Enzymatic Synthesis of 2-(β-Galactosyl)-ethyl Methacrylate by β-Galactosidase from Pyrococcus woesei and Application for Glycopolymer Synthesis and Lectin Studies

Article abstract of DOI:10.1021/acs.biomac.9b01647

Glycosidases have long been used for the synthesis of glycosides by transglycosylation reactions. Especially glycosidases from hyperthermophilic bacteria are useful for reactions under extreme reaction conditions, e.g., in the presence of organic solvents. We herein report the facile enzymatic synthesis and purification of 2-(β-galactosyl)-ethyl methacrylate (Gal-EMA) with the recombinant hyperthermostable glycosidase from Pyrococcus woesei in high yields. Optimized reaction conditions resulted in gram-scale synthesis of the galactosylated monomer with 88% transglycosylation yield. The product Gal-EMA was characterized by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS), nuclear magnetic resonance (NMR) spectroscopy, and infrared (IR) spectroscopy. Gal-EMA was utilized to synthesize sugar-functionalized acrylate polymers with defined amounts of incorporated galactose (0-100%). Analysis of the binding affinity of the lectin RCA₁₂₀ from Ricinus communis to the glycopolymers using an enzyme-linked lectin assay (ELLA) revealed K_D values between 0.24 and 6.2 nM, depending on the amount of incorporated Gal-EMA. The potential of Gal-EMA for the synthesis of acrylate-functionalized glycan oligomers was demonstrated by sequential elongation of the terminal galactose by two glycosyltransferases, resulting in the terminal glycan N-acetyllactosamine (LacNAc) epitope. In conclusion, the enzymatic synthesis of Gal-EMA opens new routes to a series of novel monomeric building blocks for the synthesis of glycan-functionalized polyacrylates.

Full text of DOI:10.1021/acs.biomac.9b01647

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Article
Enzymatic Synthesis of 2-(#-galactosyl)-ethyl methacrylate
by #-galactosidase from Pyrococcus woesei and
Application for Glycopolymer Synthesis and Lectin Studies
Marius Hoffmann, Elisabeth Gau, Susanne Braun, Andrij Pich, and Lothar Elling
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b01647 • Publication Date (Web): 15 Jan 2020
Downloaded from pubs.acs.org on January 17, 2020
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Enzymatic Synthesis of 2-(β-galactosyl)-ethyl
methacrylate by -galactosidase from Pyrococcus
woesei and Application for Glycopolymer Synthesis
and Lectin Studies
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a,1
b,c1
Susanne Braun, b,c Andrij Pich, b,c,d,
*
Marius Hoffmann, Elisabeth Gau,
and Lothar Elling a,*
a
Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical
Engineering, RWTH Aachen University, Pauwelsstraße. 20, 52074 Aachen, Germany
b
Functional and Interactive Polymers, Institute of Technical and Macromolecular Chemistry, RWTH
Aachen University, Worringerweg 2, 52074, Aachen, Germany
c
DWI – Leibniz-Institute for Interactive Materials e.V., RWTH Aachen University, Forckenbeckstraße
50, 52074, Aachen, Germany
d
Aachen Maastricht Institute for Biobased Materials (AMIBM), Maastricht University, Brightlands
Chemelot Campus, Urmonderbaan 22, 6167 RD Geleen, The Netherlands
1
these authors contributed equally
*corresponding authors
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ABSTRACT
Glycosidases have long been used for the synthesis of glycosides by transglycosylation reactions.
Especially glycosidases from hyperthermophilic bacteria are useful for reactions under extreme
reaction conditions, e.g. in the presence of organic solvents. We herein report the facile enzymatic
synthesis and purification of 2-(β-galactosyl)-ethyl methacrylate (Gal-EMA) with the recombinant
hyperthermostable glycosidase from Pyrococcus woesei in high yields. Optimized reaction
conditions resulted in gram-scale synthesis of the galactosylated monomer with 88 %
transglycosylation yield. The product Gal-EMA was characterized by HPLC-ESI-MS, nuclear
magnetic resonance (NMR) and infrared (IR) spectroscopy. Gal-EMA was utilized to synthesize
sugar-functionalized acrylate polymers with defined amounts of incorporated galactose (0-100 %).
Analysis of the binding affinity of the lectin RCA120 from Ricinus communis to the glycopolymers
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using an enzyme-linked lectin assay (ELLA) revealed K -values between 0.24-6.2 nM depending
D
on the amount of incorporated Gal-EMA. The potential of Gal-EMA for the synthesis of acrylate-
functionalized glycan-oligomers was demonstrated by sequential elongation of the terminal
galactose by two glycosyltransferases resulting in the terminal glycan N-acetyllactosamine
(LacNAc) epitope. In conclusion, the enzymatic synthesis of Gal-EMA opens new routes to a series
of novel monomeric building blocks for the synthesis of glycan-functionalized polyacrylates.
