Donor Amine Salt-Based Continuous in situ-Product Crystallization in Amine Transaminase-Catalyzed Reactions

Article abstract of DOI:10.1002/adsc.201900217

The unfavorable reaction equilibrium of transaminase-catalyzed reactions is a major challenge for the efficient biocatalytic synthesis of chiral amines. In this study the synthetic utilization of a salt-based, continuous in situ-product crystallization is

Full text of DOI:10.1002/adsc.201900217

Title: Donor Amine Salt-Based Continuous <i>in situ</i>-Product
Crystallization in Amine Transaminase-Catalyzed Reactions
Authors: Dennis Hülsewede, Jan-Niklas Dohm, and Jan von
Langermann
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.201900217
Link to VoR: http://dx.doi.org/10.1002/adsc.201900217

Advanced Synthesis & Catalysis
10.1002/adsc.201900217
UPDATE
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Donor Amine Salt-Based Continuous in situ-Product
Crystallization in Amine Transaminase-Catalyzed Reactions.
Dennis Hülsewede, Jan-Niklas Dohm, Jan von Langermann*
University of Rostock, Institute of Chemistry, Biocatalytic Synthesis Group, Albert-Einstein-Str. 3A, 18059 Rostock,
Germany
Fax: (+49)-(0)381-498-6452; phone: (+49)-(0)381-498-6456; e-mail: jan.langermann@uni-rostock.de
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######.((Please
delete if not appropriate))
Abstract. The unfavorable reaction equilibrium of powerful tool for industrial applications.[3] Herein the
transaminase-catalyzed reactions is a major challenge for synthesis of chiral amines can be achieved either by
the efficient biocatalytic synthesis of chiral amines. In this kinetic resolution of a racemic amine mixture or
study the synthetic utilization of a salt-based, continuous asymmetric synthesis from prochiral carbonyl
in situ-product crystallization is described to overcome the substrates using donor amines, e.g. alanine or
[
4]
thermodynamic limit in amine transaminase-reactions using
only the commonly used amine donor isopropylamine. The
simultaneous dissolution of isopropylammonium 3,3-
diphenylpropionate (donor salt) in combination with the
isopropylamine. The direct asymmetric synthesis of
amines is typically preferred due to the theoretical
[5]
maximum yield of 100%.
However, a major challenge is often the
crystallization of the product salt facilitates
a
unfavorable reaction equilibrium of transaminase-
thermodynamic shift of the continuous amine transaminase-
catalyzed reaction. The main process necessity is a lower
product salt solubility in comparison to the applied donor
salt. This concept facilitates a stoichiometric use of
isopropylamine in combination with a significantly lowered
concentration of the amines in solution.
[6]
catalyzed reactions towards the desired products.
A
conventional solution for this drawback is the use of
significant excesses of the chosen amine donor, but
maintaining the quaternary and tertiary protein
structure under such non-physiological conditions
(
incl. high product concentration) becomes easily a
[2]
significant problem.
Alternatively, selective
Keywords: biocatalysis; in situ-product removal;
crystallization; amine; salts
(
bio)catalytic cascades can be used to remove by-
products from the reaction equilibrium, which
similarly pushes the reaction equilibrium towards the
[7]
product side. A well-known example is the use of
the donor amine alanine in combination with a lactate
dehydrogenase and a glucose dehydrogenase. Here
the byproduct pyruvate is reduced to lactate, while
the desired equilibrium shift is eventually achieved
by the glucose dehydrogenase-based cofactor
regeneration system. Unfortunately, these cascade
options require additional substrates and even entire
biocatalytic systems, which itself may cause
Transaminases (TAs) (E.C. 2.6.1.X) catalyze the
selective transfer of an amino group from an amine
[8]
donor to an amine acceptor using pyridoxal-5’-
phosphate (PLP) as a cofactor.[
1,2]
The ability of
transaminases to synthesize optically pure amines/
amino acids is a major advantage over conventional
(
transition) metal-catalyzed options and thus a
Scheme 1. Schematic representation of the continuous donor salt-dissolution and product salt-crystallization within
1
2
3
3 2 2
an amine transaminase-catalyzed reaction system. R = aliphatic/aromatic, R = CH , R = CH -CH(Ph) .
1
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Advanced Synthesis & Catalysis
10.1002/adsc.201900217
secondary issues with the transaminase-catalyzed
reaction itself and will always lower overall atom
efficiency of the entire process and increase costs.
