Biocatalytic Synthesis of Maltodextrin-Based Acrylates from Starch and α-Cyclodextrin
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1268
Biocatalytic Synthesis of Maltodextrin-BasedAcrylates from Starch and a-Cyclodextrina
Wouter M. J. Kloosterman, Gerda Spoelstra-van Dijk, Katja Loos*
Novel 2-(b-maltooligooxy)-ethyl (meth)acrylate monomers are successfully synthesized byCGTase from Bacillus macerans catalyzed coupling of 2-(b-glucosyloxy)-ethyl acrylate andmethacrylate with a-cyclodextrin or starch. HPLC-UV analysis shows that the CGTasecatalyzed reaction yields 2-(b-maltooligooxy)-ethyl acrylates with 1 to 15 glucopyranosylunits. 1H NMR spectroscopy reveals that the b-linkage in the acceptor molecule is preservedduring the CGTase catalyzed coupling reaction, whereas the newly introduced glucose unitsare attached by a-(1,4)-glycosidic linkages. The synthesized 2-(b-maltooligooxy)-ethyl
acrylate monomers are suc-cessfully polymerized by aque-ous free radical polymerizationto yield the comb-shaped gly-copolymer poly(2-(b-maltooli-gooxy)-ethyl acrylate).Dr. W. M. J. Kloosterman, G. Spoelstra-van DijkNijenborgh 4, 9747 AG Groningen, the NetherlE-mail: [email protected]
aSupporting Information is available from theWilefrom the author.
� 2014 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimMacromol. Biosci. 2014, 14, 1268–1279
1. Introduction
Poly(oligosaccharide acrylates) are polymers that combine
an apolar backbone with pending oligosaccharide
groups. Their structure leads to specific properties as
amphiphilicity, low toxicity, good biocompatibility, and
furthermore they are based on renewable resources. Due to
these properties there is a growing interest in the
application of these polymers as biobased non-ionic
polymeric surfactants,[1–3] and specialty chemicals in
pharmaceutical and drug targeting applications.[4–6]
Most commonly they are synthesized by free radical
polymerization of functionalized oligosaccharides. Since
the functionalized oligosaccharides are highly water
soluble, they are preferentially polymerized by solution
polymerization in water. Aqueous free radical polymeriza-
tion is cheap, very robust, low temperatures can usually
be used and over-all is an environmentally friendly
, Prof. K. Loosands
y Online Library or
wileyonlinelibr
polymerization technique. The synthesis of monomers
for poly(oligosaccharide acrylates), however, requires
mono-functionalization and well-defined positioning of
the acrylate groups on the oligosaccharides. Since oligo-
saccharides possess many hydroxyl groups with equal
reactivity towards reagents, there aremany possibilities to
form positional isomers in which different or multiple
hydroxyl groups are substituted. Alternatively, to the
conventional chemistry routes, biocatalytic pathways
have shown to be beneficial in poly(saccharide) synthesis
and modification.[7–11] Since biocatalysts are in general
highly enantio-, regio- andchemoselective,mono-function-
alized saccharides can be obtained relatively easy.[12–16]
Additionally, biocatalytic synthesis routes can be consid-
ered as green processes; the reactions can typically be
performed at atmospheric pressure, low temperatures and
neutral pH and the biocatalyst are obtained from natural
renewable resources.[17] Glycosyl hydrolases and trans-
ferases (EC 3.2 and 2.4) are typically used for the
modification of (poly)saccharides, since carbohydrates are
their natural substrate.[18] The cyclodextrin glycosyltrans-
ferases (CGTase EC 2.4.1.19) have been found to possess
unique properties, their catalytic mechanism has been
studied in detail.[19–25] CGTases are found to catalyze
three kinds of reactions with maltodextrins or starch:
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i) the formation of a-(1,4)-cyclomaltodextrins containing
six (a-cyclodextrin), seven (b-cyclodextrin) or eight (g-
cyclodextrin) anhydroglucopyranosyl units, ii) coupling
reactions between a donor (starch, maltodextrins or cyclo-
dextrins) andanacceptormolecule (saccharides, glycosides,
and many other), and iii) disproportionation reactions
between two maltodextrin chains, resulting in malto-
dextrins of different sizes.[24,25] The CGTase sometimes
catalyzes a fourth hydrolytic reaction, however this
hydrolysis reaction was found to be much slower than
the first three.[25] All these reactions catalyzed by CGTase
proceed via a covalent glycosyl-enzyme intermediate
(Figure 1). The cleavage site contains two amino acid
residues, usually aspartic acid and glutamic acid, which are
involved in the cleavage of glycosidic bonds. Next to the
cleavage site, the enzyme has six or seven sites that are
involved in binding of the donor substrate and two sites
that bind the acceptor molecules. Firstly, a donor a-(1,4)-
glucan enters the active site. An a-(1,4)-glycosidic linkage
is cleaved and a stable covalent enzyme intermediate is
formed. As a result of the cleavage, the formation of the
glycosyl-enzyme intermediate always results in a new
reducing end (C1-carbon). Even when a-cyclodextrin is
used as the glycosyl donor, the formation of the enzyme-
intermediate results in a new C1-carbon. Subsequently, the
saccharide in theacceptor sites (Figure1, labeledþ1andþ2)
can leave the enzyme, whereas the donor sites remain
Figure 1. Schematic representation of a covalent glycosyl-enzymeintermediate in the active site of CGTase from Bacillus macerans.Donor sites and acceptor sites are labeled –1 to –6 and þ1, þ2,respectively.
