Amylase catalyzed synthesis of glycosyl acrylates and their polymerization

Article abstract of DOI:10.1039/c3gc41471j

The enzymatic synthesis of novel (di)saccharide acrylates from starch and 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate and 4-hydroxybutyl acrylate (2-HEA, 2-HEMA and 4-HBA) catalyzed by various commercially available amylase preparations is demonstrated. Both liquefaction and saccharification amylases were tested as biocatalysts. Transglycosidation products were only detected in reaction mixtures containing saccharification amylases. The glycoamylase from Aspergillus niger was found to catalyze the synthesis of 2-(α-glucosyloxy) -ethyl acrylate (Glc-α-EA) from starch. The maltogenic amylase from B. stearothermophilus was found to catalyze the synthesis of 2-(α- maltosyloxy)-ethyl acrylate (Glc-Glc-EA). The transglycosidation product was isolated and purified and its structure was confirmed by ¹H-NMR, ¹³C-NMR and ESI mass spectrometry. The selectivity of the enzyme towards the glycosidation position appeared to be extremely high. Only the anomeric hydroxyl group (C₁-OH) of the disaccharide appeared to be glycosidated and the glycosidation products were anomerically pure. Subsequently, the enzymatically synthesized maltosyl acrylate was successfully polymerized by aqueous free radical polymerization to yield a novel acrylic polymer with pendant maltosyl units.

Full text of DOI:10.1039/c3gc41471j

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Amylase catalyzed synthesis of glycosyl acrylates and
their polymerization†
Cite this: DOI: 10.1039/c3gc41471j
Wouter M. J. Kloosterman, Danijela Jovanovic, Sander G. M. Brouwer and Katja Loos*
The enzymatic synthesis of novel (di)saccharide acrylates from starch and 2-hydroxyethyl acrylate,
2
-hydroxyethyl methacrylate and 4-hydroxybutyl acrylate (2-HEA, 2-HEMA and 4-HBA) catalyzed by
various commercially available amylase preparations is demonstrated. Both liquefaction and saccharifi-
cation amylases were tested as biocatalysts. Transglycosidation products were only detected in reaction
mixtures containing saccharification amylases. The glycoamylase from Aspergillus niger was found to cat-
alyze the synthesis of 2-(α-glucosyloxy)-ethyl acrylate (Glc-α-EA) from starch. The maltogenic amylase
from B. stearothermophilus was found to catalyze the synthesis of 2-(α-maltosyloxy)-ethyl acrylate
(
Glc-Glc-EA). The transglycosidation product was isolated and purified and its structure was confirmed by
1
13
H-NMR, C-NMR and ESI mass spectrometry. The selectivity of the enzyme towards the glycosidation
position appeared to be extremely high. Only the anomeric hydroxyl group (C –OH) of the disaccharide
Received 24th July 2013,
1
Accepted 25th September 2013
appeared to be glycosidated and the glycosidation products were anomerically pure. Subsequently, the
enzymatically synthesized maltosyl acrylate was successfully polymerized by aqueous free radical
polymerization to yield a novel acrylic polymer with pendant maltosyl units.
DOI: 10.1039/c3gc41471j
www.rsc.org/greenchem
1
0
functionalized saccharide acrylate monomers. Alternative to
these synthesis routes, biocatalytic pathways towards (poly)sac-
Introduction
1
1–16
Glycopolymers include both natural and artificial carbohydrate charide synthesis and modification
are beneficial and
containing polymers, as well as synthetically modified natural were explored here for the synthesis of maltosyl acrylates.
saccharide-based polymers. Poly(saccharide acrylates) are Since biocatalysts show extremely high enantio-, regio- and
special types of glycopolymers that combine an apolar acrylic chemoselectivity, mono-functionalization of saccharides can
backbone with pending saccharide groups. Since the saccha- usually be achieved easily. Moreover, biocatalysis is advan-
ride side-groups lead to water-soluble, non-toxic, biocompati- tageous with respect to the ambient reaction conditions used,
ble and biobased polymers, they have received increased i.e. atmospheric pressure, low reaction temperature, typically
1
–4
17
attention over the past decade.