A powerful alternative is the use of membrane-
assisted in situ-product removal (ISPR)-techniques,
This represents a strong contrast to our earlier
study, which aimed at specifically avoiding the
presence of this unwanted donor salt since it reduces
the purity of the crystallized product amine salt. The
main purpose of this donor salt in the process concept
is a continuous feed of isopropylamine (IPA, donor
amine) and the required counter-ion 3,3-
diphenylpropionate (3DPPA, for product amine
crystallization) (Scheme 1). This is achieved by the
continuous dissolution of the donor salt throughout
the entire enzymatic process, which constantly feeds
equimolar amounts of both reactants into the aqueous
solution. The donor salt exhibits a moderate solubility
in the aqueous solution, while any excess of solid
IPA-3DPPA remains suspended in the aqueous
reaction mixture without a direct, potentially harmful
interaction with the amine transaminase reaction
system (Figure 2). The application of this salt solves
the low solubility issue of 3,3-diphenylpropionic acid,
which is a main limiting parameter for the subsequent
product salt formation. In addition, the undesired pH-
shift by the acid is circumvented avoiding high buffer
concentrations to compensate this effect.
which target the removal of the product amine from
its reaction solution.[
9,10]
Rehn et al. introduced an
approach involving a supported liquid membrane
SLM) with undecane, which used an alkaline reactor
(
phase and an acidic stripping phase for product
isolation. The presented system achieved an
impressive final product concentration of 1 mol/L,
but the limited lifetime of the SLM was identified as
a
potential issue for industrial applications.
Interestingly, the authors similarly propose the
application of a continuous process, which includes a
continuous harvesting of the product from the
reaction solution.[
10]
A further synthetic approach is the use of tailor-
made amine donors, which undergo spontaneous
secondary reactions after deamination and thus shift
the reaction equilibrium, e.g. via dimerization and
oxidation.[
11]
Although this represents a valuable
approach, a varying amount of amine byproducts is
always obtained, which have to be removed and
discarded as waste.
During the continuous fed-batch process product
salt crystallization occurs continuously in parallel and
eventually enriches as the second suspended solid
phase. In the shown concept, the mother liquor is
recycled throughout the process, in contrast to
discarding it as waste. The continuous process also
does not require a significant excess of the donor
amine to push the reaction equilibrium towards the
product side.
As an alternative we’ve recently introduced the
use of in situ-product crystallization (ISPC) of the
product salt to shift the transaminase reaction
equilibrium.[
12,13]
The apparent equilibrium shift is
obtained by salt formation of the product with a
specifically chosen counter-ion, which eventually
crystallizes from solution. Product salt isolation is
then easily achieved by a simple filtration step.
However, a noticeable limitation of the presented
technique is still the inability to reach full or even
very high conversions since a residual solubility of
the product salt is unavoidable. In addition, a 2.5-fold
excess of the donor amine is still required to push the
reaction to the product side.
IPA-3DPPA (donor salt)
1PEA-3DPPA (product salt)
70
60
50
40
30
20
ΔL
With this study we aim to expand the recently
introduced ISPC-methodology to achieve full
conversion in amine transaminase-catalyzed reactions
with only a stoichiometric amount of the donor amine
isopropylamine. We propose the application of a
continuous fed batch-type ISPC-process, which
requires the presence of the donor salt
classical (non-ISPC)
reaction equilibrium
ISPC driving force
10
0
6
7
8
9
10
pH / -
isopropylammonium
IPA-3DPPA) (Figure 1).
3,3-diphenylpropionate
(
Figure 2. pH-dependency of salt solubilities; exemplary
product salt: (S)-1-phenyl ethyl ammonium 3,3-
diphenylpropionate (1PEA-3DPPA, 5a). Conditions of
non-ISPC-based batch reaction equilibrium: 100 mM
acetophenone, 250 mM isopropylamine, 30 °C, 100 mM
phosphate buffer pH 7.5.
Throughout the entire synthesis process both
concentrations of donor and product amine remain
constant in a steady state equilibrium with the
suspended solid salts, which is only controlled by
their respective solubilities. The amount of
undissolved and thus suspended donor and product
Figure 1. Chemical structure of the applied donor salt
isopropylammonium 3,3-diphenylpropionate (IPA-
DPPA).
