Use of Immobilized Amine Transaminase from Vibrio fluvialis under Flow Conditions for the Synthesis of (S)-1-(5-Fluoropyrimidin-2-yl)-ethanamine

Article abstract of DOI:10.1002/cctc.201902080

We report on the covalent immobilization of the (S)-selective amine transaminase from Vibrio fluvialis (Vf-ATA) and its use in the synthesis of (S)-1-(5-fluoropyrimidin-2-yl)-ethanamine, a key intermediate of the JAK2 kinase inhibitor AZD1480. Immobilized Vf-ATA on glyoxyl-agarose (activity recovery: 30 %) was used in a packed-bed reactor to set-up a continuous flow biotransformation coupled with a straightforward in-line purification to circumvent the 2-step process described in literature for the batch reaction. The newly developed biotransformation was run in a homogeneous system including dimethyl carbonate as a green co-solvent. Optically pure (S)-1-(5-fluoropyrimidin-2-yl)-ethanamine (ee >99 %) was isolated in 35 % yield.

Full text of DOI:10.1002/cctc.201902080

Accepted Article
Title: Use of Immobilized Amine Transaminase from Vibrio
fluvialis under Flow Conditions for the Synthesis of (S)-1-(5-
Fluoropyrimidin-2-yl)-ethanamine
Authors: Riccardo Semproli, Gianmarco Vaccaro, Erica Elisa Ferrandi,
Marta Vanoni, Teodora Bavaro, Giorgio Marrubini, Francesca
Annunziata, Paola Conti, Giovanna Speranza, Daniela Monti,
Lucia Tamborini, and Daniela Ubiali
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ChemCatChem
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Use of Immobilized Amine Transaminase from Vibrio fluvialis
under Flow Conditions for the Synthesis of (S)-1-(5-
Fluoropyrimidin-2-yl)-ethanamine
Riccardo Semproli [a,§], Gianmarco Vaccaro [a,b,§], Erica E. Ferrandi , Marta Vanoni , Teodora Bavaro
[c]
[c]
[a], Giorgio Marrubini , Francesca Annunziata , Paola Conti , Giovanna Speranza , Daniela Monti
[a]
[b]
[b]
[d]
*[c]
, Lucia Tamborini * , Daniela Ubiali *[a]
[b]
§
These authors contributed equally to this work.
Abstract: We report on the covalent immobilization of the (S)-
selective amine transaminase from Vibrio fluvialis (Vf-ATA) and its
use in the synthesis of (S)-1-(5-fluoropyrimidin-2-yl)-ethanamine, a
key intermediate of the JAK2 kinase inhibitor AZD1480. Immobilized
Vf-ATA on glyoxyl-agarose (activity recovery: 30%) was used in a
packed bed reactor to set-up a continuous flow biotransformation
coupled with a straightforward in-line purification to circumvent the 2-
step process described in literature for the batch reaction. The newly
developed biotransformation was run in a homogeneous system
including dimethyl carbonate as a green co-solvent. Optically pure
antibiotics (e.g. moxifloxacin), anti-epilepsy agents (e.g.
levetiracetam and pregabalin), and antidiabetics (e.g.
sitagliptin).[6] Among the examples of ATAs application on an
industrial scale,[
7-12]
a milestone was placed by Merck and
Codexis 10 years ago with the synthesis by transamination of
®
sitagliptin (Januvia ), the first marketed oral antihyperglycemic
drug belonging to the gliptin family. This bioprocess allowed the
replacement of a high-pressure rhodium-catalyzed asymmetric
enamine hydrogenation step by applying a highly engineered ATA
variant.[7] Later on, a number of reports about ATA immobilization
for the synthesis of this API were released.[13-16] These
outstanding achievements have demonstrated that, if optimized,
ATA-based enzymatic platforms can be competitive with the
established manufacture processes, since align selectivity,
efficiency and sustainability.[17]
(S)-1-(5-fluoropyrimidin-2-yl)-ethanamine (ee >99%) was isolated in
35% yield.
Introduction
Another example of a pre-industrial ATA-catalyzed process
was developed by AstraZeneca for the production of (S)-1-(5-
Amine transaminases (ATAs, EC 2.6.1.x) catalyze the
enantioselective amination of ketones by utilizing simple amines
fluoropyrimidin-2-yl)-ethanamine (3, Scheme 1A),
a
key
intermediate in the synthesis of the JAK2 kinase inhibitor
AZD1480, a molecule targeting idiopathic myelofibrosis and
as amino donors and the vitamin B
PLP) cofactor as
molecular shuttle.[1-4] Since the first
characterization of these enzymatic activities in the late 90s,
6
-based pyridoxal 5-phosphate
(
a
polycythaemia rubra vera.[9,18] The well-known ATA from Vibrio
[5]
fluvialis (Vf-ATA)[19-22] was selected among 60 transaminases;
ATAs have been intensively investigated for the synthesis of chiral
amines. These molecules are valuable building blocks for the
preparation of APIs including antiarrhythmics (e.g vernakalant),
process conditions were optimized on a gram scale starting from
the fluorinated ketone (1) and using (S)-α-methylbenzylamine (2,
(
S)-MBA) as the amino donor.
The reaction was performed in a biphasic system by using
toluene to pull out the co-product acetophenone. The continuous
extraction was necessary to shift reaction equilibrium and to avoid
product inhibition effects.[18] Since the synthesized amine is water
[
a]
R. Semproli, G. Vaccaro, Dr. G. Marrubini, Dr. T. Bavaro, Prof. D.
Ubiali
soluble, the purification protocol relies on the conversion of the
amine to the corresponding Boc-derivative followed by extraction
and precipitation of the amine hydrochloride with HCl in iso-
propanol. Besides co-product removal through the use of a
biphasic system as above, unfavourable reaction equilibrium of
ATA-catalyzed amine synthesis as well as product inhibition
suffered by these enzymes have been managed in different ways.
