences the catalytic activity
of enzymes in non-aqueous media.[2b] In particular, the
effect of the pH memory of the lyophilized enzymes
was studied by tuning the ionization state of their
functional groups in aqueous buffers prior to
lyophilization and submerging them in a non-aqueous
medium.[31] Although the pH of the immobilization
buffer did not significantly affect the catalytic activity
of EziG3-AsR-ωTA immobilized from KPi buffers
(Table 1, entries 5─9), we observed a significant drop
in conversion when MOPS buffer was used for the
immobilization (21%, Table 1, entry 10). The use of
KPi buffer (100 mM, pH 8.0) led to a remarkable 96%
conversion; therefore, further studies were performed
using these conditions. Finally, EziG3-AsR-ωTA
immobilized from HEPES buffer (100 mM, pH 7.5)
and Tris buffer (100 mM, pH 7.5) resulted in 75% and
91% conversions, respectively (Table 1, entries 11–
12).
Most immobilization supports absorb water and the
use of different support materials can have a direct
effect on the catalytic activity and stability of enzymes
in non-aqueous media. For example, subtilisin
Carlsberg covalently attached to macroporous acrylic
supports was demonstrated to exhibit improved
catalytic activity at high cw as a result of water
absorption by the support material.[32] EziG-
immobilized ωTAs (EziG1-AsR, EziG2-AsR, and
EziG3-AsR, Table S3) were applied in toluene at aw
controlled by salt hydrate pairs (Table S4); no hydrate
salts were added when water-saturated solvents were
used. At comparable levels of aw, the use of EziG1-
AsR-ωTA and EziG3-AsR-ωTA resulted in higher
conversions compared with EziG2-AsR-ωTA (Figure
1A and Table S5). However, a general trend was
observed whereby all three EziG carrier materials
showed higher conversions at increased aw, with an
optimal performance at a value of 0.7 and drastically
lower conversions in water-saturated toluene. Notably,
more hydrophilic EziG1-AsR-ωTA also performed
well at low aw, thereby supporting the fact that water
adsorption by the carrier material enhances the
immobilized ωTA’s performance in solvents with low
Figure 1. Performance of immobilized ωTA in neat organic
solvents at controlled and varied aw values. A) AsR-ωTA on
three EziG carrier materials in toluene, B) EziG3-AsR-ωTA
applied in different reaction solvents and C) EziG3-Cv-ωTA
applied in toluene. Immobilization conditions: EziG (as
specified, 20 mg), AsR-ωTA (2 mg, 54 nmol) or Cv-ωTA
(2 mg, 38 nmol); enzyme loading: 10% w w-1, KPi buffer (1
mL, 100 mM, pH 8.0), PLP (0.1 mM), 4 °C, 120 rpm;
incubation time: 3 h. Reaction conditions: EziG3-AsR-ωTA
or EziG3-Cv-ωTA (22 mg, enzyme loading: 10% w w-1),
hydrate salts (ca. 25 mg), reaction solvent (1 mL, aw as
4
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10.1002/adsc.201901274
Advanced Synthesis & Catalysis
specified), 1a (50 mM), 2b (150 mM), 25 °C, 900 rpm;
reaction time: 72 h. Values are depicted with standard
deviations over three experiments. All reactions afforded
>99% enantiomeric excess of the amine product.
100
90
80
70
60
50
40
30
20
10
0
Another important parameter that determines the
hydration state of immobilized enzymes in non-
aqueous media is the solvation of water molecules by
the reaction solvent. In general, hydrophobic solvents
(i.e., solvents with a high log P value) serve as better
candidates because they lack the ability to strip the
[31b, 33]
enzyme of essential water molecules.
EziG3-
0
10
20
30
40
50
AsR-ωTA was tested in different organic solvents at
controlled aw in order to define the optimal solvent for
our system (Figure 1B). Organic solvents were chosen
with log P values ranging from 0.7 to 5.6 (Table S6).
EtOAc and MTBE with log P values of 0.7 and 0.9
respectively showed moderate to good conversions,
whereas n-heptane and n-decane (log P of 4.0 and 5.6
resp.) performed significantly better. A recent
assessment of the “greenness” of these solvents
equally defined them as usable but not recommended
for chemical manufacturing, with the only exception
being EtOAc that was classified as recommended.[34]
Conversions of 86-96% were obtained with the least
polar solvents at 0.4 ≤ aw ≤ 0.8, whereas water-
saturated solvents generally showed very poor
performance. Toluene at controlled aw of 0.7 was a
better solvent than n-heptane for our system because
of the higher volatile of the latter that complicates its
use particularly in a flow apparatus. Conversely, n-
decane proved to be unsuitable due to its difficult
removal from the final product by evaporation. Thus,
full conversion was observed within 48 h reaction time
using toluene as solvent (Figure 2 and Table S10).