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occupied by the covalent bound enzyme intermediate.[25]
Lastly, the covalent glycosyl-enzyme intermediate under-
goes a reaction with an acceptor. The own C4-hydroxyl
group of the non-reducing end of the glycosyl-enzyme
intermediate canact as theacceptor, yieldingacyclodextrin
(Cyclization reaction). When the acceptor is a maltodextrin
or a glycoside, the intermediate reacts to an extended
maltodextrin or glycosides with a longer glucan moiety
(Disproportionation and coupling reaction respectively).
Lastly, water can be the acceptor molecule yielding a linear
a-(1,4)-glucan (Hydrolysis reaction), but the hydrolysis
reactionwas found tobemuch less favorable. Especially the
coupling reactionbetweenaglycosyl donorandanacceptor
has opened doors towards the synthesis of unique
maltodextrin derivatives. CGTase catalyzed coupling of
maltodextrins (glycosyl donor) to sucrose laurate,[26] a-
arbutin (4-hydroxyphenyl a-D-glucopyranoside),[27] sali-
cin,[28] curcumin b-D-glucopyranoside,[29] 1-O-methyl glu-
coside,[30] 1-O-phenyl glucoside,[30] hesperin, ningin, andrutin,[31] have been reported. In all cases, the C4-hydroxyl
group of the glucopyranose ring reacts with the
covalent enzyme intermediate to obtain the maltodextrin
derivatives. We present here the successful synthesis of
2-(b-maltooligooxy)-ethyl acrylate and -methacrylate
monomers from starch and a-cyclodextrin by using the
CGTase catalyzed transglycosidation reaction.
2. Experimental Section
2.1. Materials
Hydroquinone (HQ), monomethyl ether hydroquinone (MEHQ),
maltose (G2), maltotriose (G3), 2-hydroxyethyl acrylate 96%
containing 200ppmMEHQ (2-HEA),a-cyclodextrin (a-CD), calcium
chloride (CaCl2), sodium azide (NaN3), potassium metabisulfite
(K2S2O8) and diphenylamine (DPA) are obtained from Sigma-
Aldrich. Maltotetraose (G4), 2-hydroxyethyl methacrylate 97%
containing 200ppmMEHQ (2-HEMA), acetic acid 99.5% (HAc) and
butan-1-ol 99% (n-But) are obtained from Acros. Tris(hydroxy-
methyl)aminomethane (Tris), glucose anhydrous (G1), ammonium
persulphate (APS), ethyl acetate (EtAc), and aniline are obtained
from Merck. Soluble starch is supplied by Difco. Silica 230–
400mesh is obtained from Silicycle. Chloroform (CHCl3), propan-
2-ol (IPA), ethyl acetate (EtAc), methanol (MeOH) and other
mentioned organic solvents were obtained as analytical reagents
from Labscan. All chemicals were used as received.
