Chemo-enzymatic synthesis route to poly(glucosyl-acrylates) using glucosidase from almonds

Article abstract of DOI:10.1039/c3gc41115j

Novel types of glucosyl-acrylate monomers are obtained by β-glucosidase from almond catalyzed glycosidation reaction. The saccharide-acrylate monomers were synthesized by reaction of d-glucose with hydroxyl functional acrylates: 2-hydroxyethyl acrylate (2

Full text of DOI:10.1039/c3gc41115j

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Chemo-enzymatic synthesis route to poly(glucosyl-
acrylates) using glucosidase from almonds†
Cite this: Green Chem., 2014, 16,
1837
Wouter M. J. Kloosterman, Steven Roest, Siti R. Priatna, Erythrina Stavila and
Katja Loos*
Novel types of glucosyl-acrylate monomers are obtained by β-glucosidase from almond catalyzed glyco-
sidation reaction. The saccharide-acrylate monomers were synthesized by reaction of D-glucose with
hydroxyl functional acrylates: 2-hydroxyethyl acrylate (2-HEA), 2-hydroxyethyl methacrylate (2-HEMA)
and 4-hydroxybutyl acrylate (4-HBA). The reaction products could be identified as 2-(β-glucosyloxy)-
ethyl acrylate, 2-(β-glucosyloxy)-ethyl methacrylate and 4-(β-glucosyloxy)-ethyl acrylate respectively. The
synthesis yield was optimized by variation of the 2-HEA–water ratio, the presence of water-miscible co-
solvents and the reaction time. The optimal reaction mixture was found to contain 13 vol% water, 80 vol%
2-HEA and 7 vol%; 1.4-dioxane. The maximal yield under these conditions was 50 wt% based on
D-glucose after 24 hours of reaction. The enzymatically synthesized glucosyl-acrylates were successfully
polymerized by free radical polymerization in DMF and water. The glycosidic linkage of the glycosyl-acry-
late monomers was retained during the polymerization process. The enzymatically synthesized glucosyl-
acrylates could be successfully copolymerized with vinyl monomers 2-HEA, 2-HEMA, methacryl amide
and N-vinyl imidazole.
Received 10th June 2013,
Accepted 23rd July 2013
DOI: 10.1039/c3gc41115j
www.rsc.org/greenchem
ventional synthesis routes toward vinyl saccharide
monomers1–5 require multiple reaction steps and/or protection
Introduction
Polyvinyl saccharides (a special type of glycopolymers) consist of hydroxyl groups to prevent the formation of positional
of a carbon–carbon backbone comprising sugar residues. They isomers in which different hydroxyl groups of the saccharide
combine typical properties of saccharides such as good water become involved in bond formation. As an alternative to these
solubility, low toxicity and biocompatibility with an apolar all- conventional synthesis routes, biocatalytic pathways towards
carbon backbone. This combination of both polarities within a (poly)saccharide synthesis and modification are often
polymer chain opens a variety of applications for polyvinyl sac- beneficial6–11 and were explored here for the synthesis of glu-
charides like drug carrier systems,1 and use as non-ionic poly- cosyl-acrylate monomers. In general biocatalysis is advan-
meric surfactants.2,3 The synthesis of polyvinyl saccharides is tageous with respect to the ambient reaction conditions used,
generally done by free radical polymerization (FRP) of vinyl i.e. atmospheric pressure, low reaction temperature, typically
saccharide monomers. FRP is the most versatile type of chain 37 °C and neutral pH. Most biocatalysts show an extremely
growth polymerization, it is a very robust polymerization tech- high enantio-, regio- and chemoselectivity and therefore
nique, relatively low reaction temperatures can be used and mono-functionalization of the saccharide can usually be
the polymerization often can be performed in water, which achieved easily.
makes FRP an environmentally friendly process. The synthesis
Enzymes are also very low hazard catalysts; they are
of structurally well-defined vinyl saccharide monomers is the obtained from natural renewable resources and can often be
major challenge in the synthesis of polyvinyl saccharides. Con- recycled.12
Especially enzymes from the class of hydrolases (EC 3)13
have been widely used in organic synthesis since they are
found to catalyze the reverse reaction of hydrolysis, the for-
Department of Polymer Chemistry, Zernike Institute for Advanced Materials,
mation of ester, amide, and glycosidic bonds, under specific
conditions.14 Lipases,15–18 proteases19–21 and glycosidases22–24
have mainly been used to prepare saccharide containing
monomers including saccharide-acrylates. Lipase and protease
catalyzed synthesis of saccharide acrylates is usually done by
transesterification of activated esters. Glucosidases, on the
University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
E-mail: K.U.Loos@rug.nl; Fax: +31-50-3636440; Tel: +31-50-3636867
†Electronic supplementary information (ESI) available: 13C-NMR spectra of Glc-
β-EA; Glc-β-EMA; Glc-β-BA; P(Glc-β-EA); P(Glc-β-EMA); P(Glc-β-EA-co-HEA); P(Glc-
β-EA-co-HEMA); P(Glc-β-EA-co-MAm); P(Glc-β-EA-co-VIm) and 1H-NMR spectra of
P(Glc-β-EA-co-HEA); P(Glc-β-EA-co-HEMA); P(Glc-β-EA-co-MAm); P(Glc-β-EA-co-
VIm). See DOI: 10.1039/c3gc41115j
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other hand, do not need activated substrates to obtain good conditions. An Alltech HP column (Econosphere, silica 5 μm,
yields. The reaction mixtures usually contain a certain amount 250 mm × 4.6 mm) was used for separation. The column temp-
of water to maintain enzyme activity and the substrates are erature was maintained at 40 °C and the injection volume was
limited to hydroxyl functional acrylates, but the glycosidic 4 μL. A gradient of heptane and IPA was used as the eluent
bond formation is always regio- and anomer selective. Two with a flow rate of 1 mL min−1. The heptane volume in the
methodologies can be used in glucosidase catalyzed synthesis: eluent was lowered during the measurement as follows: 95 vol%
the thermodynamically controlled reversed hydrolysis and the for 5 min, in 5 minutes to 55 vol%, maintained at 55 vol% for
kinetic controlled transglycosidation approach.25 Millqvist- 8 minutes, in 5 minutes to 10 vol% and maintained at 10 vol%
Fureby et al. used the β-glucosidase from almonds in a thermo- for 15 minutes and finally raised to the initial value of 95% in
dynamically controlled synthesis approach for the synthesis of 22 minutes. ESI-MS was performed on a Thermo Scientific
2-(β-glucosyloxy)-ethyl acrylate and methacrylate.24 Although LTQ Orbitrap mass spectrometer in positive ion mode.