Future studies aimed at large scale implementation of
this process must consider the replacement of toluene
with recommended “greener” alternatives possessing
similar physicochemical properties such as anisole, or
Reaction time [h]
Figure 2. Time study of EziG3-AsR-ωTA for the
transamination of 1a in toluene at controlled aw of 0.7.
Immobilization conditions: EziG3 (Fe Amber, 20 mg), AsR-
ωTA (2 mg, 54 nmol; enzyme loading: 10% w w-1), KPi
buffer (1 mL, 100 mM, pH 8.0), ), 4 °C, 120 rpm; incubation
time: 3 h. Reaction conditions: EziG3-AsR-ωTA (22 mg,
enzyme loading: 10% w w-1), salt hydrate pairs (ca. 25 mg),
reaction solvent (1 mL, aw = 0.7), 2b (150 mM), 1a (50 mM),
25 °C, 900 rpm; reaction time: 72 h. All reactions afforded
>99% enantiomeric excess of the amine product.
Next, the dependency of the reaction temperature
was tested by applying EziG3-AsR-ωTA at 25 °C,
40 °C and 50 °C in toluene (controlled aw). Similar
conversion trends were observed in the 0.2 ≤ aw ≤ 0.7
range, albeit conversions were lower at higher
temperatures (Table S8 and S6), as was previously
observed for the non-immobilized ωTA.[26] We then
tested the possibility of reducing the equivalents of the
amine donor for the reaction. Notably, an equimolar
concentration of 1a and 2b (50 mM) was sufficient to
reach 84±2% conversion in the organic solvent (Table
S9). The maximum productivity using an equimolar
concentration of 1a and 2b was obtained at 450 mM
substrates concentration, yielding 76±1% conversion
(equal to 344 mM, 52 mg of 1b (Table S11)). Finally,
recycling of EziG3-AsR-ωTA for reaction cycles at
substrate concentrations up to 400 mM was
investigated. After each reaction cycle (72 h, using 1
eq. of amine donor 2b), the reaction mixture was
separated from the biocatalyst and the conversion was
measured by GC. The same batch of EziG3-AsR-ωTA
biocatalyst was then re-suspended in a fresh reaction
mixture containing the reagents and another reaction
cycle was initiated (Figure S2 and Table S12; for
experimental details, see SI, section 4.9). At substrate
concentrations of 300 mM and 400 mM, a significant
drop in conversion was observed after the first reaction
cycle; however, it resulted in an overall analytical
product yield of 110 mg of 1b over four reaction cycles
(TTN = 13600). No evident enzyme leaching was
detected under the applied reaction conditions and
within the number of cycles that were run.
2-methyltetrahydrofuran
or
2,2,5,5-
tetramethyloxolane.[34-35] Notably, EziG3-Cv (ωTA
from Chromobacterium violaceum) employed in
toluene was found to be most active at aw ≤ 0.2 (Figure
1C and Table S7). This finding indicates that different
ωTAs operate in a neat organic environment at
different optimal aw values.
5
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10.1002/adsc.201901274
Advanced Synthesis & Catalysis
Figure 3. General loop set-up for flow reactions in organic solvent with EziG3-AsR-ωTA at controlled aw. (1) The reaction
mixture is directed back to the inlet of the flow reactor. (2) Product isolation. The apparatus comprises: a) a stainless steel
column (20 cm x 4 mm) filled with EziG3-AsR-ωTA (400 mg, enzyme loading: 10% w w-1) and Na2HPO3•5H2O/
Na2HPO4•7H2O (600 mg, ratio 1:1 w w-1); b) a stainless steel pre-column (5 cm x 10 mm) packed with
Na2HPO3•5H2O/Na2HPO4•7H2O (4 g, ratio 1:1 w w-1).
In general, we expected to obtain better system
durability by implementing the immobilized
biocatalysts in a packed-bed flow reactor. Performing
reactions in continuous flow has become a practical
tool for the recycling of enzymes.[36] In particular,
packed bed flow reactors have received considerable
attention in biocatalysis because they avoid additional
separation steps and prevent enzyme deactivation
caused by mechanical stirring in classical
bioreactors.[37] We previously reported the application
of EziG3-AsR-ωTA in a packed-bed reactor for the
kinetic resolution of racemic amines in aqueous buffer,
which proceeded without any detectable loss of
enzymatic activity for five days.[26]
tested with 20 mM 1a and 100 mM 2b in repetitive
cycles of 24 hours flow-time each (Table S14, entries
1–5). In practice, the flow system was operated for 24
h at a constant flow rate (0.2 mL min-1, 20 mL reaction
volume, 20 mM 1a) and the conversion was
determined by GC (production of 1b: ca. 32-33 mg
day-1). Then, a fresh reaction mixture was loaded and
the flow reaction was continued for another 24 h. The
process was continuously repeated for five days with
no observable loss in performance and the pure amine
product 1b was isolated in 84% isolated yield, 126 mg,
>99% chemical purity and >99% ee (measured by GC).