3
2
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Advanced Synthesis & Catalysis
10.1002/adsc.201900217
salts will instead change continuously due to the
Relatively strong reductions of enzymatic activity are
visible below pH 6.5 and above pH 8. This relatively
narrow window is mostly determined by the pH-
optimum of the applied enzyme, but also influenced
by the pH-dependent solubility behavior of both salts.
Slight differences were found between the common
HEPES and phosphate buffer systems. It should be
noted that the presence of the applied buffer salts also
increases the solubilities of both salts, based on the
uncommon ion-effect, basically lowering the
performance of the ISPC-based reaction system. The
buffer capacity was therefore limited to 25 mM
throughout this study to prevent any negative effects.
Even with the lowest buffer capacity no pH shift was
observed in this study. In addition, a similar effect is
also present for reaction temperature, whereas an
optimum of 35 °C was found (Figure 4). Herein the
temperature range overlaps with the increasing salt
solubility at higher temperatures. Consequently, the
choice of the buffer salts and the pH has a significant
effect on the ISPC-process and in this study HEPES-
buffer was found to be the best choice (Figure 5).
constant
dissolution
and
in
situ-product
crystallization. These interactions can be considered
as two pH-dependent secondary equilibria, which will
overlap with the amine transaminase-catalyzed
reaction. As shown in Figure 2, the solubility of the
product salt 5a is significantly lower than the donor
salt IPA-3DPPA over a broad pH range, indicated by
the solubility difference L (L = Ldonor salt – Lproduct
salt). Importantly, product salt solubility has to be
below the original amine transaminase-based product
concentration in solution (indicated by blue line) to
force product salt crystallization, which represents the
driving force of the apparent reaction equilibrium
shift. If both salt solubilities are too similar the purity
of the product salt will be affected. With the herein
applied amine transaminase from Silicibacter
pomeroyi (SpATA) the combined optimal pH for this
ISPC-system was found to be at 7.5 (Figure 3).
100
1
00
75
50
25
0
7
5
0
5
HEPES buffer
phosphate buffer
25
0
6.5
7.0
7.5
8.0
8.5
pH [-]
NaPi
Tris HEPES bicarb. CHES
pH 7.5 pH 7.5 pH 7.5 pH 9 pH 9
Figure 3. pH-dependency of the ISPC-based SpATA-
catalyzed reaction; 30 °C, 100 mM acetophenone, 125 mM
IPA-3DPPA, 125 mM IPA, 200 mM buffer, 0.5 U/mL
SpATA, 2 mM PLP, t=22h. Relative activity is normalized
to pH 7.5 = 100%.
Figure 5. Effect of the applied buffer system on the ISPC-
based SpATA-catalyzed reaction; 30 °C, 100 mM
acetophenone, 125 mM IPA-3DPPA, 125 mM IPA, 25
mM buffer pH 7.5, 0.5 U/mL SpATA, 2 mM PLP, t=22h
100
Phosphate and TRIS buffer showed moderate to
strong reductions of enzyme activity, which is mostly
based on the (conventional) effect of different buffer
salts towards the enzyme itself. CHES and
bicarbonate buffer systems are clearly not applicable
due to their high pH.
7
5
2
5
0
5
0
A further improvement of the ISPC-process was
achieved by the use of additional isopropylamine in
the reaction mixture. This strategy is based on the
HEPES buffer
phosphate buffer
relatively high K -value of isopropylamine for many
M
25
30
35
40
amine transaminases, which is hardly reached by the
solubility limit of the applied donor salt IPA-3DPPA
alone. A maximum of process performance was
found using an additional concentration of 100 mM
isopropylamine (Figure 6).
temperature [°C]
Figure 4. Temperature-dependency of the ISPC-based
SpATA-catalyzed reaction; 100 mM acetophenone, 125
mM IPA-3DPPA, 125 mM IPA, 25 mM buffer pH 7.5, 0.5
U/mL SpATA, 2 mM PLP, t=22h. Relative activity is
normalized to 35 °C = 100%.
3
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Advanced Synthesis & Catalysis
10.1002/adsc.201900217
the blue line presents a guide for the continuous product
salt formation.
100
Substrate concentration and the solid donor amine
content were periodically re-adjusted to their original
values (100 mM 1b and 125 IPA-3DPPA) after each
cycle to ensure identical process conditions
throughout the entire reaction time. After 120h an
additional 3 U/mL SpATA (+25%) were added to
compensate for losses of enzymatic activity.