they used supersaturated glucose solutions to suppress the Samples were dissolved in water and diluted with an equal
water activity, the yields obtained were rather low. Using a volume of methanol. Gel Permeation Chromatography (GPC)
kinetically controlled synthesis approach, Satin et al.26 syn- measurements were performed in N,N-dimethylformamide
thesized 2-(β-galactosyloxy)-ethyl methacrylate using β-gluco- (DMF) containing 0.01 M LiBr on a Viscotek GPCMAX
sidase from thermophilic Archeon Sulfolobus solfataricus cells equipped with TDA detectors (model 302), using PSS-Gram-
as the catalyst. In this paper, we report the synthesis of 1000/30 columns. The molar mass was calculated relative to
4-(β-glucosyloxy)-butyl acrylate, 2-(β-glucosyloxy)-ethyl acrylate polymethylmethacrylate using narrow disperse standards
and methacrylate by both the kinetically and the thermodyna- (Polymer Laboratories). FT-Infrared spectroscopy (FTIR) was
mically controlled approach using β-glucosidase from almonds performed using a Bruker JFS 88 equipped with an ATR-golden
as the catalyst. The enzymatically synthesized glucosyl-acrylates gate unit.
were subsequently polymerized by free radical polymerization
Synthesis of 2-(β-glucosyloxy)-ethyl acrylate by
transglycosidation
in DMF or water. The glycopolymers were characterized by
GPC, 1H-NMR and FT-IR spectroscopy.
The reaction mixtures contained 1.0 g cellobiose dissolved in
10 mL water. 2-HEA 1 mL (10 vol%) was added. The reaction
was started by addition of 50 mg (260 U) β-glucosidase from
almonds. The reaction mixture was incubated at 50 °C. Ali-
quots (1 mL) were removed at time intervals of an hour and
Experimental section
Materials
2-Hydroxyethyl acrylate (2-HEA) 96% containing 200 ppm
4-methoxy-hydroquinone (MEHQ), 2-hydroxyethyl methacrylate
(2-HEMA) 97% containing 200 ppm MEHQ, D-(+)-cellobiose
>98%, 4-nitrophenyl-β-D-glucopyranoside (pNPG) >98%, acrylic
acid (AA), methacrylamide (MAm), N-vinyl imidazole (VIm),
lithium bromide (LiBr) and potassium metabisulfite were
obtained from Sigma-Aldrich. β-Glucosidase from almonds
5.2 U mg−1 solid was obtained from Sigma-Aldrich (one unit
will liberate 1.0 μmol of glucose from salicin per min at pH 5.0
at 37 °C). 4-Hydroxybutyl acrylate (4-HBA), 95%, containing
200 ppm MEHQ was obtained from ABCR Chemicals. D-(+)-
Glucose (anhydrous), azobisisobutyronitrile (AIBN), potassium
persulphate (KPS), ammonium persulphate (APS) and 1,4-
dioxane 99% were obtained from Merck. Chloroform (CHCl3),
methanol (MeOH), propan-2-ol (IPA) and other mentioned
organic solvents were obtained from Lab-scan. All chemicals
were used as received.
quenched by the addition of 24 mL of ethanol. The samples
were subsequently analyzed by TLC and HPLC analysis.
Preparative synthesis of 2-(β-glucosyloxy)-ethyl acrylate
(Glc-β-EA), 2-(β-glucosyloxy)-ethyl methacrylate (Glc-β-EMA)
and 4-(β-glucosyloxy)-butyl acrylate (Glc-β-BA)
An enzyme solution was prepared by dissolving 0.070 g (364 U)
β-glucosidase in 1.0 mL water. A 20 mL flask was charged with
0.7 g (4 mmol) glucose. The glucose was dissolved in 1 ml
water. 12 mL 2-hydroxyethyl acrylate, 2-hydroxyethyl methacry-
late or 4-hydroxybutyl acrylate was added to the glucose solu-
tion followed by 1 mL 1,4-dioxane. The reaction was initiated
by adding the freshly prepared enzyme solution. The reaction
mixture was incubated for 24 hours at 50 °C. The new products
were detected by TLC: 2-(β-glucosyloxy)-ethyl acrylate (Rf 0.55),
2-(β-glucosyloxy)-ethyl methacrylate (Rf 0.59), 4-(β-glucosyloxy)-
butyl acrylate (Rf 0.69).
The reaction product was subsequently purified by column
chromatography. Fractions containing the aimed product were
Characterization
1H-NMR and 13C-NMR spectra were recorded using a 7.05 T pooled and the solvent was removed by rotary evaporation at
Varian VXR spectrometer (300 MHz and 75 MHz resolution for 30 °C.
