Full text of DOI:10.1039/c7gc03023a

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Synthesis of (meth)acrylamide-based
glycomonomers using renewable resources and
Cite this: DOI: 10.1039/c7gc03023a
their polymerization in aqueous systems†
Azis Adharis,
Dennis Vesper, Nick Koning and Katja Loos
*
In this work, we present the kinetically-controlled enzymatic synthesis of novel glycosyl-(meth)acrylamide
monomers using β-glucosidase. Cellobiose served as the glycosyl donor in the enzyme catalyzed trans-
glycosylation reaction and hydroxyl-alkyl (meth)acrylamides as the glycosyl acceptor. After optimization,
we were able to increase the glycomonomer yield up to 68% by changing the glycosyl donor to p-nitro-
phenyl β-D-glucopyranoside and adding BMIMPF6 as cosolvent. The structure of the glycomonomers was
confirmed by 1H NMR, 13C NMR, and mass spectrometry experiments. Aqueous RAFT polymerization of
the glycomonomers was successfully performed resulting in glycopolymers with molecular weights up to
30 kg mol−1 and relatively low polydispersity indices (PDI’s < 1.30). Free radical polymerization of the
glycomonomers was executed as well with the obtained glycopolymers resulting in higher molecular
weights and PDI’s than the glycopolymers prepared by RAFT polymerization. Thermal properties of the
synthesized glycopolymers were investigated via differential scanning calorimetry.
Received 6th October 2017,
Accepted 10th December 2017
DOI: 10.1039/c7gc03023a
rsc.li/greenchem
cedures. In addition, enzymes are non-toxic catalysts, derived
from renewable resources, and enzymatic reactions are gener-
Introduction
Carbohydrates are the most abundant renewable biomass pro- ally performed under relatively mild conditions.24,25
duced annually but only a small fraction of them is used by
Lipases17,20,21 and glycosidases14,18,22,23 are the most used
the chemical industry.1 In principle, carbohydrates can be biocatalysts for the synthesis of glycomonomers. Lipases cata-
further utilized as an alternative resource for fossil-based lyze the transesterification between a primary alcohol of
chemicals in the field of polymers. For example, researchers mono- and disaccharides that function as the glycosyl donor
used carbohydrates as starting materials to design polymers and an activated ester serves as the glycosyl acceptor. Such
having pendant sugar moieties called glycopolymers.2 reactions are usually carried out for at least 4 days in order to
Glycopolymers have gained much interest in the last decades obtain a good yield. In contrast to lipases that require an acti-
especially because of their properties that can mimic glyco- vated molecule, glycosidases only need a natural primary or
lipids and glycoproteins.3–5 The application of glycopolymers secondary alcohol to functionalize monosaccharides at the
has been reported mainly for drug delivery,6–8 tumor C-1 position with reasonable shorter reaction times than
targeting,9–11 immune stimulants,12 and therapeutics.13
lipase. For example, we previously used commercially avail-
Glycomonomers, the precursor of glycopolymers, can be able unmodified hydroxy-alkyl (meth)acrylates to synthesize
synthesized either by chemical or enzymatic methods.14–23 glycomonomers catalyzed by β-glucosidase under thermo-
Enzymatic reactions offer some advantages compared to dynamically-controlled reaction conditions.14 The maximum
chemical reactions; for instance, reaction specificity provided yield of the desired glycomonomers was obtained after one
by the enzyme can avoid necessary protection steps of the sac- day of reaction and a lower glycomonomer yield was obtained
charide-hydroxyl groups in the conventional synthetic pro- when the kinetically-controlled reaction conditions were
applied.
Most of reported glycopolymers, that can potentially be
applied as biomimetic materials6–13 employ an amide bond to
Macromolecular Chemistry and New Polymeric Materials, Zernike Institute for
Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The
attach a saccharide unit to the polymer backbone. This is due
to the fact that amide groups provide better stability towards
Netherlands. E-mail: K.U.Loos@rug.nl; Fax: +31-50-3636440; Tel: +31-50-3636867
†Electronic supplementary information (ESI) available: Theoretical and observed
hydrolysis of the saccharide units than ester groups in
ESI-MS spectra of Glc-β-EAAm, Glc-β-EMAAm, and Glc-β-BMAAm; NMR and DSC
results of the polymers prepared by free radical polymerization. See DOI:
aqueous media. In addition, we can hypothesize, that the
amide bond mimics the peptide bond in glycoproteins.