7
5
2
5
0
5
0
This fed batch process allowed a constant
HEPES buffer
phosphate buffer
product
formation
beyond
the
original
thermodynamic equilibrium, as highlighted by the
blue line in Figure 2. After an initial fast increase in
product formation, which is similar to conventional
batch reactions, the space-time yield remained
constant at ca. 4 g/(l·d), due to the continuous in situ-
product removal. Noteworthy, the presented ISPC-
0
100
200
300
400
500
additional isopropylamine [mM]
Figure 6. Effect of additional isopropylamine on the ISPC-
based SpATA-catalyzed reaction; 30 °C, 100 mM
acetophenone, 125 mM IPA-3DPPA, 25 mM buffer pH 7.5, process can be further optimized by increasing the
0
.5 U/mL SpATA, t=22h.
amount of applied biocatalyst, which was not the aim
of this study. The reaction byproduct acetone was
periodically evaporated to avoid accumulation in the
reaction solution. A major advantage of the shown
ISPC-concept is that the formed acetone content does
not have to be fully removed to facilitate a full
Even higher isopropylamine concentration yield
an increasing reduction of enzymatic activity based
on LE CHATELIER'S principle, or in this case the so-
called common ion-effect, since the additional
[14]
conversion in solution. A partial acetone removal
is still sufficient since pure product formation is
achieved in the crystallized solid phase (solid product
salt), while in solution an incomplete reaction is
present (see also Scheme 1). Significant losses of 3-
methoxyacetophenone due to unwanted evaporation
were not observed, but this will become more
substantial with other, more volatile substrates.
amount of isopropylamine results in
a
re-
crystallization of IPA-3DPPA. This effectively
lowers the concentration of 3DPPA in solution,
which itself gradually reduces crystallization of the
product salt from solution. However, higher IPA-
concentrations are not required by design since a high
conversion towards the product amine (within the
aqueous solution) is not required by the applied
ISPC-process (see also Figure 2).
Based on IPA-consumption, solely through IPA-
3DPPA addition, an impressive isolated yield of
91.5 % of 5b was achieved (see also Supplementary
information), which is significantly higher than a
conventional batch process without any ISPC (only
19%, see also Figure 2) and close to the proposed full
conversion. The additional excess of 125 mM IPA (≙
With the shown optimizations the presented
donor amine salt-based ISPC-process was eventually
performed continuously for substrate 1b at improved
reaction conditions (Figure 7).
stop of donor salt addition
1.25 eq) remains also constant throughout the entire
2
1
1
1
10
80
50
20
reaction in solution (not consumed) and is thus fully
recycled after in every cycle. The shown process was
eventually stopped by ceasing the addition of solid
rmation
ct fo
product salt
isolation
rodu
th
t p
donor salt into the reaction suspension after the 8
stan
cycle. Subsequently all remaining solid donor salt is
removed due to the ongoing conversion, which only
left the desired solid product salt as a single solid
phase. The pure product salt is then easily filtered off
and subjected to further downstream-processing.
Crystalline purity was checked by X-ray powder
diffraction, XRPD to ensure the absence of IPA-
con
9
6
3
0
0
0
0
+
25% biocatalyst
0
50
100
150
200
250
3
DPPA (see Supplementary information).
reaction time [h]
Noteworthy, the process can in theory be
Figure 7. preparative continuous ISPC-process for the
synthesis of (S)-1-(3-methoxyphenyl)ethylamine 3,3-
diphenylpropionate (S)-5b. Conditions: 25 mM HEPES
buffer pH 7.5, 100 mM 3’-methoxyacetophenone 1b (re-
adjusted every 24h), 125 mM IPA-3DPPA (re-adjusted
every 24h), 100 mM additional IPA, 30 °C, 12 U/mL
SpATA, 5 mL PLP, 1 h partial acetone removal every 24h,
continued indefinitely, but will be eventually limited
by the amount of formed solid product salt. This issue
can be easily solved by periodic filtration steps to
reduce the amount of its solid phase. In this case
donor salt addition has to be stopped temporarily as
described above to allow solid product salt as the
only solid phase. After filtration of the produce salt
new donor salt and substrate can be re-applied to the
4
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Advanced Synthesis & Catalysis
10.1002/adsc.201900217
1
0 mM sodium pyruvate in buffer solution and 250 μL
remaining mother liquor to resume the presented
continues ISPC process.
biocatalyst sample with suspended cells in buffer solution
with 0.1 mM pyridoxal phosphate. All measurements were
measured against a reference solution, whereas the
biocatalyst solution was replaced with 200 μL buffer
solution and 50 μL of 10 mM pyridoxal phosphate in
buffer solution. Buffer solution: 50 mM phosphate buffer
pH 8 with 0.25% DMSO.