1H- and 13C-NMR, respectively). Deuterium oxide (99.9 atom% D,
Glc-β-EA. Transparent syrup, 0.459 g, purity: 99%, yield:
Aldrich) was used as the solvent. TLC was done on aluminium 41 mol%; ESI-MS: calculated for C11H18O8 (Glc-β-EA) + Na:
sheets silica gel 60/kieselguhr F254 (Merck), and CHCl3– 301.0894, found 301.25. 1H-NMR (D2O) δ in ppm: 6.46 (H11,
MeOH 4/1 v/v was used as the eluent. Spots were visualized as cis, J = 17.3 Hz), 6.22 (H10, J = 17.3, 10.5 Hz), 5.99 (H11, trans,
described by Hansen using diphenylamine/aniline reagent.27 J = 10.5 Hz), 4.50 (H1, axial, J = 7.9 Hz), 4.39 (H8, J = 4.4,
HPLC was done using a Shimadzu HPLC under normal phase 4.4 Hz), 3.2–4.2 (H2, H3, H4, H5, H6, H7). 13C-NMR (D2O) δ in
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ppm: 168.6 (C9), 132.9 (C10), 127.6 (C11), 102.7 (C1β), 76.1 (0.2 mL) were removed at time intervals and the remaining
(C4), 75.9 (C3), 73.3 (C2), 69.8 (C5), 68.1 (C8), 64.4 (C7), 60.9 hydrolytic activity of the enzyme was determined spectrophoto-
(C6).
metrically using 4-nitrophenyl-β-D-glucopyranoside (pNPG) as
Glc-β-EMA. Transparent syrup, 0.514 g, purity: 97%, yield: a chromogenic substrate.28
43 mol%; ESI-MS: calculated for C12H20O8 (Glc-β-EMA) + Na:
315.1050, found 315.11. 1H-NMR (D2O) δ in ppm: 6.14 (H11,
cis), 5.70 (H11, trans), 4.48 (H1, J = 7.92 Hz), 4.35 (H8), 3.2–4.2
Polymerization of enzymatically synthesized
2-(β-glucopyranosyloxy)-ethyl acrylate and -methacrylate
(H2, H3, H4, H5, H6, H7), 1.91 (H12). 13C-NMR (D2O) δ in The enzymatically synthesized monomer Glc-β-EA or Glc-
ppm: 169.8 (C9), 135.9 (C10), 127.1 (C11), 102.7 (C1β), 76.1 β-EMA (1.80 mmol) was dissolved in 4 or 9 mL water or DMF
(C4), 75.8 (C3), 73.2 (C2), 69.7 (C5), 68.1 (C8), 64.5 (C7), 60.8 and purged with nitrogen for at least one hour to remove
(C6), 17.5 (C12).
oxygen from the reaction mixture. The reaction mixture was
Glc-β-BA. Transparent syrup, 0.310 g, purity >85%, 21 mol%; brought to reaction temperature in an oil bath (50 °C for KPS
ESI-MS: calculated for C13H22O8 (Glc-β-BA) + Na: 329.1207, and APS, 70 °C for AIBN). An initiator solution was prepared
found 329.12. 1H-NMR (D2O) δ in ppm: 6.41 (H11, cis, J = by dissolving 0.18 mmol of the initiator in 10 mL water or
17.3 Hz), 6.22 (H10, J = 17.4, 10.4 Hz), 5.95 (H11, trans, J = DMF. One milliliter of the initiator solution was added to the
10.4), 4.43 (H1, axial, J = 7.96 Hz), 4.21 (H8), 3.2–4.2 (H2, H3, reaction mixture to initiate the polymerization. The reaction
H4, H5, H6, H7), 1.75 (H7′, H8′). 13C-NMR (D2O) δ in ppm: mixture was purged with nitrogen for 3–4 hours and the reac-
168.9 (C9), 132.4 (C10), 127.9 (C11), 102.4 (C1β), 76.1 (C4), tion was continued for 24 hours under a nitrogen atmosphere.
76.0 (C3), 73.3 (C2), 69.9 (C5), 65.4 (C8), 61.7 (C7), 61.0 (C6), After polymerization, the reaction mixture was poured into ten
25.6 (C8′), 24.7 (C7′).
times the volume of acetone. The polymer precipitates as a
white powder, which was collected by centrifugation at
7500 rpm for 25 minutes. The polymer was dissolved in water
Time course of the reaction
The reaction mixtures contained 0.5 g glucose dissolved in and again precipitated in acetone. The precipitate was again
1 mL water (10 vol%). 2-HEA 8 mL (80 vol%) was added fol- collected by centrifugation and subsequently dried at 50 °C in
lowed by 1 mL t-butanol (10 vol%). The reaction was started by reduced pressure overnight. The polymers were characterized
addition of 50 mg (260 U) β-glucosidase from almonds. The by 1H-NMR, 13C-NMR, FTIR and GPC.
reaction mixture was incubated at 50 °C. Aliquots (1 mL) were
Poly(2-β-glucosyloxy-ethyl acrylate) (P(Glc-β-EA)). White
removed at time intervals and quenched by addition of 24 mL powder; 1H-NMR (D2O) δ in ppm: 4.50 (H1, J = 7.63 Hz),
of ethanol. The samples were subsequently analyzed by HPLC 3.2–4.4 (H2, H3, H4, H5, H6, H7, H8), 1.44–2.70 (H11b, H11b).
analysis.