2.2. Acceptor Acryl Glycosides
2-(b-glucosyloxy)-ethyl acrylate (Glc-b-EA) and 2-(b-glucosyloxy)-
ethyl methacrylate (Glc-b-EMA) were prepared by enzymatic
glycosidation using b-glucosidase as previously described.[15]
1-methacrylamido-2-D-gluconoylaminoethane (MEGA) was pre-
pared according to the procedure described by Narain and
Armes.[32]
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W. M. J. Kloosterman, G. Spoelstra-van Dijk, K. Loos
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2.3. CGTase from Bacillus macerans
Cyclodextrin glucanotransferase ‘‘Amano’’ from Bacillus macer-ans (CGTase from B. macerans) was obtained from Amano
Enzymes Europe Ltd, Oxfordshire. The enzyme had an activity of
at least 600UmL�1. One unit of enzyme was defined as the
amount of enzyme, which produces 1mg cyclodextrin from
5mL of 5% w/v soluble starch with 0.1mL CGTase at pH 6.0,
55 8C for 1 h.
2.4. Characterization
1H NMR and 13C NMR spectra were recorded using a 7.05 T Varian
VXR spectrometer (300MHz and 75MHz resolution for 1H and13C NMR, respectively). Deuterium oxide (99.9 atom% D, Aldrich)
wasusedas thesolvent. TLCwasdoneonaluminumsheetssilicagel
60/kieselguhr F254 (Merck) and two eluentmixtureswere used for
separation of the saccharide-acrylates: 5/10/4 v/v/v n-But/IPA/
water, or 2/1 v/v CHCl3/MeOHwas used as the eluent. Spots were
visualized as described by Hansen using diphenylamine/aniline
reagent.[33] Column chromatography was performed on silica gel
60 (400mesh). The samplewas dilutedwithmethanol andbrought
on the column and eluted with 4:1 v/v EtAc/MeOH. HPLC was
performed on a Shimadzu HPLC under reversed phase conditions.
An Alltech HP column (Alltima amino, 5mm, 150mm�4.6mm)
was used for separation. The column temperaturewasmaintained
at 40 8C and the injection volume was 15mL. Compounds were
detected by a SPD-M20A photodiode array detector at 205nm.
A gradient of acetonitrile and double distilled water was used as
the eluent with a flow rate of 0.5mLmin�1. The water content of
the eluent was 15 vol% for 5min followed by a linear increase
to 60 vol% in 85min (gradient A) or slowly increased from 5 to
15 vol% in 5min, followed by a linear increase to 60 vol% in
85min (gradient B). The column was brought back to initial
conditionsbya lineargradient from60vol%water to15or5vol% in
20min and subsequently equilibrated for 10min, before the next
injection. Gradient A was used for reaction mixtures containing
2-HEA and gradient Bwas used for reactionswith 2-HEMA. ESI-MS
was performed on a Thermo Scientific LTQ Orbitrap mass
spectrometer in positive ion mode. Samples were dissolved in
water and diluted with an equal volume of methanol. FT-infrared
spectroscopy (FTIR) was done on a Bruker JFS 88 equipped with an
ATR-golden gate unit.
2.5. CGTase Catalyzed Alcoholysis with 2-HEA and
2-HEMA
Alcoholysis reaction were performed with 100mL (60U) CGTase
from B. macerans, 0.15 g a-CD (0.154mmol) dissolved in 10mL
1.0 M Tris–HCl buffer containing 0.01M CaCl2, 0.02% NaN3
and 1% HQ. Various amounts (1, 5, or 10 vol%) of the acceptor
alcohol (2-HEA or 2-HEMA) were subsequently added to the
reaction mixtures. A blank reaction was performed in absence of
the acceptor alcohol. The reaction mixtures were incubated at
60 8C for 5 h. The reaction was stopped by addition of 20 vol%
MeOH. Subsequently, the samples were analyzed by TLC, using
nBut/iPA/H2O as eluent.
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2.6. CGTase Catalyzed Transglycosidation with Acryl
Glycosides
Reaction mixtures contain either 0.7 g a-CD (0.72mmol) or 0.35 g
soluble starch dissolved in 4mL Tris–HCl buffer. The acceptor
glycoside (100mg Glc-b-EA, Glc-b-EMA, or MEGA) was dissolved
in 1.0mL Tris–HCL buffer and added to the reaction mixtures
containing either a-CD or soluble starch. To all six reaction
mixtures 50mL (30U) of CGTase from B. macerans was added. The
reaction mixtures were incubated at 60 8C for 16h. The reactions
were stopped by addition of 50 vol% MeOH and analyzed by TLC,
using n-But/iPA/Water as eluent.