In summary, the presented alternative
application of a solid donor amine salt IPA-3DPPA
as a continuous ISPC-process facilitates three
significant advances over a conventional amine donor
usage. First, a constant isopropylamine and 3DPPA
concentration is achieved throughout the entire
reaction time, which is solely based on the solubility
limit of the donor salt IPA-3DPPA. A constant
addition and control of two separate IPA- and 3DPPA
streams into the ISPC-process is not required. Any
removal of IPA-3DPPA by the biocatalytic reaction
and in situ-crystallization is autonomously
compensated by the dissolution of excess solid donor
salt. Second, the use of this continuously operated
design requires in theory only 1 mol donor amine for
the synthesis of 1 mol product amine, which
represents a lower E-factor (environmental factor)
and a huge improvement over the conventional use of
isopropylamine excesses. Moreover, the presented
continuous ISPC-process closes the gap to tailor-
made donor amines or complex biocatalytic cascades
to overcome the unfavorable reaction equilibrium.
Third, the combined amine concentration of donor
and product amine remains significantly lower in
solution, which is beneficial to ensure high catalyst
activity and stability. The majority of both amines are
only present as suspended solid salts.
General reaction procedure
A typical reaction mixture was prepared by adjusting 125
mM IPA-3DPPA and 125 mM IPA in 5 mL 25 mM
HEPES buffer pH 7.5 and adjusting the pH with conc.
3 4
H PO . Afterwards 2 mM PLP, 100 mM substrate and 0.5
U/ml lyophilized cells were added and the resulting
mixture shaken horizontally at 150 rpm at 30 °C. Samples
(
1 mL) were taken directly from the suspension and
analyzed as described below. During continuous reactions
(
20 mL reaction volume, see also Supplementary
information), samples were taken and analyzed to
determine and eventually re-adjust the substrate and IPA-
3
DPPA concentration to their original values. If required,
full consumption of IPA-3DPPA was achieved (control via
x-ray powder diffraction, XRPD) by stopping the addition
of solid IPA-3DPPA. The remaining solid (mostly product
salt) was filtered off, washed with 10 mL MTBE and dried.
Sampling
Samples (500 μL) were taken periodically and thoroughly
mixed by a vortex mixer with 50 μL conc. NaOH to
quench the reaction and increase pH. Afterwards 500 μL
MTBE were added, mixed again by a vortex mixer and the
solution was centrifuged (2 min, 3000 rpm) to improve
phase separation. 200 μL were taken from the organic layer,
combined with 50 μL of a 25 mM n-decane solution in
MTBE (internal standard) and subsequently analyzed by
gas chromatography.
The presented process can be further improved
by a direct separation of both salts in divided reaction
vessels, which will allow a direct continuous
production of the product salt in pure form.
Chromatography
Conversion was measured with a Trace 1310 gas
chromatograph by Thermo Scientific (Dreieich, Germany),
equipped with a 1300 flame ionization detector and a
Agilent Capillary HP-5 19091J-433 (0.25 mm x 30 m x
Experimental Section
Chemicals
0
.25 µm). n-Decane was used as internal standard in all
measurements. Temperatures of injector and detector were
set to 250 °C. Enantiomeric excesses of the obtained
amines were measured by High Performance Liquid
All chemicals were obtained from Acros, TCI Chemicals,
Aldrich, Alfa Aesar and ABCR and used as received.
Deionized water was used throughout this study.
[12]
Chromatography (HPLC) as described earlier.
Enzymes
Solubility measurements
E. coli BL21(DE3) strains, containing the amine
transaminase gene from Silicibacter pomeroyi were
precultivated in LB-Media (5 g/L yeast extract, 10 g/L
tryptone, and 10 g/L NaCl), with 0,1 mg/ml ampicillin as
antibiotics at 37°C and 180 rpm overnight. The preculture
was transferred the next day to 500 mL of LB-medium,
with 0,1 mg/ml ampicillin and shaken at 37 °C. After an
OD600  0.6 was reached, 0.5 mM IPTG was added to the
culture broth and shaken at 20 °C and 160 rpm for 12 h.