13C-NMR (D2O) δ in ppm: 176.6 (C9), 102.7 (C1β), 76.1 (C4),
75.9 (C3), 73.3 (C2), 69.8 (C5), 67.6 (C8), 64.6 (C7), 61.0 (C6),
41.8 (C10), 34.3 (C11). FTIR-ATR (cm−1): 3400 (O–H stretch),
2940 (aliphatic C–H stretch), 1720 (CvO stretch); 1050 (C–O
Influence of organic co-solvents on the time course of the
reaction
The reaction mixtures were prepared as described above, but stretch).
t-butanol was substituted by 1 mL of 1,4-dioxane, t-amylalco-
Poly(2-β-glucosyloxy-ethyl methacrylate) (P(Glc-β-EMA)).
hol, acetonitrile or acetone. Aliquots (1 mL) were removed after White powder; 1H-NMR (D2O) δ in ppm: 4.50 (H1, J = 7.63 Hz),
24, 48 and 168 hours and quenched by addition of 24 mL of 3.2–4.4 (H2, H3, H4, H5, H6, H7, H8), 1.44–2.70 (H11b, H11b),
ethanol. The samples were subsequently analyzed by HPLC 0.95–1.15 (H12). 13C-NMR (D2O) δ in ppm: 176.6 (C9), 102.7
analysis.
(C1β), 76.1 (C4), 75.9 (C3), 73.3 (C2), 69.8 (C5), 67.6 (C8), 64.6
(C7), 61.0 (C6), 41.8 (C10), 34.3 (C11), 16.9 (C12). FTIR-ATR
(cm−1): 3400 (O–H stretch), 2940 (aliphatic C–H stretch), 1720
Optimalisation of water and 2-HEA concentration
The reaction mixtures contained 0.7 g D-glucose dissolved in (CvO ester stretch), 1050 (C–O stretch).
water (5–20 vol%). 2-HEA (48–86 vol%) was added and the reac-
Copolymerization of 2-(β-glucopyranosyloxy)-ethyl acrylate with
hydrophilic vinyl monomers
tion volume was completed by addition of 1,4-dioxane (4–42
vol%). The reaction was started by addition of 70 mg (364 U)
β-glucosidase from almonds. The reaction mixture was incu- Copolymerization reactions with hydrophilic vinyl monomers
bated at 50 °C for 24 hours. Subsequently, 1.0 mL of the reac- were performed similar to the homopolymerization described
tion mixture was taken and diluted with 24 mL ethanol for above. Equivalent molar amounts (3.6 mmol) of AA, MAm,
HPLC analysis.
2-HEA, 2-HEMA or VIm were added to the reaction mixture
containing one gram (3.6 mmol) of Glc-β-EA in 20 mL R.O.
water. The reaction mixture was purged with nitrogen and
Measurement of the enzyme stability
The reaction mixtures were prepared as described before: heated to 50 °C. The reaction is initiated by addition of 0.2 mL
0.7 gram D-glucose in 1 mL water, 2-hydroxyethyl acrylate APS solution (12 wt%) and 0.2 mL potassium metabisulfite
11 mL and 1 mL 1,4-dioxane. The reaction was started by solution (6 wt%). The reaction was continued overnight and
addition of 70 mg (364 U) β-glucosidase in 1 mL water. The the copolymers were isolated as described above for the
reaction mixture was incubated at 50 °C for 48 h. Aliquots homopolymers.
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Scheme 1 Thermodynamically controlled synthesis approach of glucoside-acrylates catalyzed by β-glucosidase from almonds. 2-Hydroxyethyl
acrylate (RvH and n = 1), 2-hydroxyethyl methacrylate (RvCH3 and n = 1) and 4-hydroxybutyl acrylate (RvH and n = 2) were used as the acceptors.
P(Glc-β-EA-co-AA): polymer network, 0.0773 g, yield; 12 wt%.
reaction mixture within the first hour of reaction. The spots
P(Glc-β-EA-co-MAm): yellow powder, 0.4865 g, yield; 70 wt%, were identified as Glc-β-EA (Rf 0.55) and 2-(β-cellobiosyloxy)-
copolymer composition Glc-β-EA : MAm = 1.0 : 1.59; 1H-NMR ethyl acrylate (Glc-β-Glc-β-EA, Rf 0.33). The reaction had to be
(D2O) δ in ppm: see Table 4. 13C-NMR (D2O) δ in ppm: 176.6 stopped after the first hour, since hydrolysis of Glc-β-EA was
(C9), 102.7 (C1β), 76.1 (C4), 75.9 (C3), 73.3 (C2), 69.8 (C5), 67.6 observed at longer reaction times. The product concentration
(C8), 64.6 (C7), 61.0 (C6), 41.8 (C10), 34.3 (C11), 17.6–19.3 of the reaction mixture after one hour was determined by
(MAm-CH3). FTIR-ATR (cm−1): 3400 (O–H stretch), 2940 (ali- HPLC. The product concentration was found to be 5.9 mg
phatic C–H stretch), 1720 (CvO stretch); 1665 (CvO Amide I), ml−1 which corresponds to a molar conversion of 8.3 mol%
1050 (C–O stretch).
with respect to the glycosyl donor cellobiose.