Notably, the flow reactor was still performing at 50%
of its initial activity after four weeks of storage at 4 °C
(Table S14, entries 6–7).
We performed the transamination of 1a in an
organic solvent in a continuous flow stainless steel
packed-bed reactor containing the immobilized ωTA
(EziG3-AsR) mixed with hydrate salt pairs (ratio 2:3 w
w-1) in order to ensure controlled aw in the system
(reactor volume: 1.82 mL, residence time, tR = 9.1 min,
flow rate: 0.2 mL min-1). The flow system was also
equipped with a pre-column containing hydrate salt
pairs in order to pre-equilibrate the organic solvent
before entering the reactor. Thus, a solution of 1a (50
mM) and 2b (150 mM) in toluene (50 mL) was
pumped through the system. In order to reduce flow
times, the outlet of the flow reactor was connected to
the inlet of the pump, thereby creating a loop (Figure
3). The flow reactor showed excellent performance in
two independent experiments and the ratio between 1a
and 1b gradually increased to ca. 70% conversion in
72 hours (STY = 1.99 g L-1 h-1) and up to above 90%
in 120 hours (Figure 4 and Table S13). In a third
independent experiment, we observed a slight decrease
in conversion over time (Table S13, entries 9–18). The
addition of a fresh aliquot of IPAm (150 mM) did not
significantly improve the performance of the flow
reactor.
Figure 4. Continuous flow reaction with EziG3-AsR-ωTA
in the transamination of 1a in toluene at controlled aw (aw =
0.7). Immobilization conditions: EziG3 (Fe Amber, 400 mg),
AsR-ωTA (40 mg, 1.07 µmol, enzyme loading: 10% w w-1)
in KPi buffer (10 mL, 100 mM, pH 8.0), 4 °C, 120 rpm;
incubation time: 3 h. Reaction conditions: EziG3-AsR-ωTA
(440 mg, enzyme loading: 10% w w-1), salt hydrate pairs (ca.
600 mg), toluene (aw = 0.7), 1a (50 mM), 2b (150 mM),
25 °C; flow rate: 0.2 mL min-1. All reactions afforded >99%
enantiomeric excess of the amine product.
In order to prove the reproducibility of the
transamination in the flow reactor, the set-up was
6
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10.1002/adsc.201901274
Advanced Synthesis & Catalysis
Analytical scale reactions in organic solvents with
immobilized ωTAs
Conclusion
We demonstrated high catalytic performance of
EziG-immobilized ωTA (total mass 20 mg, 10% w w-1
enzyme loading to support material) and hydrate salts
(Na2HPOx•yH2O/Na2HPOz•wH2O, total mass 20 mg, 1:1, w
w-1) were suspended in EtOAc (1 mL, at controlled aw) and
shaken for 15 min (900 rpm, thermomixer). The
immobilized enzyme was allowed to sediment and the
solvent was removed by pipetting. The immobilized enzyme
with hydrate salt pairs was suspended in EtOAc and the
process was repeated twice. The immobilized enzyme with
hydrate salts was washed with reaction solvent (1 mL, at
fixed aw). The immobilized enzyme was allowed to
sediment and the solvent was removed by pipetting.
Reaction solvent (900 µL, at fixed aw) was added. A 10-fold
stock of 2b was prepared in the reaction solvent and added
(final concentration: 150 mM, unless otherwise indicated).
Finally, 1a (6.89 µL, 0.05 mmol, final concentration: 50
mM) was 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 drying over MgSO4 and
analysis was performed by injection on GC with an achiral
column (see SI, Section 2.3 for details on analytical
equipment and determination). For determination of the
enantiomeric excess, the samples were derivatized using 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). After centrifugation,
the organic layer was dried over MgSO4 and analyzed by
GC with a chiral column (see SI, Section 2.3 for details on
analytical equipment and determination).
ωTAs immobilized on controlled porosity metal-ion
affinity carriers (EziG) in neat organic solvents at
controlled aw. A robust reaction system was developed
using hydrate salt pairs for the optimization and
control of cw (and hence aw) in non-polar solvents.
High catalytic activity was obtained by optimizing the
system in terms of immobilization buffer, carrier
material
and
reaction
solvent.
Significant
improvements in productivity were achieved when
applying higher substrate concentrations and only one
equivalent of amine donor was required in the reaction
to reach conversions above 80% (with >99% ee).