2.7. Effect of Soluble Starch to Acceptor Ratio
Reactionsmixturescontain100mgGlc-b-EAdissolved in5mLTris–
HCl buffer. A final donor:acceptor ratio (w:w) of 7:1 and 3.5:1 was
obtainedbyadditionof700and350mgsolublestarch, respectively.
The reaction was initiated by addition of 100mL (60U) CGTase
from B. macerans. The reaction mixtures were incubated for 1.5 h
at 60 8C. Samples of 200mLwere takenanddiluted to afinal volume
of 10mL,with aqueousMeOH (50 vol%) solution. Precipitateswere
removed by a 0.2mm filter, before HPLC analysis.
2.8. Increasing the Acceptor Conversion by Changing
the Molar Ratio a-CD:Acceptor
Reactionmixtures contain 0.5 ga-CD (0.51mmol) dissolved in 5mL
Tris–HCl buffer. To change the molar donor:acceptor ratio, the
amount of Glc-b-EAwas varied as follows: 46mg (3:1), 72mg (2:1),
150mg (1:1), or 300mg (1:2). The reactionwas initiatedbyaddition
of 100mL CGTase from B. macerans (60U). The reaction mixtures
were incubated for 1.5 h at 60 8C. Samples of 200mLwere taken and
diluted to a final volume of 10mL, with aqueous MeOH (50 vol%)
solution. Precipitates were removed by a 0.2mmfilter, before HPLC
analysis.
2.9. Preparative Synthesis of (Glc-a)2-Glc-b-O-ethyl
Acrylate by CGTase from B. macerans
A20mLflaskwaschargedwith0.6 gofGlc-b-EA,1.0 gsolublestarch
and 10mL Tris–HCL buffer. MEHQ (4mg) was added to prevent
thermal polymerization, followed by 100mL (60U) CGTase from B.macerans. The reaction mixture was incubated for 1.5 h at 60 8C.After 1.5 h two transglycosidation products were detected as blue
spots in TLC using nBut/iPA/H2O as the eluant. Following spots
were detected: Starting glycoside (Rf 0.78; Glc-b-EA); first product(Rf 0.71; Glc-a-Glc-b-EA) and secondproduct (Rf 0.65; (Glc-a)2-Glc-b-EA). The reactionmixture was purified by CC. Fractions containing
the second product (Rf 0.65) were pooled, 2mg HQ was added and
the solvent was removed by rotary evaporation at 30 8C.(Glc-a)x-Glc-b-EA: Transparent syrup, 0.241g, purity: 95%, yield:
38mol%; 1H NMR (D2O): d¼ 6.46 (d, J¼ 17Hz; H11), 6.22 (dd, J¼17,
11Hz;H10),5.99 (d, J¼11Hz,H11),5.39 (s;H10), 4.50 (d, J¼ 8Hz;H1),
4.39 (t, J¼4, 4Hz; H8), 3.2–4.2 (br; H2, H20, H3, H30, H4, H40, H5, H50 ,H6, H60 , H7); 13C NMR (D2O): d¼168.7 (C9), 132.9 (C10), 127.6 (C11),
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102.5 (C1), 99.7–100.0 (C10), 77.1–76.3 (C4,C40), 74.8–72.0 (C3,C30,C2,C20), 71.7–69.5 (C5, C50), 68.1 (C8), 64.4 (C7), 60.9–60.7 (C6, C60);ESI-MS: see Table 3.
2.10. Polymerization of 2-(b-maltooligooxy)-Ethyl
Acrylate
Polymer poly(2-(b-maltooligooxy)-ethyl acrylate) (P(2-MOEA) was
obtained via two polymerization routes.
2.10.1. Route A: Removal of the Oligosaccharides After
Polymerization
First, 2-(b-maltooligooxy)-ethyl acrylate monomers with various
saccharide lengths were synthesized according to the enzymatic
procedure described in above. Subsequently, the reaction temper-
ature was lowered to 55 8C after the enzymatic reaction. The crude
reaction mixture (5mL), containing 2-(b-maltooligooxy)-ethyl
acrylate monomers of various length and saccharide impurities,
was dilutedwith 5mLwater and purgedwith nitrogen for at least
1 h. Initiator solutions 6wt%K2S2O5 and 12wt% (NH4)2S2O8 were
prepared. The initiator solutions (0.2mL of each)were added to the
reactionmixture. Purgingwithnitrogenwas continued for 3–4h to
prevent oxygen inhibition and the polymerization reaction was
continued for 24h under nitrogen atmosphere.