P(Glc-β-EA-co-Hema): white powder, 0.8070 g, yield 60 wt%;
In the thermodynamically controlled synthesis approach
copolymer composition Glc-β-EA : HEMA = 1.0 : 1.0; 1H-NMR (Scheme 1), D-glucose was used as the donor substrate and
(D2O) δ in ppm: see Table 4. 13C-NMR (D2O) δ in ppm: 2-HEA as the acceptor alcohol. After 8 hours a spot of Glc-β-EA
176.6–180.1 (C9), 102.7 (C1β), 76.1 (C4), 75.9 (C3), 73.3 (C2), (Rf 0.55) was detected by TLC analysis. The product concen-
69.9 (C5), 67.6 (C8), 67.0 (HEMA-C8), 64.7 (C7), 59.4–61.0 (C6, tration of the reaction mixture was found to increase during
HEMA-C6), 31.4–45.2 (C10, C11), 16.4–22.4 (HEMA-CH3). the first 24 hours. The product concentration in the reaction
FTIR-ATR (cm−1): 3400 (O–H stretch), 2940 (aliphatic C–H mixture was found to be 25 mg ml−1 (32 mol% with respect to
stretch), 1720 (CvO stretch); 1665 (CvO Amide I), 1150 (C–O D-glucose) after 24 hours of reaction. Comparison of both
stretch).
approaches shows that the kinetically controlled approach had
P(Glc-β-EA-co-Hea): white powder, 0.9691 g, yield 74 wt%; some disadvantages compared to the thermodynamically con-
1H-NMR (D2O) δ in ppm: see Table 4. 13C-NMR (D2O) δ in trolled approach (as shown in Table 1).
ppm: 177.0 (C9), 102.8 (C1β), 76.2 (C4), 76.0 (C3), 73.4 (C2),
Firstly, the yield compared to the thermodynamic approach
70.0 (C5), 67.8 (C8), 66.8 (HEA-C8), 64.7 (C7), 61.1 (C6), 59.8 was rather low. Secondly, the glycosyl donor cellobiose is more
(C6-HEA), 41.8 (C10), 34.5 C11). FTIR-ATR (cm−1): 3400 (O–H expensive and less soluble in water as compared to glucose,
stretch), 2900 (aliphatic C–H stretch), 1750 (CvO stretch); which was used in the thermodynamically controlled
1665 (CvO amide I), 1160 (C–O stretch).
approach. Lastly, the kinetically controlled reaction liberates
P(Glc-β-EA-co-VIm): yellow powder, 0.6172 g, yield 48.8 wt%, one molecule of glucose per mole of product, which cannot be
copolymer composition Glc-β-EA : VIm = 1.0 : 0.5; 1H-NMR reused in the reaction. Therefore the thermodynamically con-
(D2O) δ in ppm: see Table 4. 13C-NMR (D2O) δ in ppm: 175.6 trolled method is the method of choice for β-glucosidase cata-
(C9), 136.7 (VIm NvC) 119.3–126.2 (VIm-CvC) 102.3 (C1β), lyzed synthesis of saccharide-acrylates from hydroxyl-
75.8 (C4), 75.6 (C3), 73.0 (C2), 71.0 (C5), 67.3 (C8), 64.6 (C7), functional acrylates.
60.7 (C6), 41.3 (C10), 37.1 (C11). FTIR-ATR (cm−1): 3400 (O–H
Enzymatic synthesis of glucosyl-acrylates from
hydroxyl-functional acrylates
stretch), 3125 (N–H stretch), 2900 (aliphatic C–H stretch), 1750
(CvO stretch); 1600 (N–H bend), 1160 (C–O stretch).
The thermodynamically controlled synthesis approach using
β-glucosidase as the catalyst was applied to synthesize gluco-
syl-acrylates from hydroxyl functional acrylates 2-HEA, 2-HEMA
Results and discussion
Kinetically versus thermodynamically controlled synthesis of
Glc-β-EA
Table 1 Comparison of β-glucosidase catalyzed kinetically controlled
vs. thermodynamically controlled synthesis both yielding Glc-β-EA
We have investigated the synthesis of saccharide-acrylate Glc-
β-EA using β-glucosidase from almonds as the catalyst by both
the thermodynamically controlled approach and the kineti-
cally controlled synthesis approach. The kinetically controlled
synthesis approach was performed with cellobiose as the glyco- Glycosyl donor
syl donor and 2-HEA as the acceptor. The reaction mixture was
Kinetic approach
Thermodynamic approach
Reaction time
1 hour
24 hours
Glucose
0.05 g mL−1
90 vol%
0.35 g
Cellobiose
0.10 g mL−1
10 vol%
[Donor]
[2-HEA]
Product
analyzed with TLC every hour to check the progress of the reac-
0.07 g
tion. Using TLC, two reaction products were detected in the Yield
8.3 mol%
32 mol%
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Fig. 2 1H-NMR spectrum of the enzymatically synthesized Glc-β-EMA.
Fig. 1 1H-NMR spectrum of the enzymatically synthesized Glc-β-EA.
and 4-HBA. After 24 hours of reaction the glucosyl-acrylates
were purified by column chromatography and analyzed by
1H- and 13C-NMR. Fig. 1 shows the NMR spectra of the reac-
tion product obtained by the reaction between D-glucose and
1
2-hydroxyethyl acrylate. The H-NMR spectrum of the reaction
product shows a one to one ratio between the peaks of the
acryloyl moiety at 5.99–6.46 ppm and the anomeric glucosyl
proton at 4.50 ppm (Fig. 1). Evidently, only one hydroxyl group
of the saccharide was involved in glycosyl bond formation
using β-glucosidase as the catalyst. Secondly, conventional
chemical synthesis approaches like Fischer glycosidation
result in products that contain two peaks of the anomeric
proton in the 1H-NMR spectrum.29 These two peaks corres-
pond to the glycosidic linkage in both α-configuration
(4.9 ppm) and β-configuration (4.5 ppm). In contrast, the
1H-NMR spectrum of the enzymatically synthesized saccha-
ride-acrylate shows just one peak of the anomeric proton at
4.50 ppm, indicating an anomerically pure product in β-con-
figuration. The 13C-NMR spectrum (Fig. S1†) of the product
Fig. 3 1H-NMR spectrum of the enzymatically synthesized Glc-β-BA.