10.1039/c7gc03023a
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Using the previously reported thermodynamically-driven and two analytical columns (PSS-GRAM-1000/30 Å, 10 μm,
approach and the same enzymes, we failed to obtain the 30 cm). The temperature for the columns and detectors were
desired glycomonomers with (meth)acrylamide-based alcohols at 50 °C. DMF containing 0.01 M LiBr was used as the eluent
serving as the glycosyl acceptor, but we were able to synthesize at a flow rate of 1.0 ml min−1. The samples were filtered
three glycosyl-(meth)acrylamide monomers under kinetically- through a 0.45 μm PTFE filter prior to injection. Narrow
controlled reaction conditions. Herewith, we present enzymati- PMMA standards were used for calibration and molecular
cally-synthesized N-(β-glycosyloxy)-ethyl acrylamide (Glc- weights were calculated by universal calibration method with
β-EAAm),
N-(β-glycosyloxy)-ethyl
methacrylamide
(Glc- refractive index increment of PMMA (0.063 ml g−1) was
β-EMAAm), and N-(β-glycosyloxy)-butyl methacrylamide (Glc- applied. The calculation was done using Viscotec Omnisec
β-BMAAm) monomers, and the study of improving monomer Software. Differential scanning calorimetry (DSC) was per-
yield. This is the first report on the enzymatic synthesis of formed on a DSC Q1000 from TA Instruments by heating the
these glycomonomers; Glc-β-EAAm has been synthesized samples to 200 °C with the heating and cooling rate of 10 °C
before but using a chemical approach involving 5 reaction min−1
.
steps26,27 whereas Glc-β-EMAAm and Glc-β-BMAAm have never
Preparative synthesis Glc-β-EAAm, Glc-β-EMAAm, and
Glc-β-BMAAm
been reported in the literature. Furthermore, these novel glyco-
monomers were successfully polymerized by aqueous revers-
ible addition–fragmentation chain-transfer (RAFT) polymeriz- In
ation and free radical polymerization (FRP).
a
25 ml round-bottom flask, cellobiose (0.50 gram,
1.46 mmol) was dissolved in Milli-Q water (2.25 ml) at 50 °C.
Subsequently, HEAAm (0.65 gram, 5.65 mmol), HEMAAm
(0.75 gram, 5.80 mmol), or HBMAAm (0.9 gram, 5.72 mmol)
was added into the cellobiose solution. The reaction was
started by adding enzyme solution (50 mg in 0.25 ml H2O).
After 1 hour of reaction, the flask was put on boiled water to
deactivate the enzyme and stop the reaction. TLC of the reac-
tion mixture was performed and the reaction product was
detected at retardation factor of 0.30, 0.35, and 0.37 for Glc-
β-EAAm, Glc-β-EMAAm, and Glc-β-BMAAm, respectively. The
water in the reaction mixture was evaporated and MeOH
(10 ml) was added to precipitate the glucose and unreacted
cellobiose. After centrifugation (4500 rpm, 10 min, 4 °C), the
supernatant was concentrated by evaporation followed by puri-
fication through column chromatography with silica gel served
as the stationary phase and CHCl3/MeOH (4/1) mixture as the
eluent. Eluent from the collected fractions containing the
product was evaporated by rotary evaporation (<40 °C at
reduced pressure) and the resulted product was stored in the
fridge.
Experimental section
Materials
β-Glucosidase from almonds with activity ≥2 units per mg
solid, N-hydroxyethyl acrylamide (HEAAm) 97%, 4,4′-azobis(4-
cyanovaleric acid) (ACVA) ≥98.0%, and 4-cyano-4-(phenylcarbo-
nothioylthio)pentanoic acid (CPADB) >97% were purchased
from Sigma-Aldrich. Cellobiose 98% was obtained from Acros
Organics. p-Nitrophenyl β-D-glucopyranoside (p-NPG) 98+%
and
1-N-butyl-3-methylimidazolium
hexafluorophosphate
(BMIMPF6) 98+% were acquired from Alfa Aesar. Chloroform
(CHCl3), methanol (MeOH), and ethanol (EtOH) were obtained
from Avantor. Silica gel was purchased from Silicycle. All
chemicals were used as received. N-Hydroxyethyl methacryl-
amide (HEMAAm) and N-hydroxybutyl methacrylamide
(HBMAAm) were synthesized according to literature.28 RAFT
agent, 3-benzylsulfanylthiocarbonylsufanyl-propionic acid
(BSPA), for the acrylamide-based monomer was prepared
according to literature.29
Glc-β-EAAm. Yellowish viscous liquid, 67 mg, yield: 16%,
purity: 98%. ESI-MS: calculated for C11H19NO7 + Na: 300.1054,
1
observed: 300.1052. H NMR (D2O) δ in ppm: 6.15–6.30 (H11-
Characterization
cis and H9), 5.74 (H11-trans, J = 11.6 Hz), 4.45 (H1-axial, J = 8.0
1H NMR and 13C NMR spectra were recorded on a 400 MHz Hz), 3.23–4.01 (H2, H3, H4, H5, H6, H7, H8). 13C NMR (D2O) δ
and 300 MHz Varian VXR Spectrometer, respectively, using in ppm: 169 (C12), 130 (C9), 127 (C11), 102 (C1β), 75.8 (C5),
deuterium oxide (99.9 atom % D, Aldrich) as the solvent. The 75.6 (C3), 73 (C2), 70 (C4), 68 (C7), 61 (C6), 39 (C8).
acquired spectra were processed by MestReNova Software from
Glc-β-EMAAm. Yellowish viscous liquid, 51 mg, yield: 12%,
Mestrelab Research S.L. Thin layer chromatography (TLC) was purity: 97%. ESI-MS: calculated for C12H21NO7 + Na: 314.1210,
performed on aluminum sheet silica gel 60/kieselguhr (Merck) observed: 314.1213. 1H NMR (D2O) δ in ppm: 5.69 (H11-cis),
with CHCl3/MeOH (4/1) mixture as the eluent. Spot visualiza- 5.44 (H11-trans), 4.45 (H1-axial, J = 8.0 Hz), 3.23–4.02 (H2, H3,
tion of the glycomonomers was done by spraying the TLC plate H4, H5, H6, H7, H8), 1.91 (H10). 13C NMR (D2O) δ in ppm: 172
with 5% H2SO4 in EtOH followed by heating. Electrospray (C12), 139 (C9), 121 (C11), 102 (C1β), 75.8 (C5), 75.6 (C3), 73
ionization mass spectrometry (ESI-MS) was executed on a (C2), 70 (C4), 68 (C7), 61 (C6), 39 (C8), 18 (C10).
Thermo Scientific LTQ Orbitrap Mass Spectrometer in positive
Glc-β-BMAAm. Yellowish viscous liquid, 90 mg, yield: 18%,
ion mode and Milli-Q water was used as the solvent. Size exclu- purity: 96%. ESI-MS: calculated for C14H25NO7 + Na: 342.1523,
sion chromatography (SEC) was carried out on a Viscotek observed: 342.1518. 1H NMR (D2O) δ in ppm: 5.65 (H11-cis),
GPCmax equipped with model 302 TDA detectors. Three 5.41(H11-trans), 4.43 (H1-axial, J = 8.0 Hz), 3.21–3.95 (H2, H3,
columns were used: a guard column (PSS-GRAM, 10 μm, 5 cm) H4, H5, H6, H7, H8), 1.90 (H10), 1.62 (H7′ and H8′). 13C NMR
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(D2O) δ in ppm: 172 (C12), 139 (C9), 121 (C11), 102 (C1β), 75.9 Free radical polymerization of Glc-β-EAAm, Glc-β-EMAAm, and
(C5), 75.8 (C3), 73 (C2), 70 (C4), 69.6 (C7), 61 (C6), 39 (C8), 26 Glc-β-BMAAm
(C7′), 25 (C8′), 18 (C10).
In a 10 ml round-bottom flask, Glc-β-EAAm (0.20 gram,
0.72 mmol), Glc-β-EMAAm (0.21 gram, 0.72 mmol), or Glc-
Time course of the reaction
β-BMAAm (0.23 gram, 0.72 mmol) was dissolved in Milli-Q
The reaction mixture was prepared as described in the pro-
water (2.4 ml). An initiator solution (10 mg in 1.0 ml DMF) was
cedure above with HEAAm used as glycosyl acceptor. After 1, 2,
prepared and 100 µl of it (3.57 µmol) was added into the
and 3 hours of reaction time, an aliquot (∼50 mg) was taken
and directly put on boiled water to deactivate the enzyme. The
monomer solution. The next steps use the same procedure as
in the RAFT polymerization.
Poly(N-(β-glycosyloxy)-ethyl acrylamide) (P(Glc-β-EAAm)).
RAFT product = pale yellowish powder, monomer conversion:
aliquots were dissolved in D2O (0.7 ml) and subsequently
measured by 1H NMR spectrometry.
95%, yield: 45%. Free radical product = white powder,
Improvement of yield
monomer conversion: 78%, yield: 54%. 1H NMR (D2O) δ in
In two 25 ml round-bottom flasks, p-NPG (0.50 gram,
1.62 mmol) was dissolved in Milli-Q water (4.50 ml) at 50 °C.
Subsequently, HEAAm (0.65 gram, 5.65 mmol) was added into
the p-NPG solution. Ionic liquid of BMIMPF6 (2.0 ml, 30 v%)
ppm: 7.86–8.30 (NH), 7.25–7.45 (H-aromatic from the RAFT
agent), 4.52 (H1-axial, J = 6.8 Hz), 3.26–4.08 (H2, H3, H4, H5,
H6, H7, H8), 1.94–2.33 (H9), 1.37–1.90 (H11). 13C NMR (D2O) δ
in ppm: 176 (C12), 102 (C1β), 75.9 (C5), 75.7 (C3), 73 (C2), 70
was added into one of the flasks and the reaction was started
(C4), 68 (C7), 61 (C6), 49 (C9), 42 (C11), 39 (C8).
by adding the enzyme solution (50 mg in 0.25 ml H2O). After
Poly(N-(β-glycosyloxy)-ethyl
methacrylamide)
(P(Glc-
1 hour of reaction time, an aliquot (∼50 mg) from each flask
was taken and directly put on boiled water to deactivate the
enzyme. The aliquots were dissolved in D2O (0.7 ml) and sub-
sequently measured by 1H NMR Spectrometry. The rest was
then put on boiled water to deactivate the enzyme and stop the
reaction. The reaction mixture was then centrifuged (4500
rpm, 30 min, 4 °C) and two layers were observed. The water
phase was collected and evaporated, then MeOH (10 ml) was
added to precipitate the glucose and unreacted p-NPG. After
another centrifugation (4500 rpm, 10 min, 4 °C), the super-
natant was concentrated by evaporation followed by purifi-
cation through column chromatography with silica gel served
as the stationary phase and CHCl3/MeOH (4/1) mixture as the
eluent. Eluent from the collected fractions containing the
product was evaporated and the obtained product was stored
in the fridge.
β-EMAAm)). RAFT product = pale pinkish powder, monomer
conversion: 88%, yield: 39%. Free radical product = white
powder, monomer conversion: 82%, yield: 40%. 1H NMR (D2O)
δ in ppm: 7.40–8.00 (NH and H-aromatic from the RAFT
agent), 4.45 (H1-axial), 3.15–4.00 (H2, H3, H4, H5, H6, H7,
H8), 1.23–2.05 (H11), 0.39–1.14 (H10). 13C NMR (D2O) δ in
ppm: 179 (C12), 102 (C1β), 75.9 (C5), 75.7 (C3), 73 (C2), 70
(C4), 68 (C7), 61 (C6), 49 (C9), 45 (C11), 40 (C8), 17 (C10).
Poly(N-(β-glycosyloxy)-butyl
methacrylamide)
(P(Glc-
β-BMAAm)). RAFT product = pale pinkish powder, monomer
conversion: 94%, yield: 41%. Free radical product = white
powder, monomer conversion: 90%, yield: 27%. 1H NMR (D2O)
δ in ppm: 7.34–7.92 (NH and H-aromatic from the RAFT
agent), 4.41 (H1-axial), 2.90–4.00 (H2, H3, H4, H5, H6, H7,
H8), 1.40–2.08 (H11, H7′, H8′), 0.70–1.30 (H10). 13C NMR
(D2O) δ in ppm: 179 (C12), 102 (C1β), 75.9 (C5), 75.7 (C3), 73
(C2), 70 (C4), 61 (C6), 45 (C11), 40 (C8), 26 (C7′), 24 (C8′), 17
(C10).
RAFT polymerization of Glc-β-EAAm, Glc-β-EMAAm, and
Glc-β-BMAAm
In a 10 ml round-bottom flask, Glc-β-EAAm (0.50 gram,
1.81 mmol), Glc-β-EMAAm (0.53 gram, 1.81 mmol), or Glc-
β-BMAAm (0.58 gram, 1.81 mmol) was dissolved in Milli-Q
Results and discussion
water (1.60 ml). BSPA and CPADB were used as the RAFT agent The enzymatic synthesis of glycosyl-alkyl (meth)acrylamides
for acrylamide- and methacylamide-based monomers, respect- was successfully performed using cellobiose as the glycosyl
ively. ACVA was used as the water-based initiator. The RAFT donor and HEAAm, HEMAAm, or HBMAAm as the glycosyl
agent (25 mg in 0.5 ml DMF) and ACVA (25.3 mg in 1.0 ml acceptors. Cellobiose, a disaccharide molecule consists of two
DMF) solutions were prepared and 100 µl of each solution β-glucose units, is preferred as starting materials since it is
(18 µmol of RAFT agent or 9 µmol of initiator) was added to largely derived from renewable cellulose and a good substrate
the monomer solution. The flask was then put into an ice bath for the enzyme β-glucosidase. The glycomonomers synthesis
and N2 bubbling was performed for at least
1 hour. was successfully carried out under the kinetically-controlled
Subsequently, the flask was placed in an oil bath at 70 °C to reaction conditions and we obtained three types of glycomono-
start the reaction. An aliquot solution (100 µl) was drawn at a mers namely N-(β-glycosyloxy)-ethyl acrylamide (Glc-β-EAAm),
specified time to determine the monomer conversion by 1H N-(β-glycosyloxy)-ethyl methacrylamide (Glc-β-EMAAm), and
NMR. The polymer was isolated by precipitation of the reaction N-(β-glycosyloxy)-butyl methacrylamide (Glc-β-BMAAm). The
mixture into MeOH (10× volume) and reprecipitated two times. monomer Glc-β-EAAm has been synthesized previously by a
The gel-like precipitates were dried in vacuum oven (40 °C) chemical approach involving five reaction steps26,27 while to
overnight.
the best of our knowledge, the monomers Glc-β-EMAAm and
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Glc-β-BMAAm have never been prepared before either by
chemical or enzymatic methods. As shown in Scheme 1, the
advantage of biocatalytic synthesis of glycomonomers is, that
it is performed in only one reaction step generating purer pro-
ducts and lower environmental waste than the conventional
reactions.
1
Fig. 1 shows H NMR and 13C NMR spectra of the purified
1
glycosyl-alkyl (meth)acrylamide monomers. From the H NMR
spectra, only one anomeric proton signal was observed at
4.45 ppm corresponding to the axial position. Additionally, the
13C NMR spectra demonstrating the anomeric carbon (C1) has
only one chemical shift at 102 ppm that relates to a glycoside
in β-configuration. These findings concluded that we obtained
anomerically pure products with the linkage of (meth)acryl-
amide unit toward glucose at β-position. Besides, by compar-
ing the peak integration of the anomeric proton with the vinyl
protons (H9 & H11) it suggested that each glucose unit con-
tains only one vinyl group. Consequently, the prepared glyco-
monomers were not only anomerically pure but also mono-
functional; a unique feature provided by the enzymatic reac-
tion offering high selectivity. In addition, molecular weights of
the glycosyl-alkyl (meth)acrylamide monomers obtained from
mass spectrometry experiments were almost identical to the
calculated ones (see Fig. S1–S3 in ESI†). Combination of 1H
NMR, 13C NMR, and mass spectrometry measurements con-
firmed the structure of the aimed glycomonomers as shown in
Scheme 1.
Scheme 2 Reaction mechanism of the synthesis of Glc-β-EAAm cata-
lyzed by β-glucosidase.
this mechanism, a competition between transglycosylation
and hydrolysis occurs during the enzymatic reaction.
Therefore, it is crucial to determine the time needed for the
reaction as short reaction times will lead to a low reactant con-
version and low amounts of transglycosylation products while
long reaction times risk the glycomonomers to be further
hydrolyzed. In order to find the optimum time, 1H NMR
spectra of Glc-β-EAAm reaction mixtures were measured at
certain time intervals and the results are displayed in Fig. 2.
According to Fig. 2(a), the anomeric proton peak of Glc-
β-EAAm at 4.45 ppm was observed after a one-hour reaction
and its peak area was further reduced with longer reaction
time. In addition, the anomeric proton peak of cellobiose at
4.50 ppm almost completely disappeared at a reaction time of
two hours. These observations indicated that the enzyme
starts to hydrolyze the glycomonomer following 2nd hydrolysis
pathway when almost all cellobiose is consumed. Hence, a
one-hour reaction time is the optimum condition for this bio-
Time course of the reaction
The reaction mechanism of the kinetically-driven enzymatic
synthesis of Glc-β-EAAm is shown in Scheme 2. According to
Scheme 1 Kinetically-controlled synthesis of Glc-β-BMAAm (m = 2, R = CH3), Glc-β-EMAAm (m = 1, R = CH3), and Glc-β-EAAm (m = 1, R = H) cata-
lyzed by β-glucosidase.
Fig. 1 1H NMR and 13C NMR spectra of the enzymatically synthesized (a) Glc-β-BMAAm, (b) Glc-β-EMAAm, and (c) Glc-β-EAAm in D2O.
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Fig. 2 1H NMR spectra of the solution mixtures from Glc-β-EAAm synthesis at different reaction times (a) with enzyme and (b) without enzyme in
D2O.
catalytic reaction. This result supports the advantage of a kine- mixtures than in pure water, which leads to an increase of the
tically-controlled enzymatic reaction having short reaction reaction speed and improves the conversion rate. Additionally,
times. Moreover, at a one-hour reaction, the peak area of the our experiments showed that the immiscibility of BMIMPF6
anomeric proton of glucose at 4.60 ppm is higher than the with water facilitates the separation from the reaction mixture
anomeric proton of Glc-β-EAAm indicating the 1st hydrolysis just by centrifugation. As a result, BMIMPF6 may potentially be
reaction in Scheme 2 is more favorable than transglycosylation. reused for the next experiments. We, therefore, studied the
As a result, Glc-β-EAAm is produced less than glucose with the yield improvement of Glc-β-EAAm synthesis by using p-NPG as
yield of about 16%. Furthermore, neither glycomonomers nor an alternative glycosyl donor in transglycosylation reaction (see
glucose proton peaks were observed in the control reaction Scheme 3) and the synthesis was performed with and without
(Fig. 2b) confirming the role of the enzyme in catalyzing the BMIMPF6.
reaction.
Fig. 3 compares 1H NMR spectra of the solution mixtures of
the Glc-β-EAAm synthesis with different substrate compo-
sitions after a one-hour reaction time. The peak area at
4.45 ppm increased significantly when the combination of
p-NPG and BMIMPF6 was used. It indicates that higher glyco-
monomer yields were achieved by adjusting the reaction
medium from the initial formulation that consists of only cel-
lobiose without cosolvent. The obtained yield of Glc-β-EAAm
was about 68%. In the event of using only p-NPG without
cosolvent, the peak area of the anomeric proton was still
higher than using cellobiose with a glycomonomer yield of
about 49%. The latter case is remarkable considering the fact
that the concentration of p-NPG was 40% less than cellobiose
but it was able to produce higher Glc-β-EAAm yield. This
outcome further confirms that using an activated mono-
saccharide as the glycosyl donor can facilitate the reaction to
be more preferred towards transglycosylation than hydrolysis.
Unfortunately, p-nitrophenol is generated as the side product
when p-NPG was used and this compound is recognized to be
Improvement of yield
The yield of the synthesized Glc-β-EAAm, Glc-β-EMAAm, and
Glc-β-BMAAm was quite low of around 16%, 12%, and 18%,
respectively, although the maximum concentration of cello-
biose (0.60 M at 50 °C) was used together with the 2–4 times30
concentration of glycosyl acceptor. It has been reported that
aryl or vinyl units on an activated sugar are much better
leaving-groups than glycosyl units on native sugars in transgly-
cosylation reactions.31,32 Hence, we might replace cellobiose
with an activated sugar like p-NPG to make the transglycosyla-
tion route more favorable than hydrolysis. Another way to
improve the glycomonomers yield is by adding cosolvents like
ionic liquids33–35 or organic solvents.36–38 For example, Bayón
et al.33 used the ionic liquid BMIMPF6 a a cosolvent and the
transglycosylation yields improved up to 97%. Their molecular
dynamic simulations revealed that the electrostatic interaction
between enzyme and substrate was higher in water-BMIMPF6
Scheme 3 Transglycosylation reaction of p-NPG and HEAAm catalyzed by β-glucosidase with BMIMPF6 as cosolvent.
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highly toxic to living organisms. Therefore, another activated
glucose such as vinyl-β-D-glucopyranoside may be exploited as
a promising glycosyl donor and we will further study this in
the future.
Polymerization of the prepared glycomonomers
The glycosyl-(meth)acrylamide monomers were successfully
polymerized by aqueous RAFT polymerization and free radical
polymerization (FRP). RAFT polymerization is one of the most
versatile methods for the synthesis of well-defined polymers;
on the other hand, FRP is a very robust technique for the pro-
duction of high-molar-weight polymers in industry. The
polymerization reactions are displayed in Scheme 4. Water was
chosen in order to create the glycopolymers using a green
solvent as we did with the synthesis of glycomonomers
although a minor fraction of DMF (6 v%) was still needed to
solubilize the RAFT agent. CPADB is a commercially available
RAFT agent that is known to be well-suited for methacryla-
Fig. 3 1H NMR spectra of the solution mixtures of the Glc-β-EAAm syn-
thesis after 1-hour reaction with different substrate compositions con-
taining (a) 0.34 M p-NPG and BMIMPF6, (b) 0.34 M p-NPG without
cosolvent, and (c) 0.60 M cellobiose without cosolvent in D2O.
Scheme 4 Synthesis of (a) P(Glc-β-EAAm) and (b) P(Glc-β-EMAAm) or P(Glc-β-BMAAm) by aqueous RAFT polymerization. (c) Synthesis of the pre-
pared glycomonomers by aqueous FRP.
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Fig. 4 1H NMR and 13C NMR spectra of (a) P(Glc-β-BMAAm), (b) P(Glc-β-EMAAm), and (c) P(Glc-β-EAAm) synthesized via aqueous RAFT polymeriz-
ation in D2O.
mides39,40 and BSPA, a trithiocarbonate-based RAFT agent,
was relatively easy to prepare and has been successfully uti-
lized in the preparation of polyacrylamides.41,42 The presence
of carboxylate unit on both RAFT agents maintains the solubi-
lity in aqueous medium.
Based on 1H NMR spectra of the synthesized glycopolymers
by RAFT polymerization in Fig. 4 (or Fig. S4† for the glycopoly-
mers prepared by free radical polymerization), vinyl proton
peaks of the monomers (H9 & H11 in Fig. 1) were no longer
observed. In addition, broad peaks appeared around
0.5–2.3 ppm to be related to the protons at the polymer back-
bone. In the case of P(Glc-β-EMAAm) and P(Glc-β-BMAAm),
aromatic proton peaks from the RAFT agent were mixed with a
broad N–H peak around 7.25–8.0 ppm. The latter peak was
also observed in the literature.43 In agreement with the 1H
Fig. 5 SEC measurements (RI signals) of the synthesized glycopolymers
by aqueous RAFT polymerization and FRP.
NMR spectra, 13C NMR spectra of the prepared glycopolymers
showed that the vinyl carbon peaks of the monomers (C9 &
C11 in Fig. 1) were absent. Also, new carbon peaks (C9 & C11
in Fig. 4) assigned to the polymer backbone were detected.
Fig. 5 shows SEC chromatograms of the prepared glycopoly- means that the latter polymers have higher molecular weights
mers by RAFT polymerization and FRP. Elugrams with rela- and PDI’s than the former polymers. The absence of chain
tively narrow peak and unimodal distribution were observed transfer agents during FRP allowing the polymer chains to
for the synthesized glycopolymers by RAFT polymerization. growth uncontrolled lead to high molecular weights polymers
Also, these glycopolymers had low polydispersity indices and the termination reaction of FRP typically occurred via
(PDI’s) as presented in Table 1. These results suggested that combination and disproportionation reactions causing broad
the controlled behavior has been achieved during RAFT polydispersities.
polymerization of the glycomonomers. Moreover, molecular
The glass transition temperature (Tg) of the prepared glyco-
weights of these glycopolymers were calculated theoretically polymers by RAFT polymerization was measured by differential
based on the monomer conversion obtained from 1H NMR scanning calorimetry and the thermograms are presented in
spectra of the reaction mixtures and the resulted molecular Fig. 6 (or Fig. S5† for the glycopolymers prepared by free
weights were in the range of 26–30 kg mol−1. Nevertheless, the radical polymerization). The Tg’s of respective glycopolymers
theoretical molecular weights of these glycopolymers were prepared by RAFT polymerization and FRP were similar. Even
lower than the molecular weights gained from the SEC though the glycopolymers synthesized by FRP had 7–8 times
measurements because of the differences in hydrodynamic higher molecular weight than the glycopolymers synthesized
volumes of the glycopolymers and the standard PMMA. In by RAFT, both polymers have already reached the level of mod-
comparison with the prepared glycopolymers by RAFT erate to high molecular weights. Then according to the Fluory–
polymerization, the prepared glycopolymers by FRP have lower Fox equation,44,45 the difference between the Tg’s for the same
elution volumes and broader peaks in their elugrams. It polymers are no longer significant. Besides, the measured Tg
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Table 1 Overview of the synthesized glycopolymers by aqueous RAFT polymerization and FRP at 70 °C
Polymera
tR
Conv. (%)
Mn, theory
Mn, SEC
PDI
Tg (°C)
b
c
d
P(Glc-β-BMAAm)-RAFT
P(Glc-β-EMAAm)-RAFT
P(Glc-β-EAAm)-RAFT
P(Glc-β-BMAAm)-FRP
P(Glc-β-EMAAm)-FRP
P(Glc-β-EAAm)-FRP
4
4
4
4
6
6
94
88
95
90
85
78
30.4
26.0
26.7
—
—
—
66.4
49.4
43.2
214.3
225.2
223.2
1.22
1.29
1.25
2.46
3.29
2.79
130
165
142
131
171
147
a [Monomer] for RAFT
=
1.0
M
and FRP
=
0.29 M, [Monomer] : [RAFT agent] : [Initiator]
d Calculated molecular weights (in kg mol−1).
=
100 : 1.0 : 0.5. b Reaction times in hours.
½Monomerꢀ
c Mn;theory
¼
ꢁ Conv: ꢁ MWmonomer þ MWRAFT agent
:
½RAFT Agentꢀ
Furthermore, the reaction condition for the kinetically-con-
trolled enzymatic synthesis of the glycomonomers has been
optimized to improve the glycomonomer yield. The structural
characterization of the glycomonomers was conducted by 1H
NMR, 13C NMR, and mass spectrometry.
The synthesized glycomonomers were successfully polymer-
ized by aqueous RAFT and free radical polymerization. The
SEC measurements demonstrated that the glycopolymers syn-
thesized by RAFT polymerization have lower molecular weights
and PDI’s than the glycopolymers prepared by FRP. The Tg’s of
both glycopolymers were around 142–171 °C as characterized
by differential scanning calorimetry.
The synthesis of glycomonomers and glycopolymers have
been performed in a green way, i.e. using renewable resources
as starting materials, applying enzyme as the biocatalyst, and
performing the reaction in water/water-ionic liquid mixture. In
spite of the simple synthesis route in creating the glycomono-
mers as presented in this report, producing the monomer on a
large scale remain challenging considering the price of the
enzyme, the substrates (cellobiose and p-NPG), and cosolvent.
Even though BMIMPF6 can be reused, further experiments
need to be performed to determine the number of cycles of
used BMIMPF6 that still resulting good amounts of glycomo-
nomers. Moreover, the future investigation will be carried out
in the direction of using these glycomonomers and glycopoly-
mers as bio-related application materials.
Fig. 6 DSC thermograms of (a) P(Glc-β-BMAAm), (b) P(Glc-β-EMAAm),
and (c) P(Glc-β-EAAm) prepared via RAFT polymerization (2nd heating
cycle).
of P(Glc-β-EAAm) was 142 °C, which is lower than the Tg of
polyacrylamide46 at 165 °C. This observation is reasonable
because the glycosyl units increase the free volume of P(Glc-
β-EAAm) while the polyacrylamide has a more rigid structure.
The same holds for the P(Glc-β-BMAAm) with longer alkyl side
chains resulting in bigger free volume thus lower Tg than
P(Glc-β-EMAAm). Furthermore, the Tg of P(Glc-β-EMAAm) was
higher compared to that of P(Glc-β-EAAm) as methyl group at
the P(Glc-β-EMAAm) backbone hinders polymer chain mobi-
lity. Consequently, P(Glc-β-EMAAm) requires higher energy
and higher temperature than P(Glc-β-EAAm) for the transition
from the glassy-state to the rubbery-like material.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
Conclusions
The authors kindly appreciate Albert J. J. Woortman for SEC
We have successfully synthesized three different types of glyco- measurements and Theodora Tiemersma-Wegman from
syl-(meth)acrylamide monomers using β-glucosidase from Stratingh Institute for Chemistry, University of Groningen, for
almond as the biocatalyst. Due to the enzyme selectivity, the ESI-MS analysis. Azis Adharis thanks the Indonesia
prepared glycomonomers were found to be monofunctional Endowment Fund for Education (Lembaga Pengelola Dana
and anomerically pure. The linkage of (meth)acrylamide units Pendidikan Republik Indonesia/LPDP RI) for the financial
was observed at the anomeric β-position of glucose. support during his PhD program.
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