Finally, practical applicability was demonstrated in a
continuous flow packed-bed reactor producing a chiral
amine with excellent productivity and without
observable loss in catalytic performance over several
days of operation. This work demonstrates the
potential of continuous flow biocatalysis in neat
organic solvents using selective immobilization of
ωTAs on controlled porosity metal-ion affinity
carriers.
Experimental Section
Flow reactions in organic solvents with immobilized
ωTA
Chemicals and carrier materials
EziG3-AsR (total mass 440 mg, 10% w w-1 enzyme loading
2-propylamine, phenoxypropan-2-one and pyridoxal-5’-
phosphate (PLP) were purchased from Sigma-Aldrich
(Steinheim, Germany). The following EziG enzyme carrier
material was provided by EnginZyme AB (Solna, Sweden):
EziG1 (Fe Opal), EziG2 (Fe Coral) and EziG3 (Fe Amber).
Further details for equipment and analytical determination
are available in SI, section 2. 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.
to support material) and
a
hydrate salt pair
(Na2HPO3•5H2O/Na2HPO4•7H2O, total mass 600 mg, 1:1 w
w-1) were suspended in EtOAc (10 mL, aw = 0.7) and shaken
for 15 min (120 rpm, orbital shaker). The immobilized
enzyme was allowed to sediment and the solvent was
removed by pipetting. The immobilized enzyme with
hydrate salts was suspended in EtOAc and the process was
repeated four times. The immobilized enzyme with hydrate
salts was washed with reaction solvent (10 mL, aw = 0.7).
The immobilized enzyme was allowed to sediment and the
solvent was removed by pipetting. The immobilized enzyme
with hydrate salts was packed into a stainless steel column
(20 cm x 0.4 cm, 1.82 mL), which was attached to a Dionex
HPLC pump. A stainless steel pre-column (5 cm x 1 cm)
Expression and purification of ωTAs
C-terminal
His-tagged
(R)-selective
ωTA
from
Arthrobacter sp. (AsR-ωTA) [28] and N-terminal His-tagged
(S)-selective ωTA from Chromobacterium violaceum (Cv-
ωTA) [29a, 29b, 34b] were expressed and purified as previously
reported [26]
was
filled
with
hydrate
salts
(4
g,
Na2HPO3•5H2O/Na2HPO4•7H2O, 1:1 w w-1) and attached
between the pump and the flow reactor. A solution of 1a
(375 µL, 50 mM final concentration) and 2b (640 µL, 150
mM final concentration) in toluene (50 mL, aw = 0.7) was
pumped through the flow reactor (equipped with pre-
column) at a rate of 0.2 mL min-1. The flow reactor outlet
was directed back into the reaction mixture as shown in
Figure 3. Conversions were determined by GC equipped
with an achiral column (see SI, Section 2.3 for details on
analytical equipment and determination). A work-up was
performed by evaporation of the reaction solvent and the
residue was dissolved in 2 M HCl (12 mL). The aqueous
layer was extracted with MTBE (3 x 10 mL) and then
basified to pH 12 with 10 M KOH and extracted again with
MTBE (3 x 10 mL). The second organic layer was dried
over MgSO4 and evaporated to dryness, yielding the amine
product 1b in high purity (>99% purity by GC, >99% ee).
Immobilization of ωTAs on EziG carrier materials
On analytical scale, EziG carrier material (20±0.2 mg) was
cooled down in an ice bath and suspended in an
immobilization buffer (KPi, 1 mL, 100 mM, pH 8.0)
supplemented with PLP (0.1 mM). Purified ω-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 μL) were taken before and after the
immobilization procedure; their concentrations were
determined using the Bradford assay (see SI section 2.2 for
details). Once full immobilization was obtained, the
immobilized enzyme was let to sediment and the buffer was
removed by pipetting.
Acknowledgements
The same procedure was also followed for immobilization
at a larger scale, typically using 40 mg of purified ωTA and
400 mg of EziG carrier material.
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
7
This article is protected by copyright. All rights reserved.

10.1002/adsc.201901274
Advanced Synthesis & Catalysis
No 638271, BioSusAmin). Dutch funding from the NWO Sector
Plan for Physics and Chemistry is also acknowledged.
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7336; e) G. Zhu, Q. Huang, Z. Wang, M. Qian, Y. Jia,
Y. Tang, Biochim. Biophys. Acta 1998, 1429, 142-150.
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FULL PAPER
Continuous Flow Bioamination of Ketones in
Organic Solvents at Controlled Water Activity
using Immobilized ω-Transaminases
Adv. Synth. Catal. Year, Volume, Page – Page
Wesley Böhmer, Alexey Volkov, Karim
Engelmark Cassimjee and Francesco G. Muttia*
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