They have been used in 37 °C, and neutral pH. In addition, enzymes are also non-
diverse applications including as models of biological toxic; they are obtained from natural renewable resources and
5
,6
7
17
systems, macromolecular drugs and drug delivery systems,
can often be recycled. Especially enzymes from the classes of
8
9
18
polymeric surfactants and many others. The synthesis of glycosyl transferases and hydrolases (EC 2.4 and 3.2) have
poly(saccharide acrylates) requires well-defined mono-functio- been used in organic synthesis of (poly)saccharides and glyco-
nalized saccharide acrylate monomers. The synthesis of such sides since carbohydrates are their natural substrates. α-Amy-
monomers involves a number of challenges, since there are lases have been applied as catalysts in the organic synthesis of
many possibilities for the formation of positional isomers in glycosides of various alcohols (methyl-, ethyl-, propyl-, butyl-
which different hydroxyl groups of the saccharide become and benzyl-). Glycoamylase has been used as a catalyst for
functionalized with the acrylate group. Using conventional the synthesis of glycosides from hydroxybenzyl alcohol,
synthesis routes, multiple reaction steps are required and all octanol, vanillin, eugenol and curcumin. Nevertheless,
but one hydroxyl group has to be protected to obtain mono- the amylase catalyzed synthesis of saccharide acrylates from
starch and hydroxyl functional acrylates that we present here
1
9
2
0
2
1
22
23
has not been described before. Our biocatalytic synthesis
Department of Polymer Chemistry, Zernike Institute for Advanced Materials,
University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
E-mail: K.U.Loos@rug.nl; Fax: +31-50-3634400; Tel: +31-50-3636867
routes using glycoamylase from Aspergillus niger or maltogenic
amylase from Bacillus stearothermophilus as the catalyst result
in anomerically pure, mono-functional (di)saccharide acrylate
monomers in one single reaction step from starch. The
polymerization of the enzymatically synthesized disaccharide
13
†
Electronic supplementary information (ESI) available: C-NMR spectra of Glc-
Glc-EA and poly(Glc-Glc-EA); positive ion ESI-MS spectrum of Glc-Glc-EA; initial
and remaining enzyme activities. See DOI: 10.1039/c3gc41471j
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acrylate 2-(α-maltosyloxy)-ethyl acrylate yields a novel acrylic Spots were visualized as described by Hansen using diphenyla-
2
4
polymer with pendant maltosyl units.
mine/aniline reagent. Saccharide derivatives appeared as
blue/black spots on a white background after spraying the
DPA/aniline reagent and heating at 120 °C for 1 hour. Column
chromatography was performed on silica gel 60 (400 mesh).
Experimental section
Materials
The sample was eluted with CHCl
CHCl –MeOH 3 : 1 and subsequently CHCl
3
–MeOH 4 : 1, followed by
–MeOH 2 : 1. UV/
3
3
Dinitrosalicylic acid (DNSS), hydroquinone (HQ), monomethyl
ether hydroquinone (MEHQ), potassium sodium tartrate
Vis spectrometry measurements were performed using a PYE
Unicam SP-200 UV/Vis spectrophotometer. HPLC was done
using a Shimadzu HPLC under reversed phase conditions. An
Alltech HP column (Alltima amino, 5 μm, 150 × 4.6 mm) was
used for separation. The column temperature was maintained
at 40 °C and the injection volume was 15 μL. Compounds were
detected using a SPD-M20A photodiode array detector at
(
K-Na-tartate), maltose (Glc-2), maltotriose (Glc-3), potato
starch, 2-hydroxyethyl acrylate (2-HEA) 96% containing
00 ppm MEHQ, 2-hydroxyethyl methacrylate (2-HEMA) 97%
containing 200 ppm MEHQ, lithium bromide (LiBr), sodium
azide >99% (NaN ), diphenylamine (DPA) and potassium
2
3
2 2 5
metabisulfite (K S O ) were obtained from Sigma-Aldrich.
Maltotetraose (Glc-4), 2-hydroxyethyl methacrylate 97% stabil-
ized with 200 ppm MEHQ (2-HEMA) and acetic acid 99.5%
2
05 nm. A gradient of acetonitrile and double distilled water
−1
was used as the eluent with a flow rate of 0.5 mL min . The
water content of the eluent was 15 vol% for 5 minutes followed
by a linear increase to 60 vol% in 85 minutes (gradient A) or
slowly increased from 5 vol% to 15 vol% in 5 minutes, followed
by a linear increase to 60 vol% in 85 minutes (gradient B). The
column is brought back to initial conditions by a linear gradi-
ent from 60 vol% water to 15 or 5 vol% in 20 minutes and sub-
sequently equilibrated for 10 minutes, before the next
injection. Gradient A is used for reaction mixtures containing
(
(
HAc) were obtained from Acros. 4-Hydroxybutyl acrylate
4-HBA), 95%, containing 200 ppm MEHQ was obtained from
ABCR chemicals. D-(+)-Glucose (anhydrous) (Glc), ethyl acetate
EtAc), sodium acetate trihydrate (NaAc), aniline and
(
ammonium persulphate (APS) were obtained from Merck.
Soluble starch was supplied by Difco. Silica 230–400 mesh
was obtained from Silicycle. Chloroform (CHCl
3
), methanol
(MeOH), propan-2-ol (IPA) and other mentioned organic
2
-HEA and gradient B is used for reactions with 2-HEMA.
solvents were obtained from Lab-scan. All chemicals were used
as received.