The use of amino donors in large excess combined with the in situ
removal of amine-derived by-products have been pursued, either
through an ancillary enzymatic reaction, or by exploiting specific
Department of Drug Sciences
University of Pavia
Viale Taramelli 12, I-27100 Pavia, Italy
E-mail: daniela.ubiali@unipv.it
G. Vaccaro, F. Annunziata, Prof. P. Conti, Prof. L. Tamborini
Department of Pharmaceutical Sciences
University of Milano
Via Mangiagalli 25, I-20133 Milano, Italy
E-mail: lucia.tamborini@unimi.it
Dr. E. E. Ferrandi, M. Vanoni, Dr. D. Monti
Istituto di Scienze e Tecnologie Chimiche "Giulio Natta" (SCITEC) -
CNR
[
[
[
b]
c]
d]
Via Bianco 9, I-20131 Milano, Italy
E-mail: daniela.monti@scitec.cnr.it
Prof. G. Speranza
diamines generating by-products that spontaneously undergo
cyclization/tautomerization.[23-24]
Given the increasing evidences supporting the use of
biocatalysis for industrial scale synthesis of APIs, a number of
innovative technologies have been considered as complementary
tools for the development of intensified and industrially relevant
biocatalytic processes. In particular, there is a growing interest in
performing biocatalyzed transformations in continuous flow
Department of Chemistry
University of Milano
Via Golgi 19, I-20133 Milano, Italy
Supporting information for this article is given via a link at the end of
the document.
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ChemCatChem
10.1002/cctc.201902080
FULL PAPER
reactors.[25-28] Both biocatalysis and continuous processing do
have the features that make these technologies as the foremost
key green research areas for sustainable manufacturing of APIs
and fine chemicals. Biocatalysts, either enzymes or whole cells,
are typically immobilized onto the reactor wall or on particles of a
carrier material, which are then packed into a column to form a
packed-bed reactor with a plug-flow behavior.[29] Among the
various methods for enzyme immobilization, covalent
immobilization is probably the most successful approach, allowing
a robust attachment of the enzyme. Powdery materials can be
used as carriers and packed in a fixed bed reactor as well as
porous monoliths that have been successfully used for ATA
immobilization.[30-31] Flow chemistry, along with enzyme
immobilization, can circumvent some critical issues of biocatalytic
reactions, thus positively influencing the outcome of the
biotransformation.[32]
In
particular,
whereas
enzyme
immobilization can improve enzyme stability, operating under
flow-conditions can significantly reduce substrate/product
inhibition effects and push forward unfavourable reaction
equilibria through the continuous removal of the product from the
reaction site. Moreover, a better process control makes the
reaction more efficient and minimizes waste generation.
Based on these premises, we developed a new protocol
integrating flow biocatalysis with downstream processing for the
end-to-end synthesis of (S)-1-(5-fluoropyrimidin-2-yl)-ethanamine
(3), using immobilized Vf-ATA as the biocatalyst.
Scheme 1. Synthesis of (S)-1-(5-fluoropyrimidin-2-yl)-ethanamine (3) catalyzed by Vf-ATA. A) Synthetic route as reported in ref.[18] B) Synthetic route investigated
in the present work
The immobilization of Vf-ATA on glyoxyl-agarose was
performed at pH 10 to obtain a “multi-point” interaction through
Results and Discussion
the lysine residues of the enzyme and the aldehyde groups of the
carrier. The resulting enzyme-carrier bonds are weak Schiff bases
and are thus reversible. Chemical reduction of the immobilized
The amine transaminase from Vibrio fluvialis (Vf-ATA) was
successfully produced by recombinant expression in E. coli
BL21(DE3) cells from the corresponding codon-optimized
synthetic gene (GenBank AEA39183.1). Subsequent purification
by nickel-nitrilotriacetic acid (Ni-NTA) chromatography afforded
enzyme (usually by NaBH
4
or NaCNBH3) is frequently applied[37-
to convert enzyme-carrier bonds into stable secondary amino
3
8]
groups (Scheme 2A).
80 mg of pure protein from 1 L culture (Supporting Information,
Figure S1). The specific activity of the purified enzyme was
determined spectrophotometrically in the transamination reaction
between the benchmark substrates (S)-MBA (2) and pyruvate
(
Supporting Information, Scheme S1). Two different carriers were
chosen for the immobilization: glyoxyl-agarose and Sepabeads™
EC-EP/S. Both carriers rely on the covalent interaction enzyme-
support and have been demonstrated to be versatile for the
immobilization of many enzymes.[33-34] Moreover, both glyoxyl-
agarose and Sepabeads™ EC-EP/S have been successfully
applied to biotransformations under flow conditions.[35-36]
Scheme 2. Vf-ATA immobilization. A) Immobilization on glyoxyl-agarose was
performed in sodium bicarbonate (pH 10, 50 mM) under mechanical stirring
-
1
(
protein loading: 2 mg g ; carrier/total volume:1 g/10 mL). Experiments were
performed at 4 °C or r.t. for 3 h, with or without glycerol (20% v/v). NaBH or
(1 mg mL ) was used as reducing agent. B) Immobilization on
Sepabeads™ EC-EP/S was performed in phosphate buffer (pH 8.0, 50 mM)
4
-1
NaCNBH
3
-
1
under mechanical stirring (protein loading: 1 mg g ; carrier/total volume:1 g/10
mL). Experiments were performed at r.t for 24 h. Quenching was performed with
Vf-ATA Immobilization on glyoxyl-agarose
3
M glycine in phosphate buffer (pH 8.5, 50 mM, 4 mL) under mechanical stirring
at r.t. for 20 h.
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This immobilization protocol gave quantitative yields in terms of
immobilized protein and immobilized activity: at the endpoint (3 h)
neither residual protein nor activity were found in the supernatant,
but the activity recovery was very poor (3%).