KEYWORDS: Biocatalysis, Glycosidase, Transglycosylation, Glycosides, Glycopolymers
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INTRODUCTION
Glycoconjugates, such as glycoproteins or glycolipids, are biologically relevant molecules
encoding cellular information for inter- and intracellular communication as well as in cell-pathogen
interaction and cancer metastasis.1-3 Glycan chains are attached to proteins via N- or
O-glycosylation and can serve as ligands for carbohydrate-recognizing enzymes (lectins).4
Therefore, glycoconjugates are regarded as highly interesting materials for biomedical
applications. 5,6 The synthesis of glycoconjugates, however, is still challenging, and chemical and
enzymatic strategies, as well as chemo-enzymatic routes, have been developed.7-13 In nature, the
interaction between carbohydrate-based ligands and lectins is generally very weak but can greatly
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be enhanced by several orders of magnitude via multivalent presentation of the sugar moiety.5,6,14,15
Specific attention has therefore been paid to the synthesis of multivalent glycopolymers.5,16-19 Free
radical as well as by controlled radical polymerizations have been applied for the synthesis of
glycopolmers.20-22 In a pioneering work, glucosidase-catalyzed synthesis of glycomonomers and
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subsequent horseradish peroxidase-catalyzed polymerization was reported. Glycopolymers can
ideally mimic natural multivalent glycoconjugates and thus offer a platform to tailor multivalences
for lectin binding with high affinity and avidity 24-28 or the design of toxin scavengers and
inhibitors29-34 and biosensors35-37. Important factors for the binding efficiency of glycopolymers are
the glycan density and the polymer backbone itself. Chemical synthesis of glycan-containing
monomers has seen significant progress in recent years.38-41 However; chemical synthesis remains
challenging and affords multiple reaction steps resulting in moderate overall product yields. 8,42,43
In contrast, enzymatic synthesis offers a viable alternative to chemical synthesis. For the enzymatic
synthesis of glycosylated monomers for later use in polymerization experiments, mainly
hydrolases, such as lipases, proteases or glycosidases have been used.43-45 Lipases catalyze the
transesterification of divinyl esters with the terminal alcohol at the C-6 position of
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monosaccharides in organic solvents. Lipase-catalyzed transesterification has been extensively
applied in the synthesis of glycomonomers in recent years with the immobilized lipase from
Candida antarctica (Novozyme-435) being the most prominent example.46-50 Regarding their
application in glycomonomer synthesis, proteases catalyze a similar reaction to lipases but are
generally used for more hydrophilic divinyl substrates.51-54 Recent advances and applications of
glycosidase-catalyzed synthesis of glycomonomers have been excellently reviewed very recently.55
Glycosidases are sugar-specific hydrolases and originally catalyze the hydrolytic cleavage of
glycosidic bonds. By applying adjusted reaction conditions, glycosidases are exploited for the
synthesis of glycoconjugates by transglycosylation reactions using activated donor substrates (e.g.
aryl-glycosides) and high acceptor substrate concentrations.56,57 However; transglycosylation
reactions are often accompanied by product hydrolysis, which reduces overall reaction yields.
Reaction engineering, e.g. addition of ionic liquids or co-solvents, increases the
transglycosylation/hydrolysis ratio by keeping the water activity low.58,59 Reaction conditions must
be adjusted tediously to find the balance between retaining sufficient enzymatic activity and
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achieving high transglycosylation yields. In some cases, the yield of the desired product is further
decreased by the formation of more than one product resulting from the subsequent addition of
sugar moieties to the desired glycoside or lack of absolute regioselectivity leading to
regioisomers.61,62 Several approaches towards the enzymatic synthesis of acrylate-functionalized
glycoside monomers have been undertaken so far, including enzymatic synthesis of 2-(β-
glucosyloxy)ethyl acrylate (2-GlcEA), 2-(β-glucosyloxy)-ethyl methacrylate (2-GlcEMA), 4-(β-
glucosyloxy)-butyl acrylate (4-GlcBA), 2-(-maltooligooxy)-ethyl methacrylate, N-(β-
glycosyloxy)-ethyl acrylamide, N-(β-glycosyloxy)-ethyl methacrylamide, and N-(β-glycosyloxy)-
butyl methacrylamide.59,63 Gal-EMA was synthesized previously by using commercial
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
-galactosidase from E. coli or lyophilized cells of Sulfolobus solfataricus. Most of these
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attempts used commercial enzyme sources and were conducted at ambient temperatures.
Thermostable enzymes are desirable for glycoside synthesis since the solubility of substrates and
products is enhanced at elevated temperatures, which results in increased product output per
batch.66,67 The enzyme used in this work is a β-glycosidase from the hyperthermophilic organism
Pyrococcus woesei DSM 3773 (GenBank accession number AF043283) (PwGly) and can be
recombinantly produced in E. coli.68 PwGly has so far been characterized for its hydrolytic
properties.68-70 In our previous study, we characterized and utilized recombinant PwGly for the
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synthesis of defined galacto-oligosaccharides using microwave irradiation. We herein report on
the synthesis of Gal-EMA in gram-scale by recombinant PwGly and utilization of Gal-EMA in
glycopolymer synthesis for lectin binding studies (Scheme 1).
Scheme 1. Synthesis of 2-(β-galactosyl)-ethyl methacrylate (Gal-EMA, 3) with the
hyperthermophilic glycosidase PwGly from Pyrococcus woesei. In glycosidase-catalyzed
transglycosylation reactions in aqueous media, hydrolysis and transglycosylation are competing
reactions. Subsequent glycoside hydrolysis may lead to further product loss.
EXPERIMENTAL SECTION
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Materials.
2-Hydroxyethyl
methacrylate
and
2,2′-azobis(2-methylpropionamidine)
dihydrochloride were received from Sigma Aldrich (now Merck, Darmstadt, Germany). The
purchased HEMA-solution contained 4-methoxyphenol as an inhibitor (200 ppm). All nitrophenol-
activated monosaccharides were obtained from Carbosynth (Berkshire, UK). UDP-GlcNAc and
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UDP-Gal were synthesized as published previously. All other chemicals, if not specifically
mentioned, were obtained from Carl Roth (Karlsruhe, Germany).
Heterologous Production and Purification of -Glycosidase. Production of recombinant
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PwGly was previously described by our group. After harvesting, 3 g of cells were resuspended
in 9 mL Na HPO -citrate buffer (pH 5.5) and disrupted via sonification (four cycles on ice, each
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cycle 30 s sonification with a 30 s break between cycles). After centrifugation (15,000 rpm, 4 °C,
0 min) the supernatant was heated up to 95 °C for 45 min and centrifuged again. The pellet was
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discarded and the enzymatic activity of the supernatant was determined. Purity of the enzyme
solution was checked by SDS-PAGE. Protein concentration was determined using the Bradford
assay.
Determination of Enzymatic Activity. Hydrolytic activity was measured following our
previously published protocols.61,71 In brief, 100 µL of appropriately diluted enzyme solution was
added to 900 µL of 4.4 mM para-nitrophenyl--D-galactopyranoside (pNP-Gal, 1) in
citrate-Na HPO -buffer (pH 5.5) and incubated for 5 min at 85 °C. The reaction was stopped at
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specific time intervals by the addition of 200 µL Na CO (0.2 M) to 100 µL sample solution. The
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released para-nitrophenol (pNP, 4) was measured in a photometer (Spectramax Plus 384,
Molecular Devices) at 405 nm. Quantification was achieved by using standard curves of pNP. One
unit of enzyme activity is defined as the amount of enzyme which converts 1 µmol pNP-Gal to
pNP and galactose per minute.