ESI-MS was performed using a Thermo Scientific LTQ Orbitrap
mass spectrometer in the positive ion mode. Samples were dis-
solved in water and diluted with an equal volume of methanol.
Gel permeation chromatography (GPC) measurements were
Amylases
Various types of amylases were obtained from five different performed in N,N-dimethylformamide (DMF) containing
suppliers. α-Amylases from Aspergillus oryzae were obtained 0.01 M LiBr on a Viscotek GPCMAX equipped with TDA detec-
from Genencor (Clarase L) and Sigma-Aldrich (A8220). α-Amy- tors (model 302), using PSS-Gram-1000/30 columns. The molar
lases from Bacillus subtilis were obtained from Amano mass was calculated relative to polymethylmethacrylate using
(
Neomalz H O and Kleitase T10S), α-amylases from Bacillus narrow disperse standards (Polymer Laboratories). FT-Infrared
2
licheniformis were obtained from Genencor (Spezyme FRED, spectroscopy (FTIR) was done using a Bruker JFS 88 equipped
AA, Alpha and Clearflow AA). Thermostable α-amylases were with an ATR-golden gate unit.
obtained from Novozymes (Liquozyme Supra) and AB enzymes
(
Gamalpha P120L and Spezial). α-Amylases from Bacillus
Determination of amylase stability in aqueous 2-HEA solution
amyloliquefaciens were obtained from Sigma-Aldrich (A7595)
and Amano (Biokleitase L500). β-Amylases from barley were The stability of the enzymes was determined by measurement
obtained from Sigma-Aldrich (A7130) and Genencor (Optimalt of their remaining hydrolytic activity after incubation in
BBA and Spezyme DBA). Glycoamylases were obtained from aqueous 2-HEA solution. The hydrolytic activity was deter-
Sigma-Aldrich (A7095), Novozymes (NS22035 and Spirizyme) mined spectrophotometrically using the DNSS method as
2
5
and AB Enzymes (Gammadex) and maltogenic amylase from described in the literature. Reaction mixtures for the deter-
Bacillus stearothermophilus were obtained from Novozymes mination of the amylase stability contained soluble starch
(
Novamyl L) and AB Enzymes (Veron Xtender).
(30 wt%/vol%), sodium acetate buffer (7 mL, pH 5.0, 50 mM),
-HEA (3 mL) and 284 U of the corresponding amylase. Test
blank solutions contain soluble starch (30 wt%/vol%), sodium
H-NMR and C-NMR spectra were recorded using a 7.05 T acetate buffer (10 mL, pH 5.0, 50 mM) and 284 U of the corres-
Varian VXR spectrometer (300 MHz and 75 MHz resolution for ponding amylase. The reaction mixtures were incubated at
2
Characterization
1
13
1
13
H and C-NMR, respectively). Deuterium oxide (99.9 atom% 60 °C for 15 minutes before a sample (200 μL) was taken. The
D, Aldrich) was used as the solvent. Thin layer chromatography sample was diluted with 17.5 mL sodium acetate buffer (pH
TLC) was performed on aluminium sheets Silica gel 60/Kiesel- 6.0, 16 mM) to obtain the right conditions for the determi-
(
guhr F254 (Merck). Three eluent mixtures were used for the sep- nation of the remaining hydrolytic activity. From this solution,
aration of the saccharide acrylates: 5/10/4 (v/v/v) n-But–IPA– 600 μL was used for the determination of the hydrolytic activity
water, 3/1/1 (v/v/v) EtAc–HAc–water or 2/1 (v/v) CHCl –MeOH. as described above. The remaining activity is calculated by the
3
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following equation:
Glc-Glc-EA: transparent syrup, 0.408 g, purity: >85%, yield:
mol%; ESI-MS: calculated for C H O + Na: 463.1422;
found: 463.3333. H-NMR (D O) δ in ppm: 6.44 (H11, cis, J =
2
ꢀ
ꢁ
5
1
7
28 13
ðmaltoseTest Blank ꢀ maltosetestÞ
1
R:A:ð%Þ ¼ 1 ꢀ
ꢁ 100
maltoseTest Blank
1
1
7.3 Hz), 6.21 (H10, J = 17.3, 10.45 Hz), 5.99 (H11, trans, J =
0.43 Hz), 5.35 (H1′, d, J = 3.76 Hz), 4.96 (H1, d, J = 3.75 Hz),
1
3
with R.A. = remaining hydrolytic activity of the amylase (%), 4.39 (H8), 4.2–3.2 (H2–H6, H2′–H6′, H7, H7′). C-NMR (D
2
O)
maltoseTest_Blank = released maltose in test blank reaction δ in ppm: 168.6 (C9), 132.8 (C10), 127.7 (C11), 100.1 (C1′α),
(
(
μmol) and maltosetest = released maltose in test reaction 98.2 (C1α), 77.5 (C4′), 76.3 (C4), 73.6 (C3), 73.1 (C3′), 72.9 (C2),
μmol). 72.0 (C2′), 71.2 (C5), 69.5 (C5′), 67.7 (C8), 64.1 (C7), 63.5 (C6′),
0.6 (C6).