However, the activity assay performed on the immobilization
suspension when the supernatant was already non-active, but
before adding the reducing agent, showed activity (data not
shown), thus suggesting that the reduction step might be the
cause of enzyme inactivation. To confirm whether the reduction
was the critical step, three immobilization trials were performed in
parallel by following the protocol described in the Experimental
Section. When both protein content and activity were negligible in
the supernatant (3 h), two aliquots of the same immobilized
activity recovery (around 30%, Table S1). From these results,
4 °C and NaCNBH as the reducing agent were selected for the
3
following immobilization experiments. The activity recovery (30%)
was considered suitable to switch from batch to flow applications,
in agreement with previous reports.[36] Immobilization results for
glyoxyl-agarose are summarized in Figure 1.
Stability assay of Vf-ATA immobilized on glyoxyl-agarose
The immobilized enzyme must be active at least for the time
required by the reaction to be completed. However, it is desirable
that the biocatalyst maintains its activity for a longer period, thus
allowing its reuse for more reaction cycles. Vf-ATA immobilized
biocatalyst were reduced with either NaBH
respectively, whereas the third aliquot was not reduced (Figure
4
or NaCNBH
3
,
on glyoxyl-agarose (at 4 °C and reduced with NaCNBH ) was
3
incubated with the reaction mixture containing all the reactants
with the exception of the prochiral ketone (1, amino acceptor) due
to its high cost. The immobilized biocatalyst maintained about
50% of the starting activity after 72 h (Figure 2).
1A). The presence of the cyano group in NaCNBH
3
makes this
3
is generally
reducing agent milder than NaBH ; in fact, NaCNBH
4
used to selectively reduce imines even in presence of aldehydes
and ketones under neutral or weakly acid conditions. Although in
all cases the activity recovery was <10%, the highest activity
recovery was obtained for the non-reduced biocatalyst and when
1
20
00
the reduction was performed by using NaCNBH
reversibility of the imine groups could be a limitation in the use of
the immobilized enzyme, NaCNBH was selected as the reducing
3
. Since the
1
80
60
40
20
0
3
agent for the following immobilization trials. To further increase
the activity recovery of the immobilized Vf-ATA, the effect of
glycerol and temperature were evaluated (Figure 1B).
0
20
40
60
80
time (h)
Figure 2. Stability of Vf-ATA immobilized on glyoxyl-agarose (blue) compared
to free Vf-ATA (red). Experimental conditions: phosphate buffer (pH 8.0, 100
mM) containing DMSO (2% v/v) in the presence of 10 mM (S)-MBA and 1 mM
PLP. Immobilized Vf-ATA: 50 mg, final volume: 1 mL; free Vf-ATA: 2.6 mg, final
volume: 3 mL. The residual activity was determined by the spectrophotometric
assay (see Experimental Section)
Although a striking difference of stability between native and
immobilized enzyme did not emerge, the clear advantage of the
immobilized biocatalyst is its easier separation from the reaction
medium and application for in continuum biotransformations.
Figure 1. A) Effect of the reducing agent on the activity recovery (observed
activity/starting activity)x100. B) Effect of glycerol and temperature on the
activity recovery (NaCNBH
3
). These immobilization experiments were
Vf-ATA Immobilization on Sepabeads™ EC-EP/S
performed with 0.5 g of glyoxyl-agarose in a final volume of 5 mL by using the
-
1
-1
same protein loading (2 mg g ), activity loading (4.58 U g ) and run time (3 h).
For the reduction step, 5 mg of the reducing agent were added and the mixture
was stirred for further 30 min.
Besides glyoxyl-agarose, epoxy carriers are probably the
most used supports to immobilize enzymes. These resins do not
need to be pre-activated since epoxides act as the reactive
groups.[34] Thus, immobilization of Vf-ATA was also performed on
Sepabeads™ EC-EP/S. Enzymes bind covalently to this support
by exploiting the nucleophilic features of lysine -amino groups,
thus generating a multi-point interaction (as for glyoxyl-agarose).
In this case, a stable secondary amine bond is formed without the
need for any further step (Scheme 2B). However, epoxides are
much more reactive than aldehydes and may still react either with
the protein, even after the immobilization, or with substrates
and/or products of the target reaction. For this reason, unreacted
epoxy groups of Sepabeads™ EC-EP/S must be quenched in
In fact, glycerol is routinely used to preserve proteins under
storage.[39] For this reason, glycerol has been used as an additive
in some immobilization procedures; typically, the amount of
glycerol does not exceed 20% v/v.[40] Furthermore, a recent report
on Vf-ATA thermostability[41] showed that this enzyme retains
100% activity at 4 °C for more than 50 h, and 80% activity when
it is incubated at 25-37 °C. In the presence of glycerol (20% v/v)
and at low temperature (4 °C), the yields in terms of immobilized
protein and activity remained quantitative and both the
immobilization experiments showed a remarkable increase of the
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ChemCatChem
10.1002/cctc.201902080
FULL PAPER
order to avoid side-reactions.[42-43] Glycine was chosen for the
post-immobilization quenching according to a literature protocol
in which a derivatized Sepabeads™ EC-EP/S was used to
immobilize the transaminase from Halomonas elongata
Table 1. Batch synthesis of (S)-1-(5-fluoropyrimidin-2-yl)-ethanamine (3)
catalyzed by Vf-ATA immobilized on glyoxyl-agarose.
Substrate
Vf-ATA
Ratio
(acceptor:
donor)
Conversion
Time (h)
a]
[b]
(mM)
(U)[
(%)
(
HEWT).[36] The efficient immobilization of an engineered ATA
mutant from Chromobacterium violaceum on a properly designed
epoxy-functionalized carrier has been also recently described for
continuous-flow applications.[44]
20
1.80
1:1
30
31
[
a]
Vf-ATA immobilization on Sepabeads™ EC-EP/S gave
quantitative yields in terms of immobilized protein and
immobilized activity after 24 h incubation. The activity of this
biocatalyst was determined by a discontinuous assay because,
unlike glyoxyl-agarose, epoxy-acrylic carriers are bead-like
materials that interfere with the spectrophotometric analysis.