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Transglycosylation Reaction - Acceptor Substrate Screening. The screening was conducted
on a 2 mL scale with 90 % [v/v] acceptor substrate. 1800 µL of acceptor substrate were mixed with
150 µL of a 600 mM lactose solution (50 mM Na PO -citrate-buffer, pH 5.5) and heated to 85 °C.
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0 µL of pre-heated enzyme solution (50 mM Na PO -citrate-buffer, pH 5.5) were added to start
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the reaction. The final enzyme concentration in the reaction was 1.8 U/mL. The screening included
the acceptor substrates hexanol, octanol, phenylmethanol, phenoxyethanol, isobutanol and HEMA.
If the acceptor substrate was not miscible with water, the reaction was stirred at high frequencies.
Samples of 50 µL were taken at different time points and stopped by the addition of a 3-fold volume
of ice-cold ethanol. Analysis of product conversion was done via Thin Layer Chromatography
(TLC). The solvent systems varied with the acceptor substrates: Hexanol: CHCl /MeOH/H O
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2
(
80/35/5); octanol: CHCl /MeOH/H O (80/25/5); phenylmethanol: CHCl /MeOH/H O (80/45/5);
3 2 3 2
phenoxyethanol: CHCl /MeOH/H O (80/45/5); isobutanol: First separation EtOAc/MeOH/H O
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(
16/6/1), second separation 2-Propanol/EtOH/H O (5/1/2); HEMA: EtOAc/MeOH/H O (72/5/4).
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Transglycosylation Reaction - Donor Substrate Screening. Analysis of the enzyme’s donor
substrate spectrum was done on a 1 mL scale at 85 °C. The screening included the donor substrates
pNP-Gal, pNP-Glc, pNP-Xyl, cellobiose and lactose. Donor substrates were dissolved in 900 µL
HEMA previous to the reaction to a final concentration of 11 mM. The reaction was started by the
addition of 100 µL enzyme solution (50 mM citrate-Na PO -buffer, pH 5.5). The final enzyme
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concentration was 7.5 U/mL. Samples of 50 µL were taken at time intervals and stopped by the
addition of a 3-fold volume of ice-cold ethanol. They were then analyzed via TLC with
EtOAc/MeOH/H2O (72/5/4) as a solvent.
Optimization of HEMA Amount. All reactions were carried out on a 1 mL scale at 85 °C. pNP-
Gal was dissolved in 900 mL of 70 – 90 % [v/v] HEMA mixed with 50 mM phosphate-citrate
buffer (pH 5.5) to a concentration of 55 mM. The final concentration of buffer varied with the
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added volume of HEMA. 100 µl of enzyme solution (50 mM citrate-Na PO -buffer, pH 5.5) was
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added to start the reaction. The final concentration of pNP-Gal was 50 mM. The final
concentrations of HEMA in the reactions were 70 % [v/v] (5.77 M), 80 % [v/v] (6.6 M) or 90 %
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[v/v] (7.42 M). Samples were taken at time intervals and stopped by the addition of 3-fold ice-cold
ethanol. The samples were centrifuged (13,000 rpm, 4 °C, 10 min) and analysis of the supernatant
was done via HPLC as described below.
Synthesis and Purification of Gal-EMA. Gal-EMA was synthesized in batch volumes of 1 mL
to 100 mL. The reaction was carried out at 85 °C. The reaction mixture contained 85 % [v/v]
HEMA and 15 % [v/v] enzyme solution (50 mM citrate-Na PO -buffer, pH 5.5) with a final
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enzyme concentration of 7.5 U/mL. pNP-Gal was dissolved in HEMA previous to the reaction to
a concentration of 55 mM. The reaction was started by the addition of 15 % [v/v] enzyme solution.
Samples of 50 µL were taken at time intervals and stopped by the addition of a 3-fold excess of
ice-cold ethanol. They were then analyzed via TLC and HPLC as described below. After complete
consumption of the donor substrate, the reaction was stopped with ice-cold ethanol. The mixture
was centrifuged (15,000 rpm, 4 °C, 30 min) and the pellet was discarded. After the removal of
potentially remaining enzyme by ultrafiltration (Viva Cell 250, 30,000 MWCO, Sartorius Stedim),
ethanol and water were evaporated using a rotary evaporator. Afterwards, HEMA was evaporated
by applying high vacuum (50 µbar) overnight. The remaining compounds were diluted with ethanol
and Gal-EMA was purified via silica column chromatography using EtOAc/CH CN/MeOH/H O
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[50/20/4/4] as a solvent. Fractions containing the pure product were pooled and the solvent was
evaporated. Pure Gal-EMA was dissolved in water for subsequent product characterization.
Characterization of Reaction Products (TLC, HPLC, MS, NMR, IR). Qualitative analysis
was done via TLC. TLC was performed on silica-coated aluminum plates (Alugram ® Xtra SilG,
Macherey-Nagel, Düren, Germany). If not specifically mentioned, the eluent consisted of
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EtOAc/MeOH/H O [72/5/4]. Spots were developed by dipping the plate in the detection dye
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solution (ethanol/p-anisaldehyde/sulfuric acid/acetic acid [150/4/5/2]) and subsequent heating to
350 °C with a heat gun.
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Quantitative analysis of samples was carried out using reversed-phase (RP) HPLC. A gradient
of acetonitrile and water was applied for separation of the analytes on a MultoKrom 100-5 C18
resin (250 mm × 4 mm, CS-Chromatographie, Langerwehe, Germany) using a flow rate
of 1 mL/min. The ratio of the two solvents in the eluate at given time points was as follows: 90 %
H O for 15 min, decrease to 40 % H O within 5 min, steady 40 % H O for 5 min, return to 90 %
2
2
2
H O within 2 min, steady at 90 % for 3 min. Peaks were detected at 205 nm and quantification was
2
done via external calibration curves.
Analysis of the molecular weight was done via LC-ESI-MS (Finnigan Surveyor MSQ Plus,
Thermo Scientific, needle voltage = 4 kV, temperature = 400 °C, cone voltage = 100 V, negative
mode). HPLC-conditions were the same as described above.
