Chemoenzymatic Synthesis of Sialic Acid Derivatives Using Immobilized N-Acetylneuraminate Lyase in a Continuous Flow Reactor

Article abstract of DOI:10.1002/adsc.201900146

The synthesis of N-acetylneuraminic acid (Neu5Ac) derivatives is drawing more and more attention in glycobiology research because of the important role of sialic acids in e. g. cancer, bacterial, and healthy cells. Chemical preparation of these carbohydrates typically relies on multistep synthetic procedures leading to low overall yields. Herein we report a continuous flow process involving N-acetylneuraminate lyase (NAL) immobilized on Immobead 150P (Immobead-NAL) to prepare Neu5Ac derivatives. Batch experiments with Immobead-NAL showed equal activity as the native enzyme. Moreover, by using a fivefold excess of either N-acetyl-D-mannosamine (ManNAc) or pyruvate the conversion and isolated yield of Neu5Ac were significantly improved. To further increase the efficiency of the process, a flow setup was designed providing a chemoenzymatic entry into a series of N-functionalized Neu5Ac derivatives in conversions of 48–82%, and showing excellent stability over 1 week of continuous use. (Figure presented.).

Full text of DOI:10.1002/adsc.201900146

Title: Chemoenzymatic Synthesis of Sialic Acid Derivatives Using
Immobilized N-Acetylneuraminate Lyase in a Continuous Flow
Reactor
Authors: Victor Bloemendal, Sam Moons, Jurriaan Heming, Mohamed
Chayoua, Olaf Niesink, Jan van Hest, Thomas Boltje, and
Floris Rutjes
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of the final Version of Record (VoR). This work is currently citable by
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content of this Accepted Article.
To be cited as: Adv. Synth. Catal. 10.1002/adsc.201900146
Link to VoR: http://dx.doi.org/10.1002/adsc.201900146

Advanced Synthesis & Catalysis
10.1002/adsc.201900146
R
COMMUNICATION
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Chemoenzymatic Synthesis of Sialic Acid Derivatives Using
Immobilized N-Acetylneuraminate Lyase in a Continuous Flow
Reactor
†
,a
†,a
a
Victor R. L. J. Bloemendal, Sam J. Moons, Jurriaan J. A. Heming, Mohamed
a
a
b
a
Chayoua, Olaf Niesink, Jan C. M. van Hest, Thomas J. Boltje, Floris P. J. T.
a,
Rutjes *
a
Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
b
Bio-organic chemistry, Eindhoven University of Technology, P.O. Box 513 (STO 3.31), 5600 MB Eindhoven, The
Netherlands
†
These authors contributed equally * To whom correspondence may be addressed: floris.rutjes@ru.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######.((Please
delete if not appropriate))
Abstract. The synthesis of N-acetylneuraminic acid
Neu5Ac) derivatives is drawing more and more attention
health and disease and are potential targets for
neuraminic acid-based drugs.
[3]
(
in glycobiology research because of the important role of
sialic acids in e.g. cancer, bacterial, and healthy cells.
Chemical preparation of these carbohydrates typically
relies on multistep synthetic procedures leading to low
overall yields. Herein we report a continuous flow process
involving N-acetylneuraminate lyase (NAL) immobilized
on Immobead 150P (Immobead-NAL) to prepare Neu5Ac
derivatives. Batch experiments with Immobead-NAL
showed equal activity as the native enzyme. Moreover, by
using a fivefold excess of either N-acetyl-D-mannosamine
Scheme 1. The reversible aldol reaction of Neu5Ac (1) to
ManNAc (2) and pyruvate (3) catalyzed by NAL.
(
ManNAc) or pyruvate the conversion and isolated yield
of Neu5Ac were significantly improved. To further
increase the efficiency of the process, a flow setup was
designed providing a chemoenzymatic entry into a series
of N-functionalized Neu5Ac derivatives in conversions of
Neu5Ac derivatives are most often prepared using
multistep organic synthesis which is laborious,
expensive, time consuming and typically low
yielding. Alternatively, Neu5Ac derivatives can be
obtained by using N-acetylneuraminate lyase (NAL),
an enzyme which catalyzes the reversible aldol
reaction of Neu5Ac (1) to form N-acetyl-D-
mannosamine (2, ManNAc) and pyruvate (3, Scheme
4
8-82%, and showing excellent stability over 1 week of
continuous use.
Keywords: N-acetylneuraminic acid, N-
acetylneuraminate lyase, flow chemistry, enzyme
immobilization
[4]
1). It has been shown that by using an excess of
pyruvate or ManNAc derivative, the equilibrium is
shifted toward the Neu5Ac product. In contrast to
chemically derived Neu5Ac substrates, ManNAc
derivatives are synthetically readily accessible and
due to the promiscuity of NAL give straightforward
access to Neu5Ac derivatives.