Although the activity of Vf-ATA immobilized on Sepabeads™ EC-
EP/S was remarkable (activity recovery: 50%, Table S2), it was
observed that acetophenone (4), i.e. the product of the
investigated biotransformation, was adsorbed by the carrier, thus
affecting its accurate quantification. The quenching with glycine
increased the hydrophilicity of the carrier, thus decreasing (but not
eliminating) the acetophenone adsorption. This adsorption
phenomenon was confirmed by incubating Sepabeads™ EC-
EP/S (without the enzyme) with a solution of acetophenone at a
known concentration and monitoring the supernatant absorbance
at 245 nm over time (data not shown).
The biocatalyst was immobilized at 4 °C and NaCNBH
reducing agent. Protein loading: 2 mg g ; activity recovery: 30%. The
specific activity, determined by spectrophotometric assay, was 2.76 U g .
Conversion was calculated by HPLC. The reaction was performed in
duplicate
3
was used as the
-
1
-
1 [b]
After the reaction, the biocatalyst was easily separated from
the reaction mixture by filtration and showed a residual activity of
0%. This result was consistent with the stability assay of the
immobilized Vf-ATA. Furthermore, no protein release was
detected in the reaction (Bradford assay), as a consequence of
the covalent immobilization.
The data obtained in the batch reaction were considered
suitable to switch the bioconversion to a flow system and thus to
run the study both of the reaction and the downstream in such a
set-up.
5
[
46]
Flow synthesis of (S)-1-(5-fluoropyrimidin-2-yl)-ethanamine
(
3)
Taking in consideration this phenomenon, glyoxyl-agarose
appeared a more convenient carrier due to the acceptable activity
A packed bed reactor with immobilized Vf-ATA was prepared
recovery and stability. Moreover, this carrier was already shown
to be suitable for flow applications.[35]
for the continuous flow application. First, the effect of residence
time and temperature on the reaction outcome was evaluated
using a 0.65 mL volume bioreactor (immobilized Vf-ATA = 0.65 g)
and flowing through it a solution of 1-(5-fluoropyrimidin-2-yl)-
ethan-1-one (1, 20 mM), (S)-MBA (2, 20 mM) and PLP (1 mM) in
phosphate buffer (100 mM, pH 8) containing DMSO (2% v/v).
Conversions (%) were calculated by HPLC, using the
acetophenone calibration curve. As reported in Table 2, a 40%
conversion was achieved in 10 minutes of residence time (entry
2); a further increase of the residence time was not beneficial
(entries 3 and 4). Keeping constant the residence time (10 min),
concentration of the substrates in the feeding flow was varied, but
the conversion was dramatically reduced (entry 6). Also, an
increase of the temperature up to 38 °C did not speed up the
reaction.
Batch synthesis of (S)-1-(5-fluoropyrimidin-2-yl)-ethanamine
(
3)
The enzymatic synthesis of (S)-1-(5-fluoropyrimidin-2-yl)-
ethanamine (3) was performed by using Vf-ATA immobilized on
glyoxyl-agarose in the view of transferring the bioconversion from
batch to a flow system. As a preliminary approach, the amination
of the non-fluorinated ketone was carried out with the aim of
exploring the reaction conditions, testing the feasibility of the
bioconversion with the newly immobilized enzyme, and setting-up
a downstream process that might be more straightforward than
that described in literature.[18] (S)-MBA (2) was selected as the
amino donor because it was also used in the activity assay and it
is commonly used as benchmark substrate with (S)-selective
transaminases (both in batch and in flow systems).[20,36,45]
Moreover, (S)-MBA is the amino donor used in the synthesis of
Under a similar degree of conversion (i.e. c=30% after 31 h
for the batch biotransformation and c=26% in 5 minutes for the
flow biotransformation), the specific reaction rate (r) of the flow
reaction resulted to be about 22-fold higher than the batch
reaction (rflow=0.52 µmol min-1 mg ; rbatch=0.024 µmol min-1 mg-1),
probably due to the high local concentration of the biocatalyst in
the packed bed reactor.[47]
(
S)-1-(5-fluoropyrimidin-2-yl)-ethanamine reported by Meadows
et al.[ The reaction was performed at pH 8, according to the
enzymatic assay conditions (see also Figure S2). In contrast to
literature,[18] in this work a monophasic system containing DMSO
18]
-1
(
2%) as the co-solvent was used. As reported in Table 1, a 30%
To increase the sustainability of the proposed protocol, we
tested dimethyl carbonate as the co-solvent (5% v/v) for the flow
reaction and evaluated the reaction outcome under continuous
work.
To this aim, a 2 mL bioreactor was prepared and 100 mL of
the substrate solution was flowed through it under the following
conditions: residence time=10 min, T=28 °C, P=atm. Overall, a
conversion was observed which was comparable with the results
obtained with the non-fluorinated ketone (data not shown).
Conversions (%) were calculated by HPLC, using the
acetophenone calibration curve.
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ChemCatChem
10.1002/cctc.201902080
FULL PAPER
3
8% conversion was obtained. Finally, an in-line purification
buffer, pH 8.0, 5% v/v dimethyl carbonate) gave the same ee%
(90%): this result thus ruled out the influence of immobilization on
the incomplete (S)-enantioselectivity of Vf-ATA.
procedure for product isolation was developed (Scheme 1B).
The same result was obtained upon analyzing the reaction
mixtures containing two different co-solvents (DMSO and
dimethyl carbonate, respectively), thus excluding a role of these
solvents, too.
Our attention was then focused on PLP, the co-factor of ATAs.