6
Determination of the synthetic activity of α-amylases
Influence of alcohol concentration on the synthesis of
-(α-maltosyloxy)-ethyl(meth)acrylate catalyzed by maltogenic
amylase from Bacillus stearothermophilus
Reaction mixtures (total volume 15 mL) contained 3.0 g
soluble starch dissolved in 10 mL sodium acetate buffer solu-
2
tion (pH 5.0, 50 mM, 0.02% NaN
3
). Subsequently 2-HEA
The influence of the alcohol concentration (2-HEA or 2-HEMA)
on the concentration of transglycosidation product was
measured by HPLC analysis. Reaction mixtures contain
soluble starch (30 wt%/vol%), sodium acetate buffer (10 mL,
(
2
5 mL) was added. The reactions were started by addition of
000 U of the corresponding amylase and incubated at 55 °C
for 3 days. After 3 days the reaction mixtures were analyzed
with thin layer chromatography using n-But–iPA–water 5/10/4
as the eluent.
pH 5.0, 50 mM, 0.02% NaN ), maltogenic amylase from
3
B. stearothermophilus (1 mL 2000 U) and 2-HEA or 2-HEMA.
The amount of 2-HE(M)A was varied from 0.9 to 70 vol% by
the addition of 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.5, 13 or 26 mL. The
reaction mixtures were incubated at 50 °C for 3 days. One milli-
liter of the reaction mixtures was taken, diluted with 24 mL
ethanol and measured with HPLC.
Maltogenic amylase catalyzed transglycosidation using
hydroxyl functional (meth)acrylates
Reaction mixtures (total volume 15 mL) contained 3.0 g
soluble starch dissolved in 10 mL sodium acetate buffer solu-
tion (pH 5.0, 50 mM, 0.02% NaN ). Subsequently, 5 mL of the
3
acceptor alcohol (2-HEA, 2-HEMA or 4-HBA) was added. The
reactions were started by the addition of 2000 U maltogenic
amylase from Bacillus stearothermophilus. The reaction mix-
tures were incubated at 55 °C for 3 days. After 3 days the reac-
tion mixtures were analyzed with TLC using EtAc–HAc–water
Polymerization of 2-(α-maltosyloxy)-ethyl acrylate
A novel maltosyl acrylate monomer (Glc-Glc-EA) was syn-
thesized according to the enzymatic synthesis procedure
described above. The monomer (0.45 mmol) was dissolved in
1
1
0 mL water ([M] = 45 mmol L− ). The reaction mixture was
3
/1/1 as the eluent. Spots were visualized as described above.
purged with nitrogen for at least one hour. Initiator solutions
were prepared: 6 wt% K in 10 mL water and 12 wt% APS
2 2 5
S O
Preparative synthesis of 2-(α-maltosyloxy)-ethyl acrylate by
maltogenic amylase from Bacillus stearothermophilus
in 10 mL water. The reaction mixture was purged with nitrogen
and heated to 55 °C. The reaction was initiated by the addition
of 0.2 mL APS solution (12 wt%) and 0.2 mL potassium meta-
bisulfite solution (6 wt%). The polymerization reaction was
continued for 24 hours under a nitrogen atmosphere. After
polymerization, the reaction mixture was precipitated into ten
volumes of methanol and subsequently dried under vacuum
overnight.
The reaction mixtures (total volume 100 mL) contained 5.0 g
soluble starch in sodium acetate buffer solution (pH 5.0,
5
0 mM, 0.02% NaN ). Subsequently 2-hydroxyethyl acrylate
3
(30 mL) was added. The reaction was started by the addition of
maltogenic amylase solution (10 mL 20 kU). The reaction
mixture was incubated at 55 °C for 4 days. The reaction
mixture was analyzed with TLC. By using the CHCl
eluent, three spots were detected with the DPA/aniline reagent:
i) 2-hydroxyethyl acrylate (R 1.00, yellow spot); (ii) 2-(α-malto-
3
–MeOH
Poly(2-α-maltosyloxyethyl acrylate) (P(Glc-Glc-EA)): white
1
powder; 0.15 g, H-NMR (D
2
O) δ in ppm: 5.35 (H1′), 4.96 (H1),
(
f
3.44–4.34 (H2–H6, H2′–H6′, H7, H8), 1.70–2.70 (H10b, H11b).
f f
syloxy)-ethyl acrylate (R 0.45, blue spot); (iii) glucose (R
0.28, 13
2
C-NMR (D O) δ in ppm: 176.4 (C9), 100.4 (C1), 98.5 (C1′),
blue spot).
7
6
7.8 (C4, C4′), 74.8–69.5 (C2–C5, C2′–C5′), 65.7 (C8), 64.5 (C7),
0.7 (C6, C6′), 42.2 (C10b), 34.3 (C11b). FTIR-ATR (cm ): 3400
Unreacted 2-HEA was removed by extraction with diethyl
ether. Hydroquinone was added to the aqueous layer and the
solution was concentrated by rotary evaporation at 30 °C. The
unreacted starch was subsequently precipitated in isopropanol
and removed by filtration. The filtrate was concentrated again
by rotary evaporation at 30 °C and a solution containing the
product was purified by flash chromatography. Fractions con-
−1
(
O–H stretch), 2940 (Aliphatic C–H stretch), 1720 (CvO
stretch); 1050 (C–O stretch).
Results and discussion
Activity of various amylases in saccharide acrylate synthesis
f
taining the product (R 0.45) were pooled, 10 mg hydroquinone
was added to prevent thermal polymerization and the eluent We have investigated the catalytic activity of various amylases
was removed by rotary evaporation at 30 °C.
in the synthesis of saccharide acrylate monomers from
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3
Scheme 1 Amylase catalyzed synthesis of novel (di)saccharide acrylates by transglycosidation of starch with 2-HEA (m = 1, R = H), 2-HEMA (m = 1, R = CH ) and
4
-HBA (m = 2, R = H).
2
-hydroxyethyl acrylate and starch according to Scheme 1. transglycosidation products can be visually detected by TLC
α-Amylases that are commonly used for liquefaction of starch analysis, their concentration in the reaction mixture is rather
were tested as biocatalysts for the transglycosidation reaction. low, especially compared to the amount of saccharidic pro-
The “liquefaction” α-amylases that we used were endo-type ducts that result from hydrolysis. As a result the transglyco-
amylases and originated from Aspergillus oryzae, Bacillus sidation products could not be obtained pure from the
subtilis, Bacillus licheniformis and Bacillus amyloliquefaciens. reaction mixture and could therefore not be analyzed by
1
Secondly, amylases that are commonly used for the saccharifi-
H-NMR and ESI-MS. However, their retention factor in TLC
cation of starch were tested as biocatalysts for the transglycosi- provides information about the physical properties of the two
dation reaction between starch and 2-HEA as well. The transglycosidation products. First of all, the retention factors
hydrolytic activities of all amylases used in this study are listed lower than the reference glucoside acrylate indicate that the
in Fig. S4.† The “saccharification” amylases that we used were compounds were more polar than 2-(β-glucosyloxy)-ethyl acry-
f
exo-type amylases and originated from Aspergillus niger, Bacil- late. Secondly, both compounds (R 0.67 and 0.59) were more
lus stearothermophilus and barley (β-amylase). The synthetic hydrophobic than glucose, which has a retention factor of only
activity of all amylases was analyzed by TLC. Remarkably, of 0.53. Finally, both spots were colored light blue by the TLC
the ten tested “liquefaction” amylases only the reaction reagent, indicating a saccharide derivative. Based on these pro-
mixture with α-amylase from A. oryzae contained saccharide perties the transglycosidation products with the retention
acrylate glucosides as a result of transglycosidation. Compar- factors 0.67 and 0.59 were most likely 2-(α-maltosyloxy)-ethyl
able results were obtained by Santamaría in the α-amylase acrylate and 2-(α-maltotriosyloxy)-ethyl acrylate, respectively,
2
6
catalyzed transglycosidation of starch with methanol. They but their structure could not be fully characterized as a result
observed no transglycosidation products when α-amylases of the low product concentrations in the reaction mixture. In
from bacterial sources, i.e. B. licheniformis, were used as the general, we might state that the “liquefaction” α-amylases
biocatalyst. On the other hand, methyl-saccharides could be from A. oryzae, B. subtilis, B. licheniformis and B. amyloliquefa-
detected using the α-amylase from A. oryzae as the catalyst for ciens that were used in this study, were ineffective catalysts for
the transglycosidation. We could detect two spots of transgly- transglycosidation of 2-HEA and starch.
cosidation products with TLC using α-amylase from A. oryzae
Much better results were obtained using the “saccharific-
as the catalyst. The retention factors of the new spots were ation” amylases from Aspergillus niger, Bacillus stearothermophi-
found to be 0.67 and 0.59, in between the spots of the nor- lus and barley (β-amylase). Almost all commercially available
mally observed hydrolysis products glucose and maltose “saccharification” amylase that were used for the transglyco-
(
R 0.53 and R 0.43, respectively) and a reference glucoside sidation reaction between 2-HEA and starch showed new spots
f f
2
f
-(β-glucosyloxy)-ethyl acrylate (R 0.73) that was synthesized by in TLC corresponding to saccharide acrylates. Transglyco-
β-glucosidase from almond catalyzed glycosidation reaction of sidation products could be detected in all reaction mixtures
2
7
D-glucose with 2-hydroxyethyl acrylate (2-HEA). Although the with the saccharification α-amylase from B. subtilis as the only
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exception. When β-amylase was used as the biocatalyst, the (R
f
0.55). Since maltogenic amylases are known for producing
2
8
retention factor of the product (R 0.67) was lower than the high maltose syrups from liquefied starch, it could be
f
f
retention factor of 2-(β-glucosyloxy)-ethyl acrylate (R 0.73) and expected that the product contained a maltose unit joined to
thus the transglycosidation product must be more polar. Given the acceptor alcohol by a glycosidic linkage. Secondly, the
that the mechanism of β-amylase catalyzed hydrolysis (cleavage maltogenic amylase from B. stearothermophilus is known to be
of β-maltose from amylose) the transglycosidation product was a retaining amylase, resulting in a glycoside in α-configuration.
likely to be 2-(β-maltosyloxy)-ethyl acrylate. However, also for Therefore, the transglycosidation product was expected to be
the β-amylase catalyzed transglycosidation, the intensity of the 2-(α-maltosyloxy)-ethyl acrylate. Since the product concen-
spot of the new compound was very low, indicating an ineffi- tration of the maltogenic amylase catalyzed transglycosidation
cient transfer or enzyme deactivation. On the other hand, both reaction was relatively high, the product could be isolated,
reaction mixtures containing glycoamylases and maltogenic purified and its structure elucidated with NMR and ESI-MS.
amylases showed intense spots of transglycosidation products.
Structural analysis of the product synthesized by maltogenic
amylase from Bacillus stearothermophilus
In general, we found that the activity after incubation in
aqueous 2-HEA solution of glycoamylases and maltogenic amy-
lases was higher compared to β-amylase from barley (see The product of the maltogenic amylase catalyzed transglyco-
Fig. S5† for the remaining activity data). The higher enzyme sidation of starch and 2-HEA was collected by preparative
stability in the presence of 2-HEA, might explain the higher column chromatography. Relative to the starch used, about
concentration of the transglycosidation products.
8 wt% of transglycosidation product was obtained after the
In the reaction mixtures containing glycoamylase from purification steps. The chemical structure of the product was
1
A. niger a product spot with a retention factor of 0.75 was revealed by H-NMR (Fig. 1). Two different signals of the
detected. The retention factor of the new product was identical anomeric protons were found. The first type of anomeric
to the spot of the reference compound; 2-(β-glucosyloxy)-ethyl protons was found at 5.34 ppm. This chemical shift is typically
acrylate. Since the mobility of the product synthesized by observed for anomeric protons, H-1, of α-(1,4)-linked D-gluco-
glycoamylases was equal to the mobility of the reference com- pyranose residues in amylose and maltooligosaccharide
2
9
pound and the glycoamylase from A. niger is known to be a chains. The second type of anomeric protons was found at
retaining amylase, the product is very likely to be 2-(α-glucosyl- 4.95 ppm. Anomeric protons with a chemical shift around
3
0
oxy)-ethyl acrylate.
4.90 ppm are found for glycosides with α-linked alkyl chains.
The reaction mixtures containing maltogenic amylase Secondly, the low coupling constant of 3.3 Hz found for this
showed a new spot with a retention factor of 0.68. The mobility proton is characteristic of a linkage in α-configuration. Thus,
of this glycosidation product indicates a polarity higher than we can conclude that the ethyl acrylate moiety was attached to
2
-(β-glucosyloxy)-ethyl acrylate and lower than glucose the saccharide by a 1-O-α-linkage. A 1 : 1 ratio between the
1
Fig. 1 H-NMR spectra of 2-(α-maltosyloxy)-ethyl acrylate (top) and its corresponding polymer poly(2-(α-maltosyloxy)-ethyl acrylate) (bottom spectrum).
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α-linked ethyl acrylate moiety (4.95 ppm) and the α-(1,4)-linked
D-glucopyranose unit (5.34 ppm) was determined by the inte-
gration of the peak areas. This fully agreed with the expected
structure of 2-(α-maltosyloxy)-ethyl acrylate. Secondly, the
characteristic peaks corresponding to the acrylate double bond
were found at 5.97–6.47 ppm. Integration of an acrylic proton
peak and a comparison with the two anomeric protons (4.95
and 5.34 ppm) result in a 1.00 : 0.94 : 1.08 ratio (see also
Fig. 1). This proves that the disaccharide was functionalized
with just one acrylate moiety. This is crucial for saccharide
acrylate monomers that will be used for the synthesis of linear
glycopolymer by aqueous free radical polymerization as
described below. Additionally, the structure of 2-(α-malto-
1
3
syloxy)-ethyl acrylate was confirmed by C-NMR and ESI-MS
(
Fig. S1 and S2,† respectively). Signals corresponding to β-gly-
1
cosides were not present in the H-NMR spectrum or the
1
3
C-NMR spectrum. Thus the biocatalyst was found to catalyze
Fig. 2 TLC chromatogram of reaction mixtures with 2-HEA (lane 1), 2-HEMA
the transglycosidation of starch with retention of the anomeric
configuration. Furthermore, using the maltogenic amylase
from B. stearothermophilus as the biocatalyst a mono-func-
tional disaccharide acrylate was directly synthesized from
starch and 2-HEA in one single reaction step, something which
is impossible to achieve using conventional chemistry.
(lane 2) or 4-HBA (lane 3). Lane S contains saccharide standards of glucose (Glc),
maltose (Glc-2), maltotriose (Glc-3) and maltotetraose (Glc-4).
Glc-Glc-EA and Glc-Glc-EMA, was determined. The concen-
tration of the products in the reaction mixtures was deter-
mined by HPLC. Using purified products, the peaks
corresponding to Glc-Glc-EA and Glc-Glc-EMA were found at
9.25 and 14.20 min, respectively. The amount of transglyco-
sidation product was determined by integration of the corres-
ponding peak areas. Fig. 3 shows the dependence of the
volume percentage of the acceptor alcohol on the peak area of
the transglycosidation products. The concentration of Glc-Glc-
EA was found to increase by increasing the amount of 2-HEA
Maltogenic amylase catalyzed transglycosidation with other
polymerizable alcohols
The specificity of the maltogenic amylase from B. stearothermo-
philus was tested with polymerizable alcohols other than
2
-HEA as well. Other polymerizable, structurally similar, alco-
hols used were 2-hydroxyethyl methacrylate (2-HEMA) and
-hydroxybutyl acrylate (4-HBA). The reaction mixtures contain-
4
10-fold from 0.9 vol% to 37 vol%. Further increase of the
ing 30 vol% 2-HEMA and 4-HBA were analyzed by TLC analysis.
Transglycosidation products were found as well using
alcohol concentration in the reaction mixture resulted in a
decrease of the amount of product. Most likely, this decrease
was caused by the loss of enzyme activity in solutions contain-
ing higher amounts of alcohols. The curve of Glc-Glc-EMA
2
-HEMA and 4-HBA as the acceptor alcohol (see Fig. 2). The
retention factors of the transglycosidation products increased
with increasing hydrophobicity of the acceptor alcohol (2-HEA
<
2
2-HEMA
< 4-HBA). The novel disaccharide acrylates
-(α-maltosyloxy)-ethyl methacrylate (Glc-Glc-MA) and 2-(α-mal-
tosyloxy)-butyl acrylate (Glc-Glc-BA) had retention factors of
.48 and 0.52, respectively. The results showed that the malto-
0
genic amylase from B. stearothermophilus catalyzed synthesis
of saccharide acrylates was not limited to 2-hydroxyethyl acry-
late, but also other (meth)acrylates with a primary alcohol
group can be used as the acceptor alcohol.
The maltogenic amylase is the first enzyme found that cata-
lyzes the synthesis of saccharide acrylates containing more
than one glucose unit by direct glycosidation in a single reac-
tion step. Therefore, optimisation of the transglycosidation
yield was done by varying the acceptor alcohol concentration.
Influence of alcohol concentration on yield of saccharide
acrylate
A quite effective way to improve the glycosidation yield in
transglycosidation reactions is to increase the donor or the
Fig. 3 Effect of the volume fraction of 2-HEA and 2-HEMA on the concen-
tration of transglycosidation products 2-(α-maltosyloxy)-ethyl acrylate and
3
1,32
acceptor concentration.
The influence of the volume frac-
tion of alcohols 2-HEA and 2-HEMA on the concentration of 2-(α-maltosyloxy)-ethyl methacrylate, respectively.
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shows a similar trend, although the optimal product concen- (cellobiosyloxy)-ethyl methacrylate) could be obtained by using
tration was found at 20 vol% 2-HEMA rather than 37 vol%. the same initiator system in a lower initiator to monomer
3
3
Apparently, the amylase was less active when the hydroxyl ratio. Therefore, we expect that the molecular weight of
functional acceptor was more hydrophobic. A similar depen- P(Glc-Glc-EA) could easily be increased by lowering the
dence on acceptor hydrophobicity was obtained in the amylase initiator to monomer ratio.
1
9
1
13
catalyzed alkyl glucoside synthesis reported by Larsson.
Nonetheless H-NMR, C-NMR and GPC proved the suc-
Nevertheless, the concentration of Glc-Glc-EMA was increased cessful polymerization of the maltosyl acrylate monomer we
with a 13-fold increase in the amount of 2-HEMA from 0.9 to synthesized enzymatically. Successful polymerization of such a
2
0 vol%.
maltosyl acrylate was never reported before and the resulting
polymers with a high OH functionality might be very interest-
ing as dispersing agents in the field of coatings and
Aqueous free radical polymerization of maltosyl acrylate
monomer Glc-Glc-EA
34
biomedicine.
The polymerization of the enzymatically synthesized monomer
2
-(α-maltosyloxy)-ethyl acrylate was performed in aqueous solu-
Conclusions
tions using an APS/K S O initiator system according to
2
2 8
1
Scheme 2. Fig. 1 shows the H-spectrum of the starting We have tested the catalytic activity of various amylases in the
monomer Glc-Glc-EA (top spectrum) and the spectrum of the synthesis of saccharide acrylate monomers from 2-hydro-
resulting product poly(Glc-Glc-EA) (bottom spectrum). The xyethyl acrylate and starch. Liquefaction α-amylases from
three signals at 6 to 7 ppm, which correspond to the protons Aspergillus oryzae, Bacillus subtilis, Bacillus licheniformis and
of the acrylate group, were found to disappear upon Bacillus amyloliquefaciens were tested as biocatalysts for the
polymerization.
transglycosidation reaction. Similarly, saccharification amy-
The spectrum of the polymer product shows broad peaks in lases from Aspergillus niger, the maltogenic amylases from
the region 1.6 to 2.6 ppm that correspond to the aliphatic Bacillus stearothermophilus and β-amylase from barley were
protons in the backbone of P(Glc-Glc-EA), proving that the tested as biocatalysts for the transglycosidation reaction.
maltosyl acrylate monomer was successfully polymerized. In Liquefaction amylases were found to be more stable in
addition, the spectra show that the signals of the anomeric aqueous 2-HEA compared to saccharification amylases, but
protons 1 and 1′ do not shift, disappear or change in ratio. they do not catalyze the transglycosidation of starch and
This proves that the glycosidic part of the monomer Glc-Glc-EA 2-HEA. In contrast, the saccharification amylases do catalyze
was not changed during the polymerisation and thus, the the transglycosidation reaction between starch and 2-HEA. The
polymer consists of a carbon–carbon backbone with a 1-O- glycoamylases from Aspergillus niger and the maltogenic amy-
α-maltosyl group attached by a 2-oxyethyl carboxylate spacer lases from Bacillus stearothermophilus appear to be the most
(
as depicted in Scheme 2). The polymer structure was further effective as biocatalysts in the synthesis of saccharide acrylates.
1
3
confirmed by C-NMR (Fig. S3†). GPC analysis revealed that The transglycosidation product of the glycoamylase catalyzed
the molecular weight of the polymer P(Glc-Glc-EA) was in reaction was found to have the same retention factor as the
accordance with the relatively high initiator to monomer ratio reference compound 2-(β-glucosyloxy)-ethyl acrylate. Given the
−
1
−1
used; M = 4 kg mol , Mw = 15 kg mol , D = 3.9. In theory, specific properties of glycoamylases, we expect the transglyco-
n
lowering the initiator to monomer ratio or changing the type sidation product to be 2-(α-glucosyloxy)-ethyl acrylate. Simi-
of initiator should be effective to increase the molecular larly, the maltogenic amylase from B. stearothermophilus was
weight of the polymer. For example, we have already reported found to catalyze the synthesis of a new kind of saccharide
that the polymerization of 2-glucosyloxyethyl acrylate acrylate monomer that was identified as 2-(α-maltosyloxy)-ethyl
monomers using APS or KPS as initiator yields higher mole- acrylate. Other hydroxyl functional acrylates 2-HEMA
cular weight saccharide acrylate polymers (M = 150 kg and 4-HBA could also be used in the transglycosidation re-
n
−
1 27
mol ). Takaragi showed that high molecular weight poly(2- action, resulting in 2-(α-maltosyloxy)-ethyl methacrylate and
Scheme 2 Synthesis of poly(2-(α-maltosyloxy)-ethyl acrylate) by free radical polymerization of 2-(α-maltosyloxy)-ethyl acrylate.
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4
-(α-maltosyloxy)-butyl acrylate, respectively. Subsequently the 14 J. van der Vlist, M. P. Reixach, M. van der Maarel,
disaccharide acrylate 2-(α-maltosyloxy)-ethyl acrylate was poly-
L. Dijkhuizen, A. J. Schouten and K. Loos, Macromol. Rapid
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type of acrylic polymer with pendant maltose groups.
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