shows a peak at 102 ppm (C1β) and no peak at 96 ppm (C1α) acrylates 2-(β-glucosyloxy)-ethyl methacrylate (Glc-β-EMA) and
confirming again that an anomerically pure product was 4-(β-glucosyloxy)-butyl acrylate (Glc-β-BA). The NMR-spectra
formed by the enzyme. Thus, we could synthesize anomerically (1H-NMR spectra Fig. 2 and 3; 13C-NMR spectra Fig. S2 and
pure mono-functionalized 2-(β-glucosyloxy)-ethyl acrylate (Glc- S3† respectively) again revealed that the enzymatic glycosi-
β-EA) in one reaction step by β-glucosidase catalyzed glycosida- dation was fully anomer selective and only mono-functiona-
tion. For comparison the synthesis of 2-(β-glucosyloxy)-ethyl lized products were obtained. The saccharide-acrylate Glc-
acrylate by conventional chemistry involves at least three reac- β-EMA was obtained in 43 mol% yield which is comparable to
tion steps.2
the yield of the glucosyl-acrylate Glc-β-EA. On the other hand,
Similarly, the β-glucosidase catalyzed synthesis of saccha- Glc-β-BA was obtained in a lower yield (21 mol%). It was
ride-acrylates from other hydroxyl-functional acrylates like reported for β-glucosidase25 and other glycosyl hydrolases30
2-hydroxyethyl methacrylate (2-HEMA) and 4-hydroxybutyl that the glycoside yield decreases with increasing length of the
acrylate (4-HBA) was investigated as well. In case 2-HEMA or alkyl alcohol. In view of this the lower glucosyl-acrylate yield of
4-HBA was used as the acceptor, new reaction products could Glc-β-BA could be explained by the longer alkyl part of 4-HBA
be detected by TLC analysis. The products were purified by compared to 2-HEA. Nevertheless the obtained 21 mol% of
column chromatography and subsequently analyzed by Glc-β-BA is about 7 times higher compared to the synthesis
1H-NMR, 13C-NMR and ESI-MS spectroscopy. The NMR and route using supersaturated solutions as published by
ESI-MS spectra confirmed the structure of the aimed glucosyl- Millquist-Fureby.24
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Influence of reaction time on substrate conversion
stant product concentration could have been enzyme de-
activation by product inhibition. Therefore the catalyst
dependence of the product concentration was investigated.
Increasing the amount of biocatalyst was expected to result in
a higher reaction rate and a higher concentration of Glc-β-EA
before inhibition of the enzyme by the reaction product.
Indeed, the glycosidation reaction proceeded faster when the
catalyst concentration was doubled (Fig. 4, triangles).
The reaction rate and substrate conversion of the β-glucosidase
catalyzed synthesis of Glc-β-EA were studied by HPLC analysis.
For calibration of the peak areas, standard solutions with a
known concentration of Glc-β-EA were prepared first. The enzy-
matically synthesized product had to be highly pure and was
therefore purified twice by column chromatography. Sub-
sequently, the pure product was used to prepare standard solu-
tions containing 0, 5, 10, 20, 30, 40 and 50 mg Glc-β-EA per ml
reaction mixture. In this range the detector response was
found to be linear and thus the calibration curve could be
used to determine the concentration of the product during the
reaction.
However, the maximal conversion was the same as that in
the previous reaction with less biocatalyst. This indicates a
thermodynamic equilibrium rather than product inhibition.
Organic solvent induced acceleration of the reaction rate
It has been reported that the synthesis yield of β-glucosidase
catalyzed glycosidation can be improved by addition of an
inert organic (co)solvent.31–33 We investigated the influence of
organic solvents that are commonly used in β-glucosidase cata-
lyzed syntheses for the synthesis of glucosyl-acrylates. The
influence of t-butanol, 1,4-dioxane, t-amyl alcohol, acetone
and acetonitrile on the reaction kinetics and the equilibrium
yield was studied. The presence of small amounts (7 vol%) of
t-butanol in the reaction mixture was found to enhance both
the reaction rate and the conversion of the donor substrate
(Fig. 5). The original conversion of 40 wt% was already
achieved in two days by the addition of t-butanol to the reac-
tion mixture. Besides acceleration of the synthesis process, the
final conversion after 7 days was increased to 53 wt% by the
addition of t-butanol.
Addition of 1,4-dioxane (7 vol%) improved the reaction
process even more. Forty percent conversion was already
achieved within 24 hours and a total synthesis yield of 50 wt%
was obtained after 48 hours. The yield obtained after 7 days
was similar, meaning that the equilibrium yield was reached
after 48 hours. The other investigated solvents t-amyl alcohol,
acetone and acetonitrile did not have a positive effect either on
the onset of the equilibrium or on the synthesis yield. Since
the addition of 1,4-dioxane had a positive effect on the
In the control reaction (without enzyme), there was no peak
of the product visible after incubation for 7 days, as shown in
Fig. 4. In contrast to this the product concentration increased
gradually during the first 4 days in the reaction mixture con-
taining the biocatalyst. After 4 days the product concentration
did not increase any further, but stayed constant for the rest of
the reaction time (Fig. 4). Possible explanations for the
observed plateau value can be: (a) deactivation of the biocata-
lyst, (b) inhibition of the catalyst by the product or (c) a
thermodynamic equilibrium was reached.
Firstly, the hydrolytic activity of the enzyme was tested with
4-nitrophenyl-β-D-glucopyranoside as the chromogenic sub-
strate. Although the enzyme activity was decreased in time, the
enzyme was found to retain 40% of its initial hydrolytic activity
after 24 hours of reaction. After 48 hours the enzyme did not
show any more significant hydrolytic activity.