PLP is an activated aldehyde and the formation of imines is its
natural role. It has been reported that PLP, upon the formation of
Table 2. Flow synthesis of (S)-1-(5-fluoropyrimidin-2-yl)-ethanamine (3)
catalyzed by immobilized Vf-ATA.[
a]
Entry
Substrate
Residence
time (min)
Temperature
(°C)
Conversion
(%)[
b]
(mM)
1
2
3
4
5
6
20
5
28
28
28
28
38
28
26
40
39
39
38
17
20
10
15
30
10
10
a Schiff base with -amino acid esters or amines, enhances
racemization through a reversible proton migration.[
51]
This
20
phenomenon has been exploited for the dynamic resolution of a
number of racemic substrates.[52-54] The hypothesized mechanism
of PLP toward (S)-1-(5-fluoropyrimidin-2-yl)-ethanamine (3) is
reported in Scheme S2 (Supporting Information).
20
20
The chiral analysis of a batch biotransformation which was
performed without adding PLP showed a complete ee%, thus
supporting our hypothesis concerning the role of PLP in the amine
racemization. In this case, the (S)-amine formation catalyzed by
Vf-ATA relies exclusively on the PLP intrinsically bound to Vf-ATA.
We concluded, indeed, that the driving force of the side-reaction
was the concentration of PLP used in the reactions (1-2 mM)
which is probably uselessly high for the biotransformation,
although consistent with other reports on transaminases.[
Furthermore, the hypothesized mechanism might be enhanced by
the alkaline pH of the reaction mixture: at this pH value (around
40
[
a]
Immobilized Vf-ATA: 0.65 g; reactor volume: 0.65 mL; ratio acceptor/donor
1
:1; PLP 1 mM; flow stream: phosphate buffer (100 mM, pH 8.0) containing
[
b]
DMSO (2% v/v); atmospheric pressure.
HPLC
Conversion was calculated by
A first attempt to extract the produced amine using an inlet
of an organic solvent (i.e. EtOAc) and a membrane phase
separator was performed. However, only a low amount of (S)-1-
55-58]
(
5-fluoropyrimidin-2-yl)-ethanamine (3) was recovered in the
organic phase (<5%). Therefore, we exploited a catch-and-
release strategy, using a column packed with a strongly acidic
resin (Dowex Marathon C) to trap any unreacted (S)-MBA and the
desired amine. Then, after a washing step with distilled water, an
inlet of aqueous ammonia (1 M) was used to release the amine
product that, due to the different pka compared to (S)-MBA, was
collected separately and isolated in 35% yield.
8-9), ATAs show the highest activity, but amines are predominant
in their neutral form. Thus, the nucleophilic reaction of amines with
the PLP aldehyde group is plausible to occur. On the other hand,
although the absence of PLP in the reaction mixture allowed to
restore the complete enantioselectivity of Vf-ATA and to achieve
a ee>99%, this condition was detrimental for the biocatalyst. After
this reaction, immobilized Vf-ATA lost almost completely its
activity. The concentration of PLP is therefore a crucial parameter
which deserves to be further investigated.
Determination of the enantiomeric excess of (S)-1-(5-
fluoropyrimidin-2-yl)-ethanamine (3)
In this context, we tested the reaction without PLP under
continuous flow using the conditions reported above. However, a
very low conversion (<5%) was obtained. Therefore, increasing
amounts of PLP (i.e., 0.1 mM and 0.25 mM) were added to the
substrate solution that was flowed through the bioreactor and the
performance of the biotransformation was evaluated. Using 0.1
mM PLP, again, a very low conversion in 10 minutes of residence
time was observed, whereas, using 0.25 mM PLP, a conversion
comparable to the previous experiments (38%) was achieved.
The amine was isolated as reported above and the enantiomeric
excess was assessed by chiral HPLC, resulting to be >99%.
It is worth reporting that the use of tailor-made carriers which
can enable the co-immobilization of transaminase and PLP has
been recently described.[38] Through this strategy, the exogenous
addition of cofactor is avoided, since a self-sufficient source of
PLP is ensured by the co-immobilized preparation for an efficient
catalysis.
Upon in-line purification, the target amine was submitted to a
HPLC chiral analysis to assess the enantiomeric excess. The
absolute configuration of a chiral amino donor has to match the
stereospecificity of the ATAs in order to be accepted: this feature
explains why transaminases can be employed in the kinetic
resolution of racemic amines.[48] For this reason, Vf-ATA, which is
a (S)-selective ATA, should not be able to produce amines with
(
R)-configuration. Surprisingly, the chiral HPLC analysis of the
amine synthesized enzymatically did result in a 90% enantiomeric
excess for the S-enantiomer which was totally unexpected. To
shed light on this result, new experiments were performed. Taking
into account that both immobilization and the presence of organic
co-solvents can alter enzyme selectivity,[49-50] the reaction was
repeated both by using the non-immobilized enzyme and either
DMSO or dimethyl carbonate as co-solvent. However, it is worth
to underline that, to the best of our knowledge, decrease and/or
inversion of enantioselectivity have never been reported for
transaminases.
The biotransformation performed in batch by using the soluble
Vf-ATA under the same conditions of the flow reaction (donor and Conclusions
acceptor: 200 µmol; PLP: 20 µmol; medium: 100 mM phosphate
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201902080
FULL PAPER
A new protocol for the synthesis of (S)-1-(5-fluoropyrimidin-2-yl)-
ethanamine (3), which integrates flow biocatalysis with an in-line
downstream processing, was successfully developed.