Carbohydrates constitute an important class of
biomolecules with a wide array of biological
functions due to the complexity of individual
[5]
saccharide moieties and the numerous branching
possibilities.[
1]
A unique carbohydrate that is
generally encountered at the outer surface of glycans
is the sialic acid N-acetylneuraminic acid (Neu5Ac,
The immobilization of NAL has been reported by
several research groups and shows promise for large
1). Neu5Ac has over 50 structurally related
[6]
scale Neu5Ac production. More recently, also
analogues, which can be recognized by specific
crosslinked examples have been reported including
CLEA, CLEC and CLIB which are notably stable,
receptors.[ These receptors play a central role in
2]
1
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Advanced Synthesis & Catalysis
10.1002/adsc.201900146
but challenging to prepare.[7] To the best of our
knowledge, immobilized NAL has never been used in
a continuous flow setup for the preparation of
synthetic Neu5Ac derivatives. Based on the broad
experience with chemoenzymatic reactions in our
a
Conditions: ManNAc (2, 450 µmol) and sodium pyruvate
(
90 or 45 or 23 µmol) in H
2
O (1.8 mL) at 38 °C for 24 h. b
1
Determined by H-NMR analysis of the crude reaction
mixture.
group, both in batch[ and in flow systems, and on
8]
[9]
Table 2. The effect of stoichiometry and concentration on
our expertise in the chemistry and biology of sialic
the preparation of Neu5Ac (1) using Immobead-NAL in a
acids,[
2,
10]
we herewith report a straightforward
continuous flow system. a
continuous flow system containing immobilized NAL
providing a new and efficient chemoenzymatic entry
into biologically relevant N-functionalized Neu5Ac
derivatives.
First, we investigated the immobilization of NAL
to increase stability, shelf life, and ease of application
in flow chemistry. Our initial attempt using an
aqueous NAL solution in combination with oxirane-
functionalized Immobead 150P (corresponding to 62
mg NAL per g Immobead 150P) was successful
providing immobilized NAL (Immobead-NAL) with
immobilization efficiencies of up to 74% (see:
Supporting Information 1).[
In order to test the activity of Immobead-NAL, the
chemoenzymatic synthesis of Neu5Ac (1) was
investigated in batch experiments using ManNAc (2),
pyruvate (3) and native NAL in a homogeneous
solution and Immobead-NAL in a heterogeneous
formulation. In literature most often an excess of
pyruvate is used, but this requires tedious purification
pyruvate
[mM]
1000
ManNAc
[mM]
100
conversion
b
entry
1
(%)
52
36
19
15
19
40
41
71
76
82
2
3
4
5
6
7
8
9
5
00
100
100
100
100
100
100
250
400
500
300
100
33
11]
20
10
50
80
1
0
100
a
in which residual buffer salts and pyruvate have to be
Conditions: The solution (900 µL) was injected in a
sample loop (1 mL) and pumped (0.05 mL·min ) over the
removed.[
3d, 5d, 12]
-1
In contrast, we showed that a
O led to
b
Immobead-NAL column at 38 °C and collected for 1.5 h.
fivefold molar excess of ManNAc in H
2
1
Determined by H-NMR analysis of the crude reaction
conversions of up to 77% (Table 1), while the excess
of ManNAc could be easily removed by anion
exchange. All Immobead-NAL formulations were
shown to be effective in the preparation of Neu5Ac
with comparable conversions. Interestingly, three
months old Immobead-NAL (stored at 0 °C) showed
only a slight decrease in activity, indicating that the
immobilized N-acetylneuraminate lyase can be stored
mixture.
for an extended period of time. Finally, increasing the
molar excess of ManNAc did not lead to higher
conversions and isolated yields, therefore a molar
ratio of 5 of ManNAc and pyruvate was considered
optimal in this setup.
We then applied Immobead-NAL in a flow system
to efficiently screen reaction parameters and optimize
conversions (Table 2). A flow reactor was designed
using an HPLC cartridge, filled with freshly prepared
Immobead-NAL, as a packed-bed reactor. Prior to
administering the substrates to the packed-bed
reactor, it was preheated to 38 °C using a water bath.
Perfluoroalkoxy (PFA) tubing (OD 1/16”, ID 1/50”)
and flangeless fittings (1/16”) were used to connect
the linear flow setup.