The obtained cells were subsequently harvested by
centrifugation, washed with phosphate buffer (50mM, pH
Solubility data were measured with 10 mL 100 mM
phosphate buffer and an excess of the respective solid salt
to create a stable suspension. The resulting mixture was
shaken at 300 rpm and 30 °C for 7 days and the pH re-
adjusted daily, if required. Afterwards the resulting
mixture was filtered to obtain a saturated crystal-free
solution. For 1PEA-3DPPA solubility a 500 µl sample was
drawn and analyzed via GC as mentioned above. For IPA-
3
DPPA analysis a sample was diluted (typically 20x), the
absorbance of 3DPPA at 249 nm measured and compared
against a calibration curve of 3DPPA in 100 mM
phosphate buffer at the identical pH (3DPPA-concentration
range: 3 – 0.5 mM, 1 cm cuvette path length).
7
.5) and lyophilized. Activity was tested via the amine
transaminase assay, as described below. Typically, 0.2-0.3
U/mg were obtained.
XRPD
Amine transaminase activity assay
Solid samples were measured via x-ray powder diffraction
Catalytic activity was measured at a wave length of 245
nm using the spectrophotometer Specord 200 from
Analytik Jena (Jena, Germany) and the extinction
(
XRPD) to discriminate the solid composition of donor salt
and/or product salt. Powder x-ray diffraction data were
collected on
a
Stoe Stadi-P with germanium-
-
1
coefficient of acetophenone: 11.852 (mM∙cm) .
monochromatised Cu-Kα-radiation (λ=1.5418 Å) in
horizontal transmission/Debey-Scherrer geometry. The x-
rays were detected with a position-sensitive detector in the
Composition of the assay: 250 μL buffer solution, 250 μL
1
0 mM (S)-1-phenylethylamine in buffer solution, 250 μL
5
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Advanced Synthesis & Catalysis
10.1002/adsc.201900217
2
Θ range from 5 to 100 °. The 40 kV high voltage and 40
Biotechnol. Bioeng. 2011, 108, 1479–1493; d) P.
Tufvesson, M. Nordblad, U. Krühne, M. Schürmann, A.
Vogel, R. Wohlgemuth, J. M. Woodley, Org. Process
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mA current were generated by a Seifert high voltage
generator (ID 3003). The equipment was controlled and the
raw data were handled with the software STOE
WinXPOW (version 2.25, 2009). The position of the
2
Theta and ω-circle were adjusted with the (111)-reflex of
crystalline silicon (2Theta=28.44°). All samples were
measured as flat preparation in poly acetate foils with
minimum amount of silicone-based grease. The sample
was spinned around its center during the measurement and
the ω-circle was spinned as well with ½ 2Theta.
[
7] a) M. Höhne, U. T. Bornscheuer, ChemCatChem 2009,
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Acknowledgements
The authors thank Prof. Dr. Udo Kragl for his ongoing support,
Dr. Dirk Michalik for his assistance with NMR measurements
and Prof. M. Köckerling and Niels Ole Giltzau for their
assistance with XRPD-measurements. Funding by German
[8] D. Koszelewski, I. Lavandera, D. Clay, D. Rozzell, W.
Kroutil, Adv. Synth. Catal. 2008, 350, 2761–2766.
[
9] a) G. Rehn, P. Adlercreutz, C. Grey, J. Biotechnol.
014, 179, 50–55; b) T. Börner, G. Rehn, C. Grey, P.
Federal Ministry of Education and Research (BMBF
–
2
Bundesministerium für Bildung und Forschung; project number:
Adlercreutz, Org. Process Res. Dev. 2015, 19, 793–
799;
0
31A123) and German Research Foundation (DFG – Deutsche
Forschungsgemeinschaft;
acknowledged.
LA
4183/1-1)
is
gratefully
[
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Advanced Synthesis & Catalysis
10.1002/adsc.201900217
UPDATE
Donor Amine Salt-Based Continuous in situ-
Product Crystallization in Amine Transaminase-
Catalyzed Reactions.
Adv. Synth. Catal. Year, Volume, Page – Page
Dennis Hülsewede, Jan-Niklas Dohm, Jan von
Langermann*
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