The main benefits of the set-up herein described include the
increased specific reaction rate in comparison with the batch
protocol and a significantly reduced time of the overall process
37°C. Colonies were aseptically removed, inoculated in 50 mL of LBKAN30
medium and incubated overnight at 37 °C and 220 rpm. The pre-cultures
were used for the inoculation of 500 mL of LBKAN30 medium, then incubated
at 37 °C and 220 rpm. When the OD600 value was between 0.5 and 1, IPTG
(0.5 M stock solution) was added till a final concentration of 1 mM to induce
the expression. The Vf-ATA cultures were incubated at 30 °C and 220 rpm
for 24 h. At the endpoint of the expression, the cells were recovered by
centrifugation at 5000 rpm and 4 °C for 30 min and resuspended in 20 mL
of washing buffer (500 mM NaCl, 20 mM imidazole, 20 mM potassium
phosphate buffer, pH 7.0). Cells were lysed by three sonication cycles at
(
reaction and product recovery). Interestingly, a possible role
played by the cofactor PLP in transaminase-catalyzed amination
of ketones was highlighted; albeit essential for the catalysis, PLP
seemed to affect the enantioselectivity of the transamination
reaction and, indeed, its concentration needs to be finely tuned.
These results demonstrated that the combination of biocatalysis
and flow chemistry should be regarded as a truly competitive
approach with established fully chemical protocols.
0
°C and the cell lysate was centrifuged at 10000 rpm and 4 °C for 30 min.
The transaminase was purified by IMAC using Ni-NTA Sepharose 6 Fast
Flow resin (GE Healthcare, Italy) as the stationary phase according to the
manufacturer's instruction. The eluates containing the target protein were
collected and dialyzed for 16 h at 4 °C in 50 mM phosphate buffer pH 9.
After the dialysis, samples were stored at -80 °C. Protein content was
measured using the Bio-Rad Protein Assay according to the method of
Bradford, bovine serum albumine (BSA) was used as standard protein.
Protein purity was verified by SDS-PAGE analysis (10% T, 2.6% C). The
molecular weight protein standard mixture from BioRad (Karlsruhe,
Germany) was used as reference. Gels were stained for protein detection
with Coomassie Brilliant Blue.
Experimental Section
General
All reagents and solvents were purchased from Sigma-Aldrich® Srl (Milan,
Italy) unless otherwise stated. 1-(5-Fluoropyrimidin-2-yl)-ethan-1-one (1)
was purchased from Fluorochem (Hadfield, UK). For the production,
purification and characterization of the enzyme, the following instruments
were used: Thermoshaker INNOVA 42 (New Brunswick); Sorvall RC6 Plus
Glyoxyl-agarose preparation
Glyoxyl-agarose was prepared as reported by Guisan.[59] Briefly, agarose
(
10 g) was suspended in deionized water (2.8 mL) at 4 °C under
mechanical stirring. A 1.7 M solution of NaOH (4.8 mL) containing NaBH
4
(Thermo) centrifuge, A.L.C. 4226 centrifuge, Omni-Ruptor 250 sonicator,
-
1
(
14.3 mg mL ) was added. Etherification was carried out in an ice bath by
UV-VIS V-530 (Jasco) spectrophotometer or
spectrophotometer (Shimadzu).
a
UV 1601
adding dropwise glycidol (3.4 mL). The reaction was carried out for 18 h.
After the incubation period, the suspension was filtered and the carrier was
washed with deionized water. Oxidation was initiated by adding 100 mM
NaIO (68 mL). The reaction was carried out for 2 h at room temperature,
4
then the carrier was filtered under reduced pressure and washed
thoroughly with deionized water and stored at 4 °C.
Agarose gel 6B-CL was purchased from Amersham Biosciences (Uppsala,
Sweden) and activated to glyoxyl-agarose as previously reported.[50]
Sepabeads™ EC-EP/S was kindly supplied by Resindion srl (Binasco,
Italy).
The flow experiments were performed by using a R2 +/R4 flow reactor
(Vapourtec).
Vf-ATA immobilization on glyoxyl-agarose
Immobilization yields
Vf-ATA (2 mg; 9.3 U) was incubated with glyoxyl-agarose (1 g) in NaHCO
3
buffer (pH 10, 50 mM, 10 mL) at 4 °C for 3 h under mechanical stirring.
Chemical reduction of imines was carried out over 30 min by adding
The immobilization yields (%) were determined as: immobilized protein,
immobilized activity and activity recovery as reported by Sheldon et al.[60]
NaCNBH
filtered under vacuum and washed thoroughly with deionized water and
HPO buffer (pH 8, 50 mM) and stored at 4 °C. The immobilization
3
(10 mg) to the mixture. The immobilized enzyme was then
according to the following equations:
Immobilized protein (%): (immobilized protein/loaded protein) x 100;
immobilized activity (%): (immobilized activity/loaded activity) x 100;
activity recovery (%): (observed activity of the immobilized enzyme/starting
activity) x 100.
K
2
4
procedure was monitored by evaluating the residual protein and the
residual activity of the supernatant by Bradford[46] and activity assay,
respectively. For the flow reactions, a protein loading of 5 mg/g was used.
Specific reaction rate
Vf-ATA immobilization on Sepabeads™ EC-EP/S
The specific reaction rate for batch (rbatch) was calculated from the amount
of the product (P, µmol), the reaction time (t, min), and the mass of
Vf-ATA (1 mg; 1.23 U) was incubated with Sepabeads™ EC-EP/S (1 g),
previously hydrated with deionized water (10 mL) under gentle shaking for
(µmol min-1 mg ). The
-1
biocatalyst employed (m
specific reaction rate for flow biotransformation (rflow) was calculated from
E
, mg): rbatch = nP/t m
E
1
h in phosphate buffer (pH 8, 50 mM, 5 mL), at room temperature for 24
-
1
-
h under mechanical stirring. The immobilization procedure was monitored
by evaluating the residual protein and the residual activity of the
supernatant by Bradford assay[ and activity assay, respectively. The
immobilized enzyme was then filtered, washed with deionized water and
the concentration of the product ([P], µmol mL ), the flow rate (f, mL min
1)
E E
, and the mass of protein employed (m , mg): rflow = [P] × f / m (µmol
46]
min-1 mg-1).
re-suspended in K
(
2
HPO
4
buffer (pH 8.5, 50 mM, 4 mL) containing glycine
Vf-ATA expression and purification
3 M) and stirred for further 20 h to quench the unreacted epoxides.[
36,43]
The biocatalyst was eventually filtered under vacuum, washed thoroughly
with deionized water and K HPO buffer (pH 8, 50 mM) and stored at 4 °C.