Table 1. The synthesis of Neu5Ac (1) in batch with
various Immobead-NAL formulations and amounts of
pyruvate.a
NAL formulation ManNAc pyruvate conversion
b
[
mM]
250
[mM]
50
25
(%)
67
59
60
77
79
74
62
65
63
58
57
63
aqueous
NAL
1
2.5
crystalline
NAL
250
250
250
50
25
1
2.5
Initially we investigated stoichiometry and
concentration as process parameters in the synthesis
of Neu5Ac (1) in flow. At a fixed concentration of
Immobead-NAL
50
25
(
1 month)
1
2.5
1
00 mM ManNAc, neither excess of pyruvate nor
ManNAc led to increase of the conversion beyond
0% (Table 2, entries 1-7). Attempts at lower flow
Immobead-NAL
3 months)
50
25
(
5
1
2.5
speed did also not improve the conversion when
2
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Advanced Synthesis & Catalysis
10.1002/adsc.201900146
using a fivefold excess of ManNAc (2) with respect
to pyruvate (3, see: Supporting Information 2).
Interestingly, use of higher concentrations of
ManNAc (2) led to higher conversions of up to 82%
(Table 2, entries 8-10). This result is supported by
mechanistic insights of Wong et al. and by Groher
and
[4a]
Scheme 2. The use of ManNAc derivatives 4-10 in the synthesis of Neu5Ac derivatives 11-17.
Hoelsch[13] showing a correlation in increased
substrate concentration and its positive influence on
Neu5Ac production. The upper limit for ManNAc
concentrations was set to 500 mM due to its solubility
in aqueous medium.
produced. Even after 168 hours of continuous
-1
pumping at 0.05 mL·min marginal decrease in
enzyme activity was observed (Scheme 3, see also:
Supporting Information IV). The crude mixture was
purified using anion exchange chromatography
resulting in effective recovery of ManNAc (2) and
production of Neu5Ac acid (1) in 58% isolated yield
between time interval 0-120 h. These results clearly
underline the stability of Immobead-NAL and the
feasibility to scale up the flow process.
To broaden the scope of the chemoenzymatic
process, synthetic ManNAc derivatives 4-10 were
prepared in batch and subjected to the flow
conditions (Scheme 2, see also: Supporting
Information). In this reaction, the NAL appeared
sufficiently promiscuous to successfully convert all
synthetic ManNAc derivatives into the corresponding
Neu5Ac analogues 11-17. Thus, the enzyme shows a
relatively large substrate scope, which is not too
much influenced by the fact that the water solubility
of the ManNAc derivatives decreases with increasing
size of the protecting group. In addition to monitoring
conversions, the sialic acid products 11-17 were also
obtained in fairly reasonable isolated yields
confirming the synthetic feasibility of this system
100
80
(
(
Scheme 2). This includes the propargyloxycarbonyl
Poc)-functionalized Neu5Ac derivative 16 which has
60
been extensively used in our group and was shown to
be an effective substrate in bioorthogonal
glycobiology research.[
Finally, we were interested in the robustness of
the system and studied a seven-day continuous flow
experiment. By pumping a stock solution of ManNAc
4
0
2b, 10]
20
0
0 12 24 36 48 60
6
84
120
168
Time (h)
(
2, 55.7 g, 500 mM) and pyruvate (3, 5.5 g, 100 mM)
over the Immobead-NAL reactor (approximately 35
mg NAL), more than 10 g of Neu5Ac (1) was
3
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Advanced Synthesis & Catalysis
10.1002/adsc.201900146
[
11]
during enzyme immobilization and measured in triplo.
Figure 1. Preparative scale formation of Neu5Ac (1) in a
continuous flow setup. Conditions: ManNAc (2, 500 mM),
The concentration of free protein was determined,
effectively providing information about the total amount of
protein coupled to the beads. The standard curve for
protein determination was conducted using solutions of
2
pyruvate (3, 100 mM), H O, Immobead-NAL, flow 0.05
-
1
mL·min , 38 °C.
-
1
-1
0
(
.02 mg·mL to 0.1 mg·mL of Bovine Serum Albumin
BSA) purchased from Sigma-Aldrich. Hereafter the
protein concentration of aqueous N-acetylneuraminate
lyase (100 µL) was determined in triplo, and used to
determine the protein content of the immobilization
washings.
In summary, N-acetylneuraminate lyase (NAL)
was immobilized and used to optimize the conversion
of ManNAc and pyruvate into Neu5Ac. The oxirane-
containing resin Immobead 150P was coupled to
NAL with a binding efficiency of up to 74%. The
resulting Immobead-NAL showed to be stable in
aqueous solution over at least three months of
storage. Optimal conversions were obtained using
Anion exchange purification of Neu5Ac (1)
A short cylindrical column (± 5 × 1.5 cm; h × d) was
–
charged with DEAE-Sephadex A-25 Cl (± 4.5 g) and
4 3
swollen using aqueous NH HCO (1.5 M, 15 mL). The
5
00 mM ManNAc, 100 mM pyruvate and a flow rate
resin was filtered and washed thrice with water (3 × 15
mL) before addition of the reaction mixture. The crude
mixture was added to the resin and washed with water,
followed by an increasing v/v% of formic acid in water, up
to 20%. The obtained products were concentrated in vacuo.