After polymerization, the reactionmixture was poured into ten
volumes of MeOH. A highly hygroscopic solid was obtained (�1 g).
The material was dissolved in 10mL of water and dialyzed
against water to remove saccharides from the polymer. Water
was removed from the dialyzed solution by rotary evaporation
yielding 0.48 g of polymer P(2-MOEA).
2.10.2. Route B: Removal of the Oligosaccharides Before
Polymerization
In the second approach, 2-(b-maltooligooxy)-ethyl acrylate mono-
mers with various saccharide lengths were again first synthesized
according to the enzymatic procedure described above. After the
enzymatic reaction, the reaction temperature was cooled down to
25 8C. Subsequently, the crude reaction mixture (5mL), containing
the 2-(b-maltooligooxy)-ethyl acrylate monomers and saccharide
impurities, was mixed with an equal volume MeOH to
precipitate high molecular weight saccharides. The precipitation
was removed by filtration and the supernatant was put in a three
neckflask.Theflaskwasputinanoilbathandbroughtto55 8Cwhile
purging the solution for at least 1 h with nitrogen. Subsequently,
the initiator solutions (0.2mL of both 6wt%K2S2O5 and 12wt%
(NH4)2S2O8) were added and the reaction was further continued
and purified as described above.
Route
� ðb�
Route
� ðb�
www.M
A : Yield : 0:48g; 36:9mol% in respect to 2
glucosyloxyÞ � ethyl acrylate ðGlc� b� EAÞ
B : Yield : 0:19g; 14:6mol% in respect to 2
glucosyloxyÞ � ethyl acrylateðGlc� b� EAÞ
1H NMR (D2O): d¼5.41 (s, 2H; H10), 4.51 (s, 1H; H1), 4.33 (br; H8),4.12–3.33 (br; H2, H20 , H3, H30, H4, H40, H5, H50 , H6, H60, H7), 2.48–
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1.70 (br; H10b, H11b); 13C NMR (D2O): d¼176.8 (C9), 102.7 (C1),
100.0 (C10), 77.3–69.6 (C4, C40, C3, C30 ,C2, C20, C5, C50), 67.8 (C8),
64.7 (C7), 60.9–60.8 (C6, C60) 41.9 (C10b), 34.4 (C11b); FTIR (ATR):
v¼3400 (br; vs(OH)), 2940 (w; vs(CH)), 1720 (s, vs(C¼O)); 1050 cm�1
(m; vs(C–O)).
3. Results and Discussion
We have investigated the synthesis of maltodextrin
acrylate monomers by CGTase from B. macerans catalyzedcoupling of various acceptors to a-CD and starch. Initially,
reactions were performed in aqueous buffer solutions
containing1, 5, or10vol%of theacceptoralcohol2-HEMAor
2-HEA and a-CD as the glycosyl donor. The aimed products
were saccharide (meth)acrylatemonomers that consist of a
linear a-(1,4)-glucan and a polymerizable acrylate moiety.
The reaction mixtures were analyzed by TLC analysis. All
reactionmixturesshowedthepresenceofmaltodextrins (as
a resultofhydrolysis/transglycosidation), butmaltodextrin
acrylates could not be detected in any of the reaction
mixtures. The catalytic mechanism of CGTases might be
unfavorable for the alcoholysis reaction, similar to what
was found for the hydrolysis reaction. Uitdehaag et al.[25]
found that binding of a small water molecule at the
subsite þ1 of CGTase from Bacillus circulans does not
induce the structural rearrangements necessary to activate
the enzyme in catalysis. This effect explains the low
hydrolytic activity and thus the transglycosidation speci-
ficity of CGTase. This phenomenonmight also apply for the
relatively small acceptor alcohols 2-HEA and 2-HEMA.
Since CGTase catalyzed hydrolysis was the less favorable
reaction, the similar alcoholysis reaction might neither be
very favorable.