2 4
The amine transaminase from Vibrio fluvialis (Vf-ATA) was produced as
previously reported.[57] Briefly, E. coli BL21(DE3) cells carrying the
expression plasmid for Vf-ATA were streaked on LB-Agar plates
Vf-ATA activity assay
-
1
containing kanamycin 30 µg mL (LBKAN30) and incubated overnight at
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201902080
FULL PAPER
Enzyme specific activity was determined by a spectrophotometric assay
(
was directed into a column packed with Dowex Marathon C resin (reactor
volume: 5 mL) that was previously activated with 2 M HCl and washed with
Scheme S1) as previously reported.[45] Briefly, phosphate buffer (pH 8, 50
mM,
3
mL) containing sodium pyruvate (2.5 mM), (S)-α-methyl
H
2
O. The desired amine was trapped by the resin. At the end of the
reaction, the column packed with Dowex Marathon C resin was washed by
flowing H
O at 0.25 mL min-1 for 20 min. (S)-1-(5-fluoropyrimidin-2-yl)-
ethanamine 3 was then released using a 1 M solution of NH OH at 0.25
benzylamine ((S)-MBA, 2.5 mM) and DMSO (0.25 % v/v) was poured in a
quartz cuvette. Soluble Vf-ATA (6 µL) was added and the absorbance of
acetophenone (ε=12 mM-1 cm ) at 245 nm was detected for 60 s under
the kinetic mode. One unit (U) is defined as the amount of enzyme which
produces one µmol of acetophenone per minute under the above
conditions. For the immobilized Vf-ATA, the same assay was performed
by using 10-15 mg of the immobilized biocatalyst under magnetic stirring
for 180 s. Enzymatic activity was determined according to Equation 1. The
measurements were performed at least in duplicate.
2
-1
4
mL min-1 for 30 min. The flow stream was collected in fractions of 2.0 mL;
TLC assays were performed to select the samples containing the pure
product. After evaporation of the solvent, the pure amine was recovered in
35% isolated yield (99 mg). H NMR (CDCl
4.07-4.15 (m, 1H), 1.20 (d, 3H).
1
3
) δ 8.60 (s, 2H), 4.65 (br s 2H),
Chemical synthesis of racemic 1-(5-fluoropyrimidin-2-yl)-ethanamine
U
ΔA/minx Vs
ε x E
=
mL or g
The chemical synthesis of racemic 1-(5-fluoropyrimidin-2-yl) ethanamine
was performed slightly modifying a previously reported procedure.[61] i) 1-
(5-Fluoropyrimidin-2-yl)-ethan-1-one 1 (200 mg, 1.43 mmol) was dissolved
in MeOH (5 mL) at room temperature. The solution was cooled at 0 °C and
-
1
Equation 1. ∆A/min= slope of Abs vs time curve (mAU min ), Vs = total assay
volume (mL), ε = acetophenone molar absorbivity (12.000 mM cm ), E =
amount of soluble enzyme (mL) or immobilized enzyme (g)
-
1
-1
4
NaBH (54 mg) was added. The resulting mixture was stirred at room
temperature for 1 h. Water (2 mL) was added and the organic solvent was
removed under reduced pressure; the aqueous phase was extracted with
The activity of Vf-ATA immobilized on Sepabeds™ EC-EP/S was
determined by a discontinuous assay. Three aliquots of immobilized Vf-
ATA (15 mg) were placed in plastic tubes and the assay solution (2 mL)
was added. The samples were kept under gentle stirring (rotary shaking);
every two minutes, a sample was quenched with HCl (2 M, 2 mL) and the
absorbance at 245 nm was registered, as a single reading. Acetophenone
EtOAc (3 x 3 mL). The organic extracts were dried over Na
evaporation of the volatiles under reduced pressure, 1-(5-fluoropyrimidin-
-yl)ethan-1-ol was obtained as a red oil (75% yield). ii) The alcohol
obtained from the previous step (152 mg, 1.07 mmol) was dissolved in
CH Cl (2 mL) and triethylamine (297 µL, 2.14 mmol) was added at room
2 4
SO and, after
2
2
2
(µmol) was quantified by a calibration curve and used to determine the
temperature. The solution was cooled to 0 °C, and methanesulfonyl
chloride (100 µL, 1.28 mmol) was added dropwise. The resulting mixture
was allowed to stir at r.t. for 2 h. The mixture was washed with 1 M HCl (2
enzyme activity.
Residual activity of Vf-ATA immobilized on glyoxyl-agarose
x 2 mL), the organic phase was dried over Na
reduced pressure to give 1-(5-fluoropyrimidin-2-yl)ethyl methanesulfonate
90% yield). iii) The residue (212 mg, 0.96 mmol) was dissolved in DMF (5
2 4
SO and evaporated under
A stock solution containing (S)-MBA (64.5 µL, 500 µmol), DMSO (1 mL) in
phosphate buffer (pH 8, 100 mM, 49 mL) and PLP (12.4 mg, 50 µmol) was
prepared. Aliquots (50 mg) of immobilized Vf-ATA were placed in
eppendorf micro tubes and 1 mL of the stock solution was added to each
eppendorf. The samples were kept under stirring (rotary shaker) at 25 °C.
At fixed times, each sample was filtered under vacuum, rinsed thoroughly
with distilled water, conditioned with phosphate buffer (pH 8, 50 mM) and
(
mL) and treated with sodium azide (62 mg, 0.96 mmol). The resulting
mixture was stirred at r.t. for 72 h and was then partitioned between EtOAc
(
5 mL) and brine (10 mL). The organic layer was collected, dried over
Na SO , and evaporated under reduced pressure. The crude material was
purified by flash column chromatography as a colorless oil (55% yield). iv)
-(1-Azidoethyl)-5-fluoropyrimidine (88 mg, 0.53 mmol) was dissolved in
2
4
2
-
1
dried for 5 min. Residual activity (U g ) was measured by submitting the
filtered immobilized enzyme (10-15 mg) to the spectrophotometric assay
MeOH (5 mL) and submitted to a hydrogenation reaction in a Thales-Nano
-1
H-Cube Mini (cartridge: Pd/C 10%, flow rate: 1 mL min ). The exiting
-
1
as reported above and compared with the activity (U g ) of the enzyme
before incubation, which was considered as 100%.
solution was evaporated under reduced pressure to afford racemic 1-(5-
fluoropyrimidin-2-yl) ethanamine as a pale yellow oil in quantitative yield.
R
HPLC chiral analysis retention times (t ): (S)-MBA (2) = 3.8 min, (S)-1-(5-
Batch synthesis of (S)-1-(5-fluoropyrimidin-2-yl)-ethanamine (3)
fluoropyrimidin-2-yl)-ethanamine (3) = 5.9 min; (R)- 1-(5-fluoropyrimidin-2-
yl)-ethanamine = 6.1 min; 1-(5-fluoropyrimidin-2-yl)-ethan-1-one (1) = 8.0
min.
1-(5-Fluoropyrimidin-2-yl)-ethanone 1 (28.0 mg, 200 µmol) was dissolved
in DMSO (200 µL) and added to phosphate buffer (pH 8, 100 mM, 9.8 mL)
containing PLP (4.9 mg, 20 µmol) and (S)-MBA 2 (25.8 µL, 200 µmol) in a
plastic flask. Immobilized Vf-ATA (652 mg, 1.80 U) was added and the
mixture was kept under mechanical stirring. Samples were taken, diluted
with bidistilled water (1:10) and analyzed by HPLC. Once the highest
conversion was achieved, the biocatalyst was recovered by filtration under
vacuum.
Analytical methods
HPLC analyses of batch reactions were performed on a HPLC Chromaster
00 bar System, Merck Hitachi VWR, equipped with a Purospher® STAR
6
RP-18 endcapped (3 µm), Hibar HR 100-2.1, UHPLC column, Sorbent Lot
No. TA 1822868 (Merck KGaA, Darmstadt, Germany). Injection volume: 2
µL; mobile phase: H
2
O + TFA 0.05% (A)/ACN +TFA 0.05% (B); gradient
Flow synthesis of (S)-1-(5-fluoropyrimidin-2-yl)-ethanamine (3)
conditions: 0-5 min 100% A; 520 min 50% (A)/50% (B); flow rate: 0.3 mL
-
1
min ; λ: 245-254 nm. Retention times (t
ethanamine (3)=3.60 min, 1-(5-fluoropyrimidin-2-yl)-ethan-1-one (1)=
3.40 min, (S)-MBA (2)= 14.37 min, acetophenone (4)= 20.90 min.
R
): (S)-1-(5-fluoropyrimidin-2-yl)-
Immobilized Vf-ATA (2.0 g) was packed into a glass column (Omnifit, 6.6
mm i.d. x 100 mm length). The packed bed reactor was washed with a 100
mM phosphate buffer pH 8 at 100 µL min-1 for 10 min. The obtained reactor
volume was 2.0 mL. A solution (100 mL) of 1-(5-fluoropyrimidin-2-yl)-
ethan-1-one (1, 20 mM), (S)-MBA (2, 20 mM) and PLP (1 mM) in
1
HPLC analyses of the flow reactions were performed on a Breeze 2 HPLC
System, Waters, equipped with a Waters 2489 UV–vis detector (Waters,
Milford, MA) and a Phenomenex Synergi column (150 mm × 4.6 mm, 4
μm). Injection volume: 10 µL; mobile phase: H
2
O + 0.05% TFA(A)/ACN +
phosphate buffer (pH 8, 100 mM) with 5% v/v dimethyl carbonate was
prepared. The solution was pumped at 0.20 mL min-1 (residence time = 10
min) through the bioreactor maintained at 28 °C. The exiting flow stream
0.05% (B); gradient conditions: 0-9 min 90% (A)/10% (B), 914 min 50%
-
1
(
A)/50% (B); flow rate: 1.0 mL min ; λ: 254 nm. Reaction samples were
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201902080
FULL PAPER
diluted with a solution 1:10 H
2
O/ACN + 0.05% TFA. Retention times (t
R
):
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S)-1-(5-fluoropyrimidin-2-yl)-ethanamine (3) = 2.7 min, (S)-MBA (2)= 5.6
min, 1-(5-fluoropyrimidin-2-yl)-ethan-1-one (1)= 7.0 min, acetophenone (4)
16.4 min.
HPLC chiral analyses were performed using a Jasco PU-980 pump
PerkinElmer, Inc., Waltham, MA) equipped with a UV–vis detector Jasco
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Riccardo Semproli, Gianmarco Vaccaro,
Erica E. Ferrandi, Marta Vanoni,
Teodora Bavaro, Giorgio Marrubini,
Francesca Annunziata, Paola Conti,
Giovanna Speranza, Daniela Monti*,
Lucia Tamborini*, Daniela Ubiali*
The amine transaminase from Vibrio fluvialis (Vf-ATA) was covalently immobilized
and used in the synthesis of (S)-1-(5-fluoropyrimidin-2-yl)-ethanamine (3) in a flow
system. Coupling of the continuous flow biotransformation with an in-line purification
step resulted in a highly productive and straightforward bioprocess.
Page No. – Page No.
Use of Immobilized Amine
Transaminase from Vibrio fluvialis
under Flow Conditions for the
Synthesis of (S)-1-(5-Fluoropyrimidin-
2-yl)-ethanamine
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