-1
of 0.05 mL·min , giving conversions of up to 82%.
We also demonstrated that the system was
effective in the synthesis of a library of differently N-
substituted Neu5Ac derivatives. Finally, the reactor
was subjected to a durability test of 168 hours
Flow chemistry setup
showing
a
relatively small decrease of N-
acetylneuraminate lyase activity over a week time.
Thus, using this immobilized lyase, an efficient and
stable continuous flow process has been developed
for the preparation of neuraminic acid derivatives.
The Immobead-NAL resin (700 mg) was loaded directly in
an HPLC cartridge (±1.5 mL) using vacuum suction
followed by washing with copious amounts of
demineralized water. The ManNAc/pyruvate solution was
prepared in demineralized water, and the pH adjusted to
7
.0-7.5 using 1 M aqueous NaOH or HCl and a digital pH
indicator. The HPLC cartridge was connected to the HPLC
pump, and heated to 38 °C in a water bath. After 30 min at
-
1
Experimental Section
38 °C, the pump was started at 0.05 mL·min . After the
reaction mixture was pumped, additional demineralized
water (±5 mL) was used to remove remaining reactants.
The product was concentrated in vacuo and the conversion
1
Chemicals and enzymes
determined using H-NMR in D
2
O.
Chemicals used in this study were purchased from
Fluorochem, Sigma Aldrich and Carbosynth with a purity
grade of >95%. Demineralized water was used during all
experiments. N-Acetylneuraminate lyase (NAL) was used
Preparative scale experiment
-
1
as a crystalline powder (8.7 U·mg ) or a suspension in
The Immobead-NAL resin (700 mg) was loaded directly in
an HPLC cartridge (±1.5 mL) using vacuum suction
followed by washing with copious amounts of
demineralized water. ManNAc (55.7 g, 252 mmol) and
sodium pyruvate (5.55 g, 50.4 mmol) were added to water
-
1
aqueous solution (111 U·mL ). The crystalline powder
and aqueous solution were kept cool at 4 °C, and the latter
was shaken before use.
(
504 mL) and the pH was adjusted to 7.0-7.5 using 1 M
aqueous NaOH or HCl using a digital pH indicator. The
HPLC cartridge was connected directly to the HPLC pump
without the sample loop, and heated to 38 °C in a water
bath. After 30 min at 38 °C, the pump was set to 0.05
Immobilization of NAL
The immobilization procedure comprised incubation of
Immobead-150P (300 mg) and N-acetylneuraminate lyase
-
1
mL·min . After the indicated time intervals, the reaction
mixture was separately collected for 10 min (3 ×). The
-
1
(
crystalline: 50 mg, 8 U mg or solution: 900 µL, 111
-1
U·mL ) dissolved in aqueous K
mL) in a falcon tube. The mixture was shaken for 24 h at
0 °C, and thereafter filtered using a cellulose paper. The
obtained grain was washed twice with demineralized water
2
PO
4
(1.25 M, pH = 8.0, 45
samples were concentrated in vacuo and the conversion
1
determined using H-NMR in D
2
O.
2
(
45 mL), and collected again. The beads were diluted in a Acknowledgements
glycine solution (2.0 M, pH = 8.5, 45 mL) and shaken for
2
4 h at 20 °C, and thereafter filtered using cellulose paper.
We kindly acknowledge the funding by the H2020-FETOPEN-
The Immobead-150P was washed twice with
demineralized water (30 mL), and twice with aqueous
2
016-2017 programme of the European Commission (Grant
agreement number: 737266-ONE FLOW). This work was also
supported by a European Research Council (ERC) Starting Grant
2 4
K PO (0.1 M, pH = 7.4, 30 mL). The obtained
immobilized lyase was stored at 4 °C in a falcon tube, and
used within three months of preparation.
(Grant agreement number: 758913) awarded to T. J. Boltje.
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4
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Advanced Synthesis & Catalysis
10.1002/adsc.201900146
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10.1002/adsc.201900146
COMMUNICATION
Chemoenzymatic Synthesis of Sialic Acid
Derivatives Using Immobilized N-
Acetylneuraminate Lyase in a Continuous Flow
Reactor
Adv. Synth. Catal. Year, Volume, Page – Page
Victor R. L. J. Bloemendal, Sam J. Moons, Jurriaan
J. A. Heming, Mohamed Chayoua, Olaf Niesink,
Jan C. M. van Hest, Thomas J. Boltje, Floris P. J.
T. Rutjes*
6
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