In order to rule out enzyme deactivation as the cause of the
constant product concentration, freshly active enzyme was
added to the reaction mixture after 4 days.
It was found that the concentration of Glc-β-EA did not sig-
nificantly increase after the addition of the second portion of
active enzyme. Thus, enzyme deactivation was not the cause of
the constant product concentration. Another cause of the con-
Fig. 4 Time course of β-glucosidase catalyzed synthesis of Glc-β-EA
followed by HPLC analysis. Open circles: reaction without enzyme; solid
squares: normal biocatalyst level (26 U mL−1); solid triangles: high bio-
catalyst level (52 U mL−1).
Fig. 5 Influence of organic co-solvents on the reaction rate and
product concentration.
1842 | Green Chem., 2014, 16, 1837–1846
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corresponds to a yield of 50
8 wt% with respect to the
Table 2 Effect of the 1,4-dioxane content on the yield of Glc-β-EA
initially added amount of glucose.
Dioxane content (vol%)
[Glc-β-EA] (mg mL−1
)
Yield (wt%)
Besides the upper reaction parameters, the influence of
other reaction parameters was investigated as well. The influ-
ence of the reaction temperature, the amount of the enzyme
and the amount of glucose was investigated. However, none of
these reaction parameters leads to a higher conversion or
could reduce the time needed to set the equilibrium.
0
7
13
28
35
15
26
16
10
7
1
3
2
1
1
28
50
34
24
19
3
8
6
2
1
Polymerization of enzymatically synthesized
2-(β-glucopyranosyloxy)-ethyl acrylate and -methacrylate
synthesis yield, the amount of 1,4-dioxane was increased to
further improve the conversion. The synthesis yield after
24 hours decreased with increasing amount of 1,4-dioxane
(shown in Table 2). Besides the lower product concentration,
aggregation of the enzyme powder was observed with increas-
ing the 1,4-dioxane content. It was found that aggregation of
the enzyme powder led to a lower conversion, because the
enzyme was not dispersed homogeneously throughout the
solution. Our attempts to keep the biocatalyst dispersed homo-
geneously in the solution by non-covalent immobilization of
the enzyme on an inert carrier could not increase the synthesis
yield.
Both enzymatically synthesized monomers Glc-β-EA and Glc-
β-EMA were polymerized by free radical polymerization in
1
water or DMF according to Scheme 2. The H-NMR spectra of
both poly(2-β-glucosyloxy-ethyl acrylate) and poly(2-β-glucosyl-
oxy-ethyl methacrylate) showed that the glucosyl monomers
could be successfully polymerized by free radical polymeri-
zation in solution (1H-NMR spectra Fig. 7, left and right,
respectively; 13C-NMR spectra Fig. S4 and S5,† respectively).
The characteristic signals from the acryloyl moiety of the
monomer Glc-β-EA at 6.0–6.5 ppm disappear from the respect-
ive spectra as a result of the polymerization reaction. The
signals corresponding to the protons of the polymer backbone
appear between 1.5 and 2.6 ppm. Likewise, polymerization of
the monomer Glc-β-EMA shows the disappearance of the
double bond signals at 5.7 and 6.2 ppm and the appearance of
the methylene signals from the polymer backbone in the
1.5–2.6 ppm region. In addition, the signal of the methyl
group of Glc-β-EMA at 1.9 ppm shifts to 0.9–1.1 ppm upon
polymerization, due to the change in electron density upon
polymerization. The glycosidic signals in the region
3.2–4.6 ppm of both polymers P(Glc-β-EA) and P(Glc-β-EMA)
can easily be correlated to those of the corresponding mono-
mers. The anomeric configuration of the monomers, which
was introduced by the biocatalyst, was found to be preserved
during the polymerization.
Optimization of the amount of alcohol and water
Since the β-glucosidase catalyzed synthesis of Glc-β-EA reaches
a thermodynamic equilibrium, the total conversion could be
influenced by the amount of reactants and especially the
amount of water present in the reaction mixture. The conver-
sion was optimized by changing the volume fractions of water
and hydroxyl-functional acrylates. As can be seen from Fig. 6, a
relatively low concentration of product was found in reaction
mixtures containing less than 10% water, most likely caused
by enzyme deactivation. On the other hand, the reaction
mixture containing over 18 vol% water resulted in a lower
product concentration, due to the hydrolysis of the product.
Moderate amounts of product were found in reaction mixtures
containing between 10 and 18 vol% water. The best conversion
was achieved in a reaction mixture containing 7 vol% 1,4-
dioxane, 14 vol% water and 79 vol% 2-hydroxyethyl acrylate.
The product concentration measured 26 3 mg mL−1, which
Effect of the free radical initiator on the molecular weight of
the polymers
The polymerization of monomers Glc-β-EA and Glc-β-EMA was
performed with the oil-soluble free radical initiator azobis-
isobutyronitrile (AIBN) and the water-soluble initiators
ammonium persulfate (APS) and potassium persulfate (KPS).
Scheme 2 Free radical polymerization of enzymatically synthesized
Glc-β-EA (RvH) and Glc-β-EMA (RvCH3).
Fig. 6 Influence of water and 2-HEA on the Glc-β-EA concentration.
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Fig. 7 1H-NMR spectra of P(Glc-β-EA) (left) and P(Glc-β-EMA) (right).
The polymerizations were performed at two monomer con- solvent for the polymerization is preferred over the much more
centrations. The polymer yields and the molecular weights toxic DMF.
(obtained by GPC) are summarized in Table 3. Relatively low
molecular weight polymers of Glc-β-EA were obtained by
polymerization in DMF with AIBN as the initiator (entries 1
Copolymerization of 2-glucosyloxyethyl acrylate with
hydrophilic vinyl monomers
and 2). On the other hand, both monomers Glc-β-EA and Glc- Further application of the above described poly(saccharide-
β-EMA were easily polymerized to relatively high molecular acrylates) might require control of the amount of saccharide
weight polymers in water using APS and KPS as initiators. The units per chain or modification of the physical properties of
corresponding acrylic polymers P(Glc-β-EA) and P(Glc-β-EA) the desired polymers. The most commonly used approach to
were obtained in good yields and their molecular weights were achieve such modifications is copolymerization with other
in the range 75–155 kg mol−1 (entries 3–6). Increasing the (hydrophilic) vinyl monomers. Therefore, the copolymerization
monomer concentration in the reaction mixture (entries 4 and 6) of the synthesized monomer Glc-β-EA with commonly used
resulted in higher molecular weight polymers and broadening hydrophilic vinyl monomers (acrylic acid, methacrylamide,
of the polydispersity. Using the water soluble initiators, the 2-hydroxyethyl (meth)acrylate and N-vinyl imidazole) was inves-
polymer yields of the methacrylic polymers (entries 7–10) are tigated. The copolymers are synthesized starting from equal
comparable to the acrylic polymers. The number average mole- molar amounts of the listed co-monomers and the saccharide-
cular weights (Mn) of the methacrylic polymers P(Glc-β-EMA) acrylate Glc-β-EA. The 1H-NMR spectra of the copolymers of
and P(Glc-β-EMA) were found to be slightly higher (171–518 kg Glc-β-EA and the tested hydrophilic co-monomers show the
mol−1) than those of the acrylic glucoside polymers. Up to characteristic peaks of both the glucoside monomer residues
quantitative yields of both glycopolymers P(Glc-β-EMA) and and the co-monomer residues (signals listed in Table 4;
P(Glc-β-EMA) were obtained in aqueous solution, indicating 1H-NMR spectra Fig. S6, S8, S10 and S12;† 13C-NMR spectra
that the polymerization was neither inhibited nor hindered by Fig. S7, S9, S11 and S13† respectively). As in the case of
the bulky saccharide groups. Secondly, the use of water as the the homopolymerization of Glc-β-EA, the co-polymerization
Table 3 Results of polymerization of Glc-β-EA and Glc-β-EMA
Entry
Polymer
[M] (mol L−1
)
Initiator
Yield (wt%)
Mn (kg mol−1
)
Mw (kg mol−1
)
Mw/Mn
1
2
3
4
5
6
7
8
9
10
P(Glc-β-EA)
P(Glc-β-EA)
P(Glc-β-EA)
P(Glc-β-EA)
P(Glc-β-EA)
P(Glc-β-EA)
P(Glc-β-EMA)
P(Glc-β-EMA)
P(Glc-β-EMA)
P(Glc-β-EMA)
0.37
0.73
0.37
0.73
0.37
0.73
0.37
0.73
0.37
0.73
AIBNa
AIBNa
APS
57
39
73
80
57
99
77
62
74
63
9
17
76
155
115
150
356
171
192
518
11
81
328
1116
446
740
1332
1019
1014
2524
1.2
4.8
4.3
7.2
3.9
4.9
3.7
6.0
5.3
4.9
APS
KPS
KPS
APS
APS
KPS
KPS
a Polymerization performed in DMF at 65 °C.
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Table 4 1H-NMR shifts of synthesized saccharide-acrylate copolymers
Protonsa
P(Glc-β-EA)
-co-MAm
-co-2-HEA
-co-2-HEMA
-co-VIm
–CH3
10, 11
2–8
HOCH2CH2
HOCH2CH2
1ax
—
0.60–1.34
1.44–2.70
3.20–4.40
—
—
4.50
—
0.92–1.11
1.49–2.47
3.30–4.30
3.80
4.10
4.47
—
1.44–2.70
3.20–4.40
—
—
4.50
—
1.67–2.47
3.21–4.32
3.80
4.20
4.50
1.50–2.47
3.30–4.35
—
—
4.50
N–CHvN–CHvCH–
—
—
—
6.96–7.59
a Proton numbering according to Scheme 2.
reaction with MAm, 2-HEA, 2-HEMA or VIm was not affected copolymer structure was confirmed by 1H-NMR and FT-IR
by the pendant glucose group; only the copolymerization of spectroscopy.
Glc-β-EA with AA resulted in the formation of an insoluble
hydrogel. The polymer network was most probably caused by
intermolecular crosslinking between the acrylic acid residues
and the hydroxyl groups of the glucosyl-acrylate residue.
Acknowledgements
This research was financed by BASF, Ludwigshafen, Germany.
We kindly acknowledge Theodora Tiemersma–Wegman for
help with HPLC and ESI-MS analysis.
Conclusions
β-Glucosidase from almonds was found to catalyze the syn-
thesis of saccharide-acrylate monomers from D-glucose and
hydroxyl functional acrylates. The monomers 2-(β-glucosyloxy)-
Notes and references
ethyl acrylate (Glc-β-EA), 2-(β-glucosyloxy)-ethyl methacrylate
(Glc-β-EMA) and 4-(β-glucosyloxy)-butyl acrylate (Glc-β-BA) were
synthesized from 2-hydroxyethyl acrylate, 2-hydroxyethyl
methacrylate and 4-hydroxybutyl acrylate, respectively. The
almond β-glucosidase showed a high selectivity for the glyco-
sidation position. The anomeric (C-1) hydroxyl group was
exclusively involved in the glycosidation reaction using β-gluco-
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were found to be mono-functionalized, which is essential for
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equilibrium was reached after about 72 hours. However, it was
found that the addition of an appropriate organic co-solvent
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