3.1. CGTase Catalyzed Synthesis of Maltodextrin
(meth)acrylates by Transglycosidation
Transglycosidation of glycosides is a more common
synthesis approach to obtain maltodextrin derivates
and has been the topic of multiple reviews.[19,22,23,34] We
have investigated theCGTase catalyzed transglycosidation
of a-CD and several saccharide acrylates as depicted in
Scheme 1.
The glycoside acceptors 1-methacrylamido-2-D-gluco-
noylaminoethane (MEGA), 2-(b-glucosyloxy)-ethyl meth-
acrylate (Glc-b-EMA) and 2-(b-glucosyloxy)-ethyl acrylate
(Glc-b-EA) that were used as the acceptors, were synthe-
sized by conventional or enzymatic synthesis routes
(Table 1).
After 24h, the reaction mixtures were analyzed by TLC.
The TLC chromatogram (Figure S1, Supporting Information)
shows novel (maltooligooxy)-ethyl acrylates (Rf 0.71 and
0.65) and methacrylates (Rf 0.75, 0.69, and 0.64) in the
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Scheme 1. Synthesis of 2-(b-maltooligooxy)-ethyl acrylates of various saccharide length x by CGTase catalyzed transglycosidation of a-CDand 2-(b-glucosyloxy)-ethyl acrylate and their subsequent polymerization using aqueous free radical polymerization.
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W. M. J. Kloosterman, G. Spoelstra-van Dijk, K. Loos
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reactionmixtures containingGlc-b-EA (Table2, row1and2)
and Glc-b-EMA (Table 2, lane 3 and 4). Both a-CD (Table 2,
lane 1 and 3) and soluble starch (Table 2, lane 2 and 4) were
found to be good glycosyl donors for the CGTase from B.macerans. Remarkably, no spots corresponding to trans-
glycosidation products were found using MEGA as the
acceptor (Table 2, lane5and6). Compared toGlc-b-EMA, the
molecular structure of MEGA differs only in two aspects.
Firstly, theaglyconpart ofMEGAcontains twoamidebonds
that make the molecule more polar than Glc-b-EMA. The
higher polarity of MEGA leads to different interactions in
Table 1. Glycosides used as acceptor in the CGTase catalyzed transg
Chemical structure
1-methacry
2-(b-glucos
2-(b-glucos
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the active site of CGTase, whichmight disturb the catalytic
mechanism. Secondly, the glycon part of MEGA cannot
adopt the pyranose conformation. It was found for CGTase
from B. circulans, that binding of the acceptor changes
the conformation of Tyr195 and induces structural
rearrangements that activate the enzyme in transfer of
the reaction intermediate to the acceptor.[21] It is likely
that the absence of a pyranose ring does not induce this
structural rearrangement necessary for transfer of the
acceptor to the enzyme-intermediate. The absence of
literature on transglycosidation of glucosides that are
lycosidation.
Name Refs.
lamido-2-D-gluconoylaminoethane (MEGA) [32]
yloxy)-ethyl methacrylate(Glc-b-EMA) [15]
yloxy)-ethyl acrylate (Glc-b-EA) [15]
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Table 2. TLC results of reaction mixtures containing Glc-b-EA, Glc-b-EMA, and MEGA as the glycosyl acceptors (Rf values given betweenbrackets).
Lane Donor Acceptor First transfer product Second transfer product Third transfer product
1 a-CD Glc-b-EA(0.78) Glc-a-Glc-b-EA(0.71) (Glc-a)2-Glc-b-EA(0.65) –
2 Starch Glc-b-EA(0.78) Glc-a-Glc-b-EA(0.71) (Glc-a)2-Glc-b-EA(0.65) –
3 a-CD Glc-b-EMA(0.82) Glc-a-Glc-b-EMA(0.75) (Glc-a)2-Glc-b-EMA(0.69) (Glc-a)3-Glc-b-EMA(0.64)
4 Starch Glc-b-EMA(0.82) Glc-a-Glc-b-EMA(0.75) (Glc-a)2-Glc-b-EMA(0.69) (Glc-a)3-Glc-b-EMA(0.64)
5 a-CD MEGA(0.69) – – –
6 Starch MEGA(0.69) – – –
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not in the pyranose form or similar derivatives supports
our idea that derivatives with a linear glucose chain or
aliphatic (poly)alcohols cannot be used as the acceptor
in CGTase from B. macerans catalyzed transglycosidation.