1
13
The H-NMR and C-NMR spectra of the Gal-EMA monomer were recorded on a Bruker DPX-
4
00 FT-NMR spectrometer at 400 MHz using deuterated water (D O), deuterated methanol
2
(
MeOD-d ) and deuterated dimethyl sulfoxide (DMSO-d ) as solvent.
4
6
FT-IR spectroscopy was performed with lyophilized samples on a Thermo Nicolet Nexus 470
FTIR with Si-ATR or as KBr pellet on a Nicolet 5700 FTIR.
Synthesis of GlcNAc1,3Gal-EMA with Recombinant 1,3GlcNAcT from H. pylori. The
reaction mixture (final volume 100 µL) consisted of 15 mM purified Gal-EMA, 30 mM
UDP-GlcNAc, 5 mM DTT, 20 mM MgCl and 25 mM KCl in 100 mM HEPES buffer (pH 7.2).
2
Alkaline Phosphatase (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was added prior
to the reaction to a final concentration of 10 U/mL. 1,3GlcNAcT was expressed and purified as
described previously, and the activity was determined.73 The enzyme was added to a final
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concentration of 500 mU/mL to start the reaction. The reaction progress was monitored via
RP-HPLC as described above. Analytes were detected at 205 nm. The reaction product was verified
by LC-ESI-MS.
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Synthesis of Gal1,4GlcNAc1,3Gal-EMA with Recombinant Human 1,4GalT. The
reaction mixture (final volume 100 µL) consisted of 5 mM purified GlcNAc1,3Gal-EMA, 10 mM
UDP-Gal, 5 mM MnCl and 25 mM KCl in 100 mM HEPES buffer (pH 7.2). Alkaline Phosphatase
2
(Thermo Fisher Scientific, Waltham, Massachusetts, USA) was added prior to the reaction to a
final concentration of 10 U/mL. 1,4GalT was expressed and purified, and the activity was
6
9
determined as described previously. The enzyme was added to a final concentration of 480
mU/mL to start the reaction. The reaction progress was monitored via reversed-phase HPLC as
described above. Analytes were detected at 205 nm. The reaction product was verified by
LC-ESI-MS.
Hydrolytic Cleavage of Gal1,4GlcNAc1,3Gal-EMA with 1,4-Galactosidase. 100 µL of 1
mM Gal1,4GlcNAc1,3Gal-EMA in H O were evaporated to dryness. The analyte was dissolved
2
in 90 µL of 50 mM citrate-Na PO -buffer (pH 5.5). Commercial 1,4-galactosidase from Bacillus
2
4
circulans (BIOLACTASA, Biocon, Spain) was diluted 1:10000 in 50 mM citrate-Na PO -buffer
2
4
(pH 5.5), and 10 µL of the diluted enzyme preparation were added to start the reaction. Samples of
2
5
0 µL were taken at different time points. The enzyme was removed by heat-inactivation (95 °C,
min) and subsequent centrifugation. The supernatant was analyzed via HPLC as described above.
Synthesis of PHEMA and PGal-EMA Homopolymers and PHEMA/PGal-EMA
Copolymers. PHEMA and PGal-EMA homopolymers as well as PHEMA/PGal-EMA copolymers
were synthesized by free radical polymerization in aqueous medium. The respective amounts of
the monomers HEMA and Gal-EMA were solved in distilled water (see Table 1 for detailed
information) in a round flask equipped with a stirrer. The solutions were heated up to 70 °C and
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stirred under nitrogen for 30 min. Afterwards the thermal initiator 2,2’-azobis(2-
methylpropionamidine)dihydrochloride (AMPA) was solved in 1 mL of distilled water and added
to the solutions which were then stirred at 70 °C for five hours. The polymers were obtained in
their solid form by lyophilization after synthesis. No purification was performed to investigate the
whole molecular weight distribution of the product without any loss. Gel permeation
chromatography (GPC) was used to analyze the molecular weight distribution of the obtained
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
polymers. H-NMR spectroscopy was measured to investigate if monomer was left after the end of
the polymerization.
Table 1. Amounts of chemicals for the synthesis of PHEMA/PGal-EMA homo- and copolymers.
Sample Gal-
EMA
HEMA m(Gal-EMA m(HEMA) m(AMPA)
content solution) [mg] [mg]
V(H Ototal) [mL]
2
content [mol%] [mg]
[mol%]
1
11
1
1
1
0
0
5
10
50
100
100
95
90
50
0
0
29
58
292
584
651
16.7
6.7
6.7
3.3
3.3
50
20
20
10
10
247
234
130
0
2
3
4
Gel Permeation Chromatography (GPC). Number average molecular weights (푀 ), weight
n
average molecular weights (푀 ) and dispersities (D, 푀 /푀 ) were determined by GPC using
w
w
n
dimethylformamide (DMF) as eluent for the PHEMA homopolymer 10 as well as the PHEMA
based copolymers containing 5 mol% Gal-EMA 11, 10 mol% Gal-EMA 12 and 50 mol% Gal-EMA
13. PGal-EMA 14 was not soluble in DMF, therefore it was measured in H O. The measurements
2
of the samples dissolved in DMF were performed using an Agilent 1260 system equipped with a
refractive index detector (SECurity2, PSS). The eluent was containing 1 g/L lithium bromide (LiBr,
≥
99 %, Sigma-Aldrich). The solvent of the samples was containing traces of toluene as an internal
1
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standard. The used sample concentration was ca. 5 mg/mL. One precolumn (8 x 50 mm) and three
PSS GRAM gel columns (8 x 300 mm, Polymer Standards Service) were applied at a flow rate of
1.0 mL/min at a temperature of 60 °C. The diameter of the gel particles was 10 μm and the nominal
pore widths were 30, 1000, 1000 Å. The calibration was achieved using narrow distributed
Polymethylmethacrylate standards.
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The measurement of PGal-EMA 14 dissolved in H O was performed using an Agilent 1200
2
system equipped with a refractive index detector and a UV detector (UV-VWD, Agilent) at a
wavelength of λ = 280 nm. The eluent was containing both 0.1 M sodium nitrate (NaNO , ≥ 99 %,
3
Merck KGaA) and 0.01wt% sodium azide (NaN , extra pure, Merck KGaA). The solvent of the
3
samples was containing traces of ethylen glycol as an internal standard. The used sample
concentration was ca. 5 mg/mL. One precolumn (8 x 50 mm) and three Suprema Lux gel columns
(8 x 300 mm, Polymer Standards Service) were applied at a flow rate of 1.0 mL/min at 40 °C. The
diameter of the gel particles was 5 µm and the nominal pore widths were 30, 1000 and 1000 Å.
The calibration was achieved using narrow distributed Pullulan standards. In both cases the results
were evaluated using the PSS WinGPC UniChrom software (Version 8.3.2).
Enzyme-linked Lectin Assay (ELLA). Due to their varying hydrophilicity caused by the
incorporation of different amounts of galactose, the polymer samples were solved in DMSO (14),
a mixture of 50 % DMSO/PBS (13, 12) or pure phosphate-buffered saline (PBS, 50 mM, pH 7.2)
(11, 10) to a final concentration of 50 µg/mL. 100 µL of this solution was used for immobilization
in microtiter plates (MaxiSorp, Nunc, Wiesbaden, Germany) overnight. Non-occupied binding
sites were blocked by 2 % bovine serum albumin (BSA) in PBS for 1 h, followed by 1 h incubation
with 100 µL of varying concentrations of biotinylated RCA120 (Vectorlabs, Burlingame, USA) in
PBS. Subsequently, 100 µL of a streptavidin-coupled peroxidase (Roche, Basel, Austria; 1:5000
in PBS) was applied for 1 h. 50 µL of the substrate TMB One was added and converted by the
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peroxidase for 40 s. After stopping the reaction by addition of 50 µL of 3 M HCl, samples were
analyzed at 450 nm. After each step, wells were washed three times with 200 µL of PBS-Tween
(0.05 % [v/v]). Binding curves were fitted and K -values were calculated using the software
D
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
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7
8
9
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1
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4
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6
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1
2
3
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2
3
4
5
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9
0
1
2
3
4
5
6
7
8
9
0
SigmaPlot (Equation 1).
Bmax ∗ abs(x)
f(x) =
(
KD + abs(x))
Equation 1. Calculation of binding curves for the analysis of RCA120 binding to glycopolymers
0-14. Bmax: Maximum binding signal.
1
Inhibition of RCA120 Binding with Galactose. D-Galactose was used as binding inhibitor of
RCA120 to the glycopolymers. Glycopolymer samples were immobilized in the wells of a microtiter
plate as described above. After blocking with BSA, 50 µM RCA120 was mixed with D-galactose in
varying concentrations up to 1 M and incubated on the plate for 1 h. Binding signals were obtained
at 450 nm as described above. The binding curves were fitted and IC -values were calculated using
5
0
SigmaPlot software (Equation 2).
(
퐵푚푎푥 ― 퐵푚푖푛
1 + 10(푥 ― 푙표푔퐼퐶50)
)
f(x) = 퐵푚푖푛
+
Equation 2. Calculation of inhibition curves for the analysis of the inhibition of RCA120 binding
to glycopolymers 10-14 by galactose. B : Minimal binding signal; Bmax: Maximum binding
min
signal.
RESULTS AND DISCUSSION
Substrate spectrum of PwGly. We previously demonstrated the hydrolytic activity of PwGly
with a range of aryl-glycosides including the sugar moieties β-galactose, β-glucose, -mannose and
71

-xylose. We here investigated 2-hydroxy-ethyl methacrylate (HEMA, 2) as well as some aryl-
and alkyl-alcohols as non-sugar acceptors in transglycosylation reactions with lactose as donor
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substrate (Table S1 and Figure S1). Generally, the enzyme displayed a good capability for the
glycosylation of the tested acceptor substrates in mono- and biphasic systems. The highest product
formation was observed with 2 as acceptor substrate (Table S1). HEMA 2 is miscible with water
due to its hydrophilic structure. As a result, it acts both as a solvent for the donor substrate and as
an acceptor, thus providing the possibility to reduce the water activity by increasing the
HEMA/water-ratio. In glycosidase-catalyzed glycosylation reactions, the desired aglycone and
water compete for the role as acceptor substrate (Scheme 1). In order to reach a high
transglycosylation/hydrolysis ratio, the aglycone concentration should be kept high and the water
activity in the reaction mixture should be minimized. However, a low water activity often causes a
loss in enzymatic activity and stability. In order to find a compromise between high enzymatic
activity and a high transglycosylation/hydrolysis ratio using PwGly, volumetric amounts between
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0 – 90 % [v/v] of 2 in water were tested (Figure 1; Figure S2). Previous experiments indicated
that amounts of 2 below or equal to 60 % [v/v] resulted in unfavorable product formation (data not
shown). Similar reaction velocities and product yields were obtained for transglycosylation
reactions with 70 – 90 % [v/v] of 2. Therefore, PwGly appears to be stable and active at high
HEMA/water ratios. Product formation reached equilibrium after approximately 7 h. Most
importantly, product 3 (Gal-EMA) is stable within 7 h reaction time. Unlike in other glycosidase-
catalyzed transglycosylation reactions, a longer incubation time did not result in much product
hydrolysis as 3 remains at a high concentration in the reaction mixture after 24 h. Control reactions
with 3 as a substrate for hydrolysis in aqueous reaction conditions with the same enzyme resulted
in full hydrolytic conversion by the enzyme after 24 h (Figure S3). The hydrolytic activity of PwGly
for 3 is with 4.2 mU/mL far lower compared to 1 (75 U/mL), which indicates that the galactosyl-
acrylate is the less-preferred hydrolytic substrate of the enzyme. In addition to 3 being a poor
substrate, another potential reason for the low hydrolytic activity observed here is the low water
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activity of the reaction. The concentration of water is not sufficient to compete with 2 as an acceptor
substrate in the catalytic center of the enzyme, thus hydrolysis is not the predominant reaction. At
the same time, the lack of water most probably causes the enzyme to precipitate. This assumption
was further reinforced by the observation that, after approx. 1 h, a brown substance started to
precipitate at the bottom of the reaction vessel. The deactivation of the catalyst can, in this case, be
regarded as an advantage because it prevents hydrolytic product degradation.
1
1
1
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1
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Figure 1. Synthesis of Gal-EMA (3) with different volumetric amounts [% v/v] of HEMA (2). The
synthesis was conducted with a final concentration of 50 mM pNP-Gal (1) and 70 – 90 % [v/v] of
2
in a total reaction volume of 1 mL. The amount of added buffer volume (50 mM citrate-
phosphate-buffer, pH 5.5) and the final buffer concentration varied according to the concentration
of 2. The final enzyme concentration in each reaction was 7.5 U/mL. The reactions were analyzed
by HPLC.
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2
2
2
2
2
2
2
2
2
3
3
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Having optimized the reaction conditions for transglycosylation, a set of potential donor
substrates was tested (Table 2 and Figure S4). Concerning the nitrophenol-activated
monosaccharides, pNP-Gal 1 and para-nitrophenyl-D-glucopyranoside (pNP-Glc) showed
comparable hydrolysis/transglycosylation ratios and conversion rates with pNP-Glc being the
slightly better substrate. However, para-nitrophenyl--D-xylopyranoside (pNP-Xyl) indicated
lower conversion. The naturally occurring disaccharides lactose and cellobiose indicated similar
conversion to pNP-Gal 1 and pNP-Glc but were both limited in their solubility in a 90 % [v/v]
solution of 2. Furthermore, the use of lactose resulted in a product mixture of 3 and Glc-EMA,
which is probably caused by the enzyme’s ability to use glucose as donor substrate by conducting
reverse hydrolysis. Transglycosylation products with cellobiose, pNP-Glc and pNP-Xyl indicate
potential access to 2-(β-glucosyl)-ethyl methacrylate (Glc-EMA) and 2-(β-xylosyl)-ethyl
methacrylate (Xyl-EMA). We focused here on the synthesis of 2-(β-galactosyl)-ethyl methacrylate
0
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2
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2
3
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5
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2
3
4
5
6
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0
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2
3
4
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0
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2
3
4
5
6
7
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0
(Gal-EMA, 3). Since solubility of lactose in 2 is limited and because of the formation of a product
mixture using this substrate, 1 was chosen for the production of sugar-functionalized acrylate
monomers on a larger scale (100 mL). 1 is well soluble in 2 (55 mM at 85 °C) and the side product
pNP (4) can be easily detected.
Table 2. Screening of donor substrates in the transglycosylation reaction of the -glycosidase from
P. woesei.a
Entry
Donor Substrate
Product
Conversion
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OH OH
OH OH
1
2
NO2
+++
+++
O
O
O
HO
O
O
HO
OH
OH
O
pNP-Gal
Gal-EMA
1
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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OH
OH
NO2
O
O
HO
HO
HO
HO
O
O
O
OH
OH
O
O
pNP-Glc
Glc-EMA
NO2
OH
OH
O
3
4
HO
HO
+
O
O
HO
O
HO
O
OH
Xyl-EMA
OH
pNP--D-Xyl
OH
OH
OHOH
OH
HO
+++
O
O
O
O
O
HO
HO
O
OH
OH
OH
O
Lactose
Gal-EMA
3
OH
OH
OH
5
6
+++
n.d.
O
O
O
O
HO
HO
HO
HO
O
O
HO
OH
OH
OH
O
Cellobiose
Glc-EMA
OH OH
O
OH OH
O
O
HO
OH
HO
O
OH
OH
O
Galactose
Gal-EMA
3
OH
O
OH
7
+
O
HO
HO
HO
HO
O
OH
O
OH
OH
O
Glucose
Glc-EMA
a
HEMA (2) was used as acceptor substrate in each of the reactions. Donor substrates were
dissolved in 900 µL 2 to a concentration of 11 mM (entries 4, 5) or 22 mM (entries 1, 2, 3, 6, 7)
and pre-heated to 85 °C. The addition of 100 µL pre-heated enzyme solution (citrate-Na HPO -
buffer, pH 5.5) started the reaction. The final enzyme concentration was 7.5 U/mL. Thin layer
chromatography (TLC) was used for qualitative analysis of conversion (Figure S4 in supporting
information). +: low conversion to product; ++: medium conversion to product; +++ high
conversion to product, n.d.: Not determined.
2
4
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Enzymatic synthesis and purification of Gal-EMA. The synthesis was conducted using a large
excess of 2 (7.01 M; 140-fold molar excess) with regard to the donor substrate. 1 was used as a
donor in the reaction in order to monitor both donor and acceptor substrates via HPLC analysis
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(Error! Reference source not found.).
Figure 2. A: Synthesis of 2-(β-galactosyl)-ethyl methacrylate (Gal-EMA, 3) with -glycosidase
from P. woesei. B: RP-HPLC-analysis of the synthesis at 0 h and 5 h reaction time. The product
peak is indicated by a black arrow; 5: 4-methoxyphenol; HPLC analysis was conducted at 205 nm.
C: LC-ESI-MS analysis of 3 (Error! Reference source not found.B). The calculated molecular
weight of 3 is 292.1 g/mol. The expected m/z ratio is 291.2 [M-H].
Since 2 is used in large excess, it appears as saturated peak after 19 min (Figure 2B). Peak 5
represents the polymerization inhibitor 4-methoxyphenol which was included in the commercial
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solution of 2. After 5 h, 1 is converted to compounds 4 and 3 which elute after approx. 23 min and
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1 min, respectively. Isolation and analysis via LC-ESI-MS of 3 revealed an m/z ratio of 291.1 [M-
H] in the negative detection mode, which corresponds to the calculated molecular weight of Gal-
EMA (292.1 g/mol) (Figure 2C). After stopping the reaction by the addition of EtOH and removal
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of the precipitated enzyme, 3 was purified by evaporation of EtOH, H O and HEMA and
2
subsequent silica column chromatography. The purified product was obtained as a transparent,
highly viscous liquid. The final yield was measured gravimetrically and amounted to 82 %
1
[
mol/mol] with regard to 1. The purified product was further analyzed via H-NMR (Figure 3) and
1
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1
C NMR (Figure S5). Regarding the H-NMR spectrum, all hydrogen-atoms in 3 can be assigned
to signals in the spectrum, which shows much more peaks compared to the spectrum of 2 (Figure
S6) due to the additional H-atoms of the galactose ring. However, although the product was purified
carefully, some signals not belonging to 3 probably resulting from impurities were observed.
Similar yields for 3 of up to 85 % were reported at a comparable transglycosylation/hydrolysis ratio
6
5
using whole-cell synthesis with lyophilized cells of S. solfataricus. Various glucosylated acrylate
monomers, such as 2-GlcEA (41 % [mol/mol]), 2-GlcEMA (43 % [mol/mol]) and 4-GlcBA (21 %
[mol/mol]) were produced in lower yields using reverse hydrolysis with commercial -glucosidase
59
from almonds. With the same enzyme, a kinetically driven approach with cellobiose as donor
substrate and 10 % [v/v] of 2 as acceptor yielded 8 % [mol/mol] of the product.59
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Figure 3. H-NMR of purified Gal-EMA (3) (solvent D O).
2
For further analysis of the chemical composition, the purified product was analyzed via infrared
(IR) spectroscopy (Figure 4).
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Figure 4. IR-spectrum of purified Gal-EMA (3) and HEMA (2). 3: red signal line; 2: blue signal
line.
For reasons of comparison, the spectra of 3 and 2 are displayed. 3 shows an increase in
-1
absorbance at the characteristic region for the stretching vibration of O-H bonds (3250 cm ) when
compared to 2. This indicates the presence of additional OH-groups which are incorporated into
the coupled galactose. Furthermore, 3 shows an increased absorbance band at approx. 1000 cm-1
that is not present in the spectrum of 2. This signal belongs to the C-O stretching vibration. Due to
the high number of C-O bonds in the attached galactose molecule, it is predominant in 3. Both
-1
-1
samples contain the characteristic bands at approx. 1600 cm and 1700 cm of the C=O bond and
the C=C bond stretching vibrations in the HEMA moiety, respectively.
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Having confirmed the correct mass, structure and presence of functional groups in the product,
a calibration curve for the absorbance of 3 at 205 nm was established using HPLC (Figure S7).
Subsequently, all analytes in the reaction except for the hydrolytic product galactose (5) could be
quantified using HPLC. The synthesis was scaled up to 100 mL and the course of the reaction over
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4 h was monitored via HPLC (Figure 5). The donor substrate 1 is almost completely converted
after 4.5 h. 3 is formed at approximately the same rate as 1 is converted. This indicates that, under
the given reaction conditions, 2 acts as the preferred acceptor substrate. The highest concentration
of 3 is reached after 4.5 h and amounts to 44 mM. The calculated yield of transglycosylation is
therefore 88 %, which means that apparently only 12 % of the initial donor substrate are subject to
hydrolysis. These numbers prove that the amount of hydrolysis is low compared to most other
74
transglycosylation reactions catalyzed by glycosidases. Evaporated 2 was collected and analyzed
1
via H-NMR (Figure S8). The spectrum indicated a pure substance which could successfully be
reused as acceptor substrate in further experiments (data not shown). The recycling of 2 therefore
reduces the amount of organic waste of this reaction and contributes to a sustainable monomer
production process.
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Figure 5. Time course of Gal-EMA synthesis catalyzed by -glycosidase from P. woesei in
100 mL reaction volume with a starting concentration of 50 mM pNP-Gal in an 85 % [v/v] HEMA
solution. The aqueous volume consisted of 15 mL enzyme solution (50 mM
citrate-phosphate-buffer, pH 5.5). The final enzyme concentration was 7.5 U/mL.
Enzymatic Elongation of Gal-EMA. So far, mainly sugar-functionalized acrylates have been
enzymatically synthesized which carry merely one monosaccharide moiety or contain a repetitive
sequence of the same monosaccharide, respectively.59,63 However, regarding potential future
applications, the elongation of the galactose-functionalized monomer synthesized in the present
work is desirable since most glycans with a significant role in biology consist of more than one
sugar building block. For instance, the precursors of the Lewis blood group epitopes consist of the
disaccharides N-acetyllactosamine (LacNAc) type 1 or type 2 in which the monosaccharides
galactose (Gal) and N-acetylglucosamine (GlcNAc) are linked by 1,3 or 1,4 glycosidic bonds,
8
respectively. Therefore, there is a need for incorporation of LacNAc-based glycan epitopes into
glycopolymers for further research of their role in pathogen adhesion and cancer metastasis.
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The galactose moiety of 3 is a potential substrate for the synthesis of LacNAc type 2 by the
reaction of glycosyltransferases. Figure 6A shows the scheme for the conversion of 3 to the new
product N-acetylglucosaminyl-1,3-2-(β galactosyl)-ethyl methacrylate (GlcNAc1,3Gal-EMA,
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) by the reaction of recombinant 1,3-N-acetylglucosaminyltransferase (1,3-GlcNAcT) from
Helicobacter pylori.
Figure 6. Synthesis of GlcNAc1,3Gal-EMA (7) by recombinant 1,3GlcNAcT from H. pylori.
A: Reaction equation. AP: Alkaline Phosphatase B: HPLC-chromatogram of the elongation of Gal-
EMA (3) to GlcNAc1,3Gal-EMA (7). x: unidentified peak. The product peak is indicated by a
black arrow. UDP-GlcNAc (6) and uridine elute in the solvent front (not shown). C: LC-ESI-MS
spectrogram of 7. The calculated molecular weight of 7 is 495.2 g/mol. The expected m/z ratio is
494.2 [M-H].
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HPLC-analysis of the reaction showed that the peak representing 3 is transformed into a second
peak (7) in the course of the reaction (Figure 6B). The product apparently elutes slightly after the
substrate, which is probably due to an overall decrease in hydrophilicity of 7 caused by the presence
of the acetyl group of the GlcNAc moiety. After 7 h, 3 is fully converted. The emerging peak was
analyzed via LC-ESI-MS and revealed an m/z ratio of 494.2, which corresponds to the calculated
molecular weight of 7 (495.2 g/mol) (Figure 6C). The catalytic reaction of the 1,3GlcNAcT used
here has been previously characterized by NMR spectroscopy with respect to its linkage specificity
on terminal galactose moieties.73,75 Therefore, we concluded that the stereo- and regioselective
chain elongation was successful. Gal-EMA represents a novel acceptor substrate for this
glycosyltransferase.
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The product peak was collected and the solvent was evaporated. Purified compound 7 was then
used for further chain elongation by recombinant human 1,4Galactosyltransferase (1,4GalT)
from human using UDP-Gal as donor substrate (Figure 7). Similar to the 1,3GlcNAcT, the
enzyme 1,4GalT has been extensively used by our group for the highly regio- and stereoselective
enzymatic production of amino-functionalized LacNAc-oligomers.73,76 The new product of this
reaction
is
termed
Galactosyl-1,4-N-acetylglucosaminyl-1,3-2-(β-galactosyl)-ethyl
methacrylate (Gal1,4GlcNAc1,3Gal-EMA).
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Figure 7. Synthesis of Galactosyl-1,4-N-acetylglucosaminyl-1,3-2-(β-galactosyl)-ethyl
methacrylate (Gal1,4GlcNAc1,3Gal-EMA, 9) by recombinant 1,4GalT from human. A:
Reaction equation. AP: Alkaline Phosphatase B: HPLC-chromatogram of the elongation of
GlcNAc1,3Gal-EMA (7) to Gal1,4GlcNAc1,3Gal-EMA (9). y: unidentified peak. The product
peak is indicated by a black arrow. UDP-Gal (8) and uridine elute in the solvent front (not shown).
C: LC-ESI-MS spectrogram of peak 9. The calculated molecular weight of 9 is 657.3 g/mol. The
expected m/z ratio is 656.3 [M-H].
HPLC analysis of the reaction indicates full conversion after approximately 1 h (Figure 7B).
Peak 9 was isolated and analyzed via LC-ESI-MS, revealing an m/z ratio of 656.2 [M-H], which
corresponds to the calculated molecular weight of 9 (657.3 g/mol) (Figure 7C). The correct regio-
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and stereoselective 1,4-linkage of Gal to the terminal GlcNAc residue was verified by the
hydrolytic activity of a commercially available 1,4 galactosidase preparation (BIOLACTASA
NTL) from Bacillus circulans (Figure S9). This enzyme has been described for its selective
hydrolysis of 1,4-linked terminal galactose.61
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In summary, the regioselective elongation of 3 to terminal LacNAc type 2 can be regarded as a
proof of principle that compound 3 can serve as a starting point in the synthesis of
acrylate-functionalized LacNAc glycan structures providing the basis for further synthesis of
LacNAc-based glycan epitopes and subsequent incorporation in glycopolymers. In this way, novel
kinds of glycopolymers can be provided for biomedical applications.
Synthesis and Characterization of PHEMA and PGal-EMA Homopolymers and
PHEMA/PGal-EMA Copolymers. In the next step, the synthesized and characterized
galactose-acrylate monomer was polymerized via free radical polymerization. Gal-EMA was
copolymerized with HEMA in different amounts leading to a series of linear PHEMA/PGal-EMA
copolymers (Figure 8, Table 3).
Figure 8. Chemical structure of PHEMA/PGal-EMA copolymers.
Also, linear PHEMA and PGal-EMA homopolymers were synthesized and investigated for a
complete analysis of the polymer system. In the final step, an Enzyme-Linked-Lectin Assay
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(
ELLA) was performed with the synthesized glycopolymers to demonstrate the application of these
glycopolymers as scaffolds for multivalency studies.
During the synthesis, a different behavior of the polymer reaction mixtures was observed. The
higher the amount of Gal-EMA in the system, the better the solubility of the synthesized polymers.
The higher water solubility of the polymers with a higher Gal-EMA content was expected, since
the hydrophilicity of the Gal-EMA due to the hydroxyl groups in the galactose moiety is higher
than the hydrophilicity of the HEMA with only one single hydroxyl group. For the PHEMA
homopolymer, the polymer chains already precipitated from the solution during the synthesis. The
amount of precipitate decreased with increasing Gal-EMA content. For the PGal-EMA
homopolymer, no precipitation was observed at all, but a clear polymer solution was obtained.
After lyophilization, the glycopolymers were obtained in form of fluffy and white solids. To
investigate the molecular weight distribution, gel permeation chromatography (GPC) was
performed (Table 3). However, due to the above described differences in hydrophilicity of the
polymers, not all samples could be solved in the same solvent for GPC measurements. The PGal-
EMA homopolymer was only soluble in water, while the other samples were solved in
dimethylformamide (DMF).
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Table 3. Molecular weights and molecular weight distributions (D) of synthesized glycopolymers
with varying Gal EMA 3 content determined by GPC.
Mn
Sample Gal-EMA content [mol%]
M [g/mol]
D
w
[g/mol]
10
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6600
17400
27700
54600
47000
14800
2.6
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From the GPC results, it can be seen that the molecular weight of the synthesized polymers
increases with increasing Gal-EMA content from ca. 6600 g/mol (PHEMA homopolymer) for the
number average to ca. 19200 g/mol (50 mol% PGal-EMA) (Table 2). However, the PGal-EMA
homopolymer has the lowest molecular weight of all samples, indicating a difference in the
polymerization process. This might be due to the sterical hindrance of the galactose moieties,
complicating the homopolymerization compared to the copolymerization with the less sterically
hindered HEMA monomer. The PGal-EMA homopolymer also has a higher MWD and thus a
broader size distribution compared to the copolymers, except for the copolymer with a Gal-EMA
content of 10 mol%, which can probably be regarded as outlier (see Figure S10 for size distribution
curves determined by GPC).
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NMR was performed to analyze if copolymers of HEMA and Gal-EMA were obtained and to
investigate the residual monomer content in the polymers (Figure 9). As described for the GPC
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measurements, also for H-NMR spectroscopy different solvents had to be used. The PHEMA
homopolymer was solved in deuterated methanol (MeOD-d ) for the NMR investigation, while all
4
other polymers were solved in dimethyl sulfoxide-d6 (DMSO-d6).
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