Development of an in situ-Product Crystallization (ISPC)-Concept to Shift the Reaction Equilibria of Selected Amine Transaminase-Catalyzed Reactions

Article abstract of DOI:10.1002/ejoc.201800323

The synthesis of enantiopure amines via amine transaminases involves several challenges including unfavorable reaction equilibria and product inhibition. Described here is a non-catalytic approach to overcome such problems by using an in situ-product crystallization (ISPC) to selectively remove a targeted product amine from an amine transaminase-catalyzed reaction. The continuous removal of the product amine from its reaction solution as a barely soluble salt effectively yields a displacement of the reaction equilibrium towards the products and facilitates a simple downstream processing approach via filtration. The targeted product amine is eventually obtained from the salt, while the counterion compound can be easily recycled.

Full text of DOI:10.1002/ejoc.201800323

A Journal of
Accepted Article
Title: Development of an in situ-Product Crystallization (ISPC)-Concept
to Shift the Reaction Equilibria of Selected Amine Transaminase-
Catalyzed Reactions
Authors: Dennis Hülsewede, Marco Tänzler, Philipp Süss, Andrea
Mildner, Ulf Menyes, and Jan von Langermann
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201800323
Link to VoR: http://dx.doi.org/10.1002/ejoc.201800323
Supported by

10.1002/ejoc.201800323
European Journal of Organic Chemistry
COMMUNICATION
Development of an in situ-Product Crystallization (ISPC)-Concept
to Shift the Reaction Equilibria of Selected Amine Transaminase-
Catalyzed Reactions
Dennis Hülsewede,[a] Marco Tänzler,[a] Philipp Süss,[b] Andrea Mildner,[a,c] Ulf Menyes[b] and Jan von
Langermann*[a]
Abstract: The synthesis of enantiopure amines via amine
transaminases involves several challenges including unfavorable
reaction equilibria and product inhibition. Described here is a non-
catalytic approach to overcome such problems by using an in situ-
product crystallization (ISPC) to selectively remove a targeted product
amine from an amine transaminase-catalyzed reaction. The
continuous removal of the product amine from its reaction solution as
a barely soluble salt effectively yields a displacement of the reaction
equilibrium towards the products and facilitates a simple downstream
processing approach via filtration. The targeted product amine is
eventually obtained from the salt, while the counter ion compound can
be easily recycled.
With this study we present a novel crystallization-based
approach for the direct removal of a desired product amine from
an amine transaminase-catalyzed reaction (Scheme 1).
NH3
*
O
TA, PLP
1a
2a
NH3
O
+ R-COO
3
4
+ R-COO
R-COO
NH3
*
R-COO
NH3
Biotransformations became over the past decades a powerful
technique for the synthesis of valuable compounds on laboratory
and industrial scale.[1-7] Herein pyridoxal 5’-phosphate (PLP)-
dependent transaminases (TAs) and especially amine
transaminases (ATAs) have gained in recent years a significant
impact in the synthesis of optically pure amines, which are
valuable building blocks for various agrochemicals and active
pharmaceutical ingredients, e.g. sitagliptin.[8-14] These enzymes
basically catalyze the deamination of a primary amine (amine
donor) with a simultaneous amination of an aldehyde or ketone
(amine acceptor). The transamination-reaction can be carried out
as a kinetic resolution of a racemic amine or an asymmetric
synthesis from the respective prochiral ketone.[15] Due to a
maximum yield of 100% the asymmetric synthesis is in theory
preferred, especially if a catalyst with high enantioselectivity is
used.[16] Unfortunately, thermodynamic limitations and certain
product inhibitions tend to limit the applicability of transaminases
in asymmetric synthesis, which needs to be overcome for
synthetic purposes.[17-21] Aside using an uneconomic excess of
amine donor, complex (co)product removal techniques are
currently considered, e.g. enzymatic cascades, membrane
processes and non-catalytic side reactions.[22-30] Such techniques
unfortunately increase (in general) overall complexity of
biocatalytic reaction systems, require additional or tailor-made co-
substrates and generate further by-products.[31-34]
5
6a
barely soluble salt
crystallization
high salt solubility
no crystallization
Scheme 1. Combination of an in situ-product crystallization (ISPC) with an
amine transaminase-catalyzed reaction to yield
1-phenylethylamine salt
a
barely soluble
The product amine 2 is herein selectively crystallized from
solution as a barely soluble amine salt 6, while all other reactants,
especially the applied donor amine 3, remain in solution. This in
situ-product crystallization (ISPC) continuously removes the
desired product amine from solution and thus yields an
equilibrium displacement towards the products.[35-37] The counter
ion (here shown as a carboxylate) is added directly to the reaction
solution and can be isolated for reuse from the formed solid salt.
A stoichiometric use of the carboxylate in comparison to all
applied amines is not required (see below).
First, the main requirement of this concept is the choice of a
specific counter ion, which generates a barely soluble salt with the
target amine for its crystallization, while the donor amine salt is
not crystallized. Unfortunately, most commonly used amine salts,
especially amine halides salts, show very high solubilities in
aqueous solutions and thus are not applicable for such an ISPC
concept. Moreover, simple and reliable prediction approaches for
amine salt solubilities are not available. Second, the used
transaminase has to tolerate the required concentration of the
chosen counter ion. Third, the formed salt has to be stable under
process conditions and should be easily recovered from the
reaction mixture (filtration).
[a]
Institute of Chemistry, Biocatalysis Group, University of Rostock
Albert-Einstein-Straße 3A, 18059 Rostock, Germany
E-mail: jan.langermann@uni-rostock.de
[b]
[c]
Enzymicals AG
Walther-Rathenau-Straße 49a, 17489 Greifswald, Germany
Institute for Chemical and Thermal Process Engineering
Braunschweig University of Technology
Consequently, commercially readily available aliphatic,
aromatic and heteroaromatic carboxylic acids were selected and
screened as their respective carboxylate salts towards common
amines from an amine transaminase-catalyzed reaction. 1-
Phenylethylamine and substituted derivatives 2a-f thereof served
as model product amines and were compared with typical donor
Franz-Liszt-Straße 35a, 38106 Braunschweig, Germany
Supporting information for this article is given via a link at the end of
the document.
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10.1002/ejoc.201800323
European Journal of Organic Chemistry
COMMUNICATION
amines such as isopropylamine 3, racemic 2-butylamine, DL-
alanine and L-alanine (see Supporting Information for further
details). Here the salt of the product amine needs to exhibit a
significant lower solubility then its donor amine salt counterpart
since the donor amine is still applied with an excess. This straight-
forward screening approach resulted in two benzylbenzene-
based acids and three benzoic acid derivatives that matched the
above mentioned criteria (as the respective carboxylate ions at
pH 7.5) (Scheme 2). Noteworthy, all three benzoic acid
derivatives show a 3,4-substitution pattern, which seems to be
beneficial for the crystallization of enantiopure 1-
phenylethylamine.
The results show that almost all investigated amine
transaminases are already strongly inhibited by 50 mM of the
more soluble acids 43CNA and DPAA. Herein MvATA and SpATA
were identified as the most stable enzymes. A noticeable
exception from these results is the acid 3DPPA, which is only
sparingly soluble in buffered solutions. This effectively limits the
carboxylate concentration in an aqueous solution to a maximum
of ≤25 mM, depending on temperature and pH. The excess of
solid 3DPPA remains suspended in the reaction mixture. Such a
low 3DPPA-concentration also does not significantly inhibit the
investigated ATAs. Fortunately, the investigated 3DPPA-salts
exhibit the lowest solubility of product amine salt 6, which fits
perfectly into the above mentioned ISPC-requirements (Table S3
and S4, supporting information). Consequently, 3DPPA was
identified as the most valuable acid for the application in a
crystallization-based in situ-product removal (ISPR) of amine 2
from an amine transaminase-catalyzed reaction.
COOH
COOH
diphenylacetic acid 3,3-diphenylpropionic acid
DPAA
3DPPA
The use of 3DPPA in combination with an exemplary
SpATA-catalyzed conversion of 100 mM acetophenone 1a to (S)-
1-phenylethylamine 2a shows clearly the synthetic advantage of
an acid-based ISPC (Figure 2). The classical reaction approach
N
N
Cl
Cl
COOH
O2
COOH
O2
COOH
N
O2
Cl
4-chloro-3-nitrobenzoic acid
3,4-dichloro-benzoic acid 3,4-dinitro-benzoic acid
34CA 34NA
43CNA
with
a low donor amine concentration of only 250 mM
Scheme 2. Investigated carboxylic acids for an in situ-product crystallization of
amines from an amine transaminase-catalyzed reaction
isopropylamine yields a non-sufficient conversion of 19%. A
simple addition of 1.25 eq. solid 3DPPA improved the overall
conversion directly to ca. 75%, regardless of the use as a whole
cell biocatalyst or partially purified cell extract.
For example, the isopropylamine salt of 4-chloro-3-nitrobenzoic
acid (43CNA) 5 shows a very high solubility of 993 mmol·L-1, while
the 1-phenylethylamine salt of 43CNA 6a is considerable less
soluble with 22 mmol·L-1. As given by LE CHATELIER’s principle,
the solubility of such amine salts can be further reduced if an over-
stoichiometric amount of carboxylate is added to the mother liquor,
which pushes the equilibrium from the dissociated forms in
solution towards its non-dissociated solid salt form (precipitated).
The applicability of the three best acids was tested with 7
exemplary amine transaminases from Aspergillus fumigates
(AfATA), Gibberella zeae (GzATA), Neosartorya fischeri (NfATA),
Aspergillus oryzae (AoATA), Aspergillus terreus (AtATA),
Mycobacterium vanbaalenii (MvATA) and Silicibacter pomeroyi
(SpATA) (Figure 1).
80
80
60
60
40
40
20
20
0
0
0
0
24
24
48
48
72
72
96
96
120
120
144
144
168
168
reaction time / h
reaction time / h
Figure 2. Time progression curves for a SpATA-catalyzed synthesis of
(S)-1-phenylethyl-amine with and without in situ-product crystallization (ISPC);
-whole cells or -cell extract with 125 mM 3DPPA, -whole cells or -cell
extract without 3DPPA; Conditions: 200 mM phosphate buffer pH 7.5,
100 mM acetophenone, 250 mM isopropylamine, 15 mg·mL-1 lyophilized cell
extract or whole cells, 30 °C
60
60
reference (no acid)
reference (no acid)
50 mM 43CNA
50 mM 43CNA
50 mM DPAA
50 mM DPAA
12.5 mM 3DPPA
12.5 mM 3DPPA
50
50
40
40
30
30
20
20
The majority of product 2 is afterward present as solid salt 6,
which can be almost quantitatively recovered by filtration after
cooling the reaction mixture to 0 °C. Thus this ISPC-concept with
3DPPA translates to a more atom-efficient synthesis since less
donor amine is required and a simplified downstream processing-
approach is facilitated (see below). Noteworthy, the low solubility
of 3DPPA does not limit the crystallization of product amine salt
since the constant removal of 3DPPA from aqueous solution is
continuously compensated back to its original solubility limit by a
simultaneous dissolution of 3DPPA (from excess solid).
10
10
0
0
AfATA GzATA NfATA
AfATA GzATA NfATA
AoATA
AoATA
AtATA MvATA SpATA
AtATA MvATA SpATA
transaminases / -
transaminases / -
Figure 1. Effects of 43CNA, DPAA and 3DPPA on selected amine
transaminases; Conditions: 200 mM phosphate buffer pH 7.5,
10 mM acetophenone 1, 500 mM (43CNA and DPAA) or 250 mM (3DDPA)
isopropylamine, 5 mg·mL-1 lyophilized cell extract, 30 °C, 22 h; the obtained
concentrations of 2 were intentionally held below the solubility limit of product
salt 6 to prevent an undesired in situ-product crystallization; 43CNA was chosen
as a representative of the identified benzoic acid derivatives
The shown ISPC-concept with acid 3DPPA was also
successfully used for the SpATA-catalyzed conversion of 100 mM
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10.1002/ejoc.201800323
European Journal of Organic Chemistry
COMMUNICATION
of selected acetophenone derivatives 1b-g and five non-aromatic
substrates 1h-l (Table 1). As expected, for all substrates low
conversions were obtained without ISPC due to the low, but still
over-stoichiometric, use of 250 mM isopropylamine. A simple
addition of 3DPPA increases product formation significantly for
almost all investigated substrates. Improvements range between
2 and 8.1-fold with yields of up to 96 % for 1l, while the products
are selectively crystallized as its 3DPPA-salts. Noteworthy, the
obtained high enantiomeric excesses of the product amines are
solely based on the high enantioselectivity of the applied enzyme
and are not affected by the crystallization of the respective amine
salt.[38] The remaining mother liquor, including excess
isopropylamine, can be directly reused for a further increase of
atom efficiency of the transaminase-catalyzed reaction. Even
higher donor amine concentrations will yield a further increase in
conversion, but include the risk of an undesired crystallization of
donor amine salt, which eventually yields a decrease in product
formation. A noticeable entry is also the conversion of 2-
pentanone 1i, which represents the lower limit of the shown
concept. Herein the respective product amine salt is too similar to
its donor amine salt and thus both salts crystallize simultaneously,
lowering the apparent conversion.
Alternatively, the respective hydrochloride salt can be directly
crystallized from the ether phase by a careful addition of HCl. In
addition, the spent acid 3DPPA can also be precipitated from the
remaining aqueous phase by acidification with concentrated HCl,
due to its low solubility at low pH.
Summarizing, the presented in situ-product crystallization of an
amine from an amine transaminase-catalyzed reaction by
addition of a selected acid/carboxylate presents a powerful
synthetic alternative to the use of tailor-made donor amines and
complex cascade reaction systems. The main advantages of this
ISPC are a more atom-efficient use of donor amines and a
simplified downstream processing-approach by filtration. The
targeted product amine can be afterwards extracted from its salt
and the applied 3DPPA acid easily recycled.
Experimental Section
General semi-preparative procedure for the amine transaminase-
catalyzed synthesis of 2a-m in combination with an in situ-product
crystallization. To 25 mL 200 mM phosphate buffer pH 7.5 532 µL
isopropylamine (≙ 250 mM) and 707 mg 3DPPA (≙ 125 mM) were given
and the resulting suspension adjusted to pH 7.5 with aqueous H3PO4-
solution. Afterwards PLP, substrate (≙ 100 mM) and biocatalyst were
added and the resulting mixture shaken at 200 rpm. After completion of
the reaction the resulting mixture was filtered to obtain the formed product
amine salt. (This solid will also contain the remaining biocatalyst and
excess 3DPPA.) Afterwards the solid fraction was washed with 10 mL
MTBE to remove remaining substrate and parts of excess 3DPPA. The
solid was then given into 5 mL water, 0.5 ml conc. NaOH was added to
increase pH and formed product 2 extracted with 5 mL MTBE. After phase
separation the product was obtained as its hydrochloride by a slow addition
of conc. HCl to the ether phase. 3DPPA can be precipitated from the
remaining aqueous solution by adding conc. HCl, e.g. for recycling
(isolated yield 71%).
Table 1. ISPC-supported SpATA-catalyzed synthesis of chiral amines
NH
NH33
NH
NH33
O
O
ISPC
ISPC
SpATA, PLP
SpATA, PLP
1
1
1
R1
R1
R1
R
R
R
3DPPA
3DPPA
x
x
x
x
+ IPA, -acetone
+ IPA, -acetone
+ 3DPPA
+ 3DPPA
x
x
1a-l
1a-l
2a-l
2a-l
6a-l
6a-l
substrate
R1
x
conversion
reference / %
conversion
ISPC / %
e.e.(S)
/ %[a]
1a
1b
1c
1d
1e
1f
Ph
0
0
0
0
0
0
0
0
2
3
4
1
19
21
11
8
75
69
61
46
65
37
8
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
>99.5
n.d.
m-F-Ph
p-F-Ph
m-Cl-Ph
p-Cl-Ph
m-MeO-Ph
p-MeO-Ph
Cy
General reaction control procedure. 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 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 (column:
CP-Chirasil-Dex CB; 25 m, 0.25 mm, 0.25 µm by Agilent, USA).
Enantiomeric excesses of the obtained amines were measured by High
Performance Liquid Chromatography (HPLC) using a Chiralcel OD-H
column with a n-heptane/ethanol eluent (see also supporting information).
8
10
4
1g
1h
1i
0
8
Me
61
37
20
36
53
72
78
96
n.d.
1j
Me
>99.5
98.7
Acknowledgements
1k
1l
Me
The authors thank Prof. Dr. Udo Kragl for his ongoing support &
fruitful discussions, Jennifer Kuziola & Jan-Niklas Dohm for their
help with the acid screening procedure, Dr. Dirk Michalik for his
assistance with NMR-measurments and Dr. Christine Fischer and
Eleonora Gerova-Martin from the analytical department of Leibniz
Institute for Catalysis, Rostock for measuring the enantiomeric
excesses of amine samples. Funding by German Federal Ministry
of Education and Research (BMBF – Bundesministerium für
Bildung und Forschung; project number: 031A123) and German
iPr
n.d.
[a] Values are given for the ISPC-supported reaction; Conditions: 200 mM
phosphate buffer pH 7.5, 100 mM substrate, 250 mM isopropylamine,
15 mg·mL-1 lyophilized whole cells, 30 °C; 125 mM 3DPPA for ISPC; n.d. –
not determined
Isolation of product amine 2 is easily realized by dissolving
product salt 6 in an aqueous solution at high pH, followed by
extraction with MTBE and a subsequent solvent evaporation.
Research
Foundation
(DFG
–
Deutsche
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COMMUNICATION
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Keywords: biocatalysis • amine synthesis • in situ-product
removal • crystallization • chiral
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10.1002/ejoc.201800323
European Journal of Organic Chemistry
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Dennis Hülsewede, Marco Tänzler,
Phillip Süss, Andrea Mildner, Ulf Menyes
and Jan von Langermann*
Page No. – Page No.
Development of an in situ-Product
Crystallization (ISPC)-Concept to
Shift the Reaction Equilibria of
Selected Amine Transaminase-
Catalyzed Reactions
Crystallization vs. equilibrium: A novel in situ-product crystallization method has
been developed to overcome the unfavorable equilibrium of transaminase-
catalyzed reactions. A specific carboxylic acid is added to the aqueous reaction
mixture to form a barely soluble salt with the targeted product amine, which pushes
the reaction to the product side and simplifies downstream-processing.
This article is protected by copyright. All rights reserved.