Nevertheless, thoroughly mapping of the effect of gluco-
sides that are not in the pyranose form on the structural
rearrangements in the enzyme needs to be performed to
verify these speculations.
The reaction product with Rf of 0.65, that was presumed
to be 2-(b-maltotriosyloxy)-ethyl acrylate ((Glc-a)2-Glc-b-
EA), was isolated from the reaction mixture by repeated
silica gel column chromatography. The purified product
was subjected to ESI-MS, 1H NMR, and 13C NMR spectrosco-
py. Even though the product was repeatedly purified, the
ESI-MS spectrum (Figure S2, Supporting Information) of
the isolated transglycosidation product showed four
peaks corresponding to molecular ion peaks (Table 3).
The signal of the transglycosidation product with Rf of
0.65, which was aimed for, was found to be the most
abundant. The found m/z values of the molecular ion
peaks correspond to sodium adducts of saccharide acrylate
monomers with respectively one, two, three or four
anhydroglucopyranose units.
The 1HNMRspectrumof the transglycosidationproducts
was compared to the spectrum of the starting glucoside
(Figure 2). Like the starting glucoside, the spectrum of
the transglycosidation product showed the characteristic
peaks of the double bond at 6.00, 6.23, and 6.46 ppm.
Table 3. ESI-MS results from the transglycosidation product withRf 0.65.
Saccharide acrylate
(sodium adducts)
Calculated
m/z
Found
m/z
Relative
abundance
NaþGlc-b-EA 301.0899 301.0899 22
NaþGlc-a-Glc-b-EA 463.1428 463.1427 20
Naþ (Glc-a)2-Glc-b-EA 625.1956 625.1957 100
Naþ (Glc-a)3-Glc-b-EA 787.2484 787.2491 7
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Similarly, the characteristic peak at 4.51 ppm of the
anomeric proton (Scheme 1, H1) of the starting glycoside
Glc-b-EA was found in the transglycosidation product.
The relatively large coupling constant (J¼ 7.9Hz) of this
proton was found to be equal in both the product and
the starting glycoside. Consequently, it could be concluded
that the anomeric configuration and the linkage to the
acrylate moiety remain unmodified during CGTase from
B. macerans catalyzed transglycosidation. On the other
hand, the proton spectrum of the transglycosidation
product showed two key differences compared to the
spectrum of the starting glycoside. First of all, a peak
at 5.39 ppm was found, that is known to correspond
to anomeric protons in a-(1,4)-linked maltodextrins.[35]
Secondly, a broad signal in the region 3.0–4.0 ppm was
observed, caused by the similar protons of multiple
glucopyranose rings. This indicates the presence of
multipleglucoseunits linkedbya-1,4-linkages. Theproduct
was found to be free of saccharidic side products since
reducing ends were detected in neither the 1H NMR
spectrum (5.20 and 4.67ppm, a and b, respectively) nor
Figure 2. 1H NMR spectrum of the enzymatically synthesized2-(b-maltooligooxy)-ethyl acrylates (top) from a-CD and2-(b-glucosyloxy)-ethyl acrylate (bottom spectrum). Protons arenumbered according to Scheme 1.
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Figure 3. HPLC chromatograms of the reaction mixture beforeincubation with CGTase from B. macerans (top) and afterincubation for 1 h (bottom). Numbers indicate the number ofglucopyranosyl units of the acrylic monomer (Glc-a)x-b-EA.
Figure 4. Effect of the donor to acceptor ratio on the size of themaltodextrin acrylate.
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the 13C NMR spectrum (92 and 96ppm, a and b,
respectively) of the purified product. Additionally, the
presence of glycosidic linkages in both a- and b-configura-
tion was confirmed by 13C NMR spectrometry (Figure S3,
Supporting Information). Two signals of anomeric carbons
of the glucopyranose units were found at 99.9 and
102.5 ppm. The signal at 102.5 ppm corresponds to the
b-linkage in the starting glycoside. The signals at 99.9 ppm
correspond to the a-linkages newly formed by the
CGTase. Thus, the isolated product consists of the starting
glycoside Glc-b-EA, extended with glucopyranose units
attached by a-1,4-glycosidic linkages (as represented in
Scheme 1). The b-linkages in the acceptor Glc-b-E(M)A
that was used in the transglycosidation reaction has two
big advantages: it is not hydrolysable by the enzyme
during the reaction and b) the acrylate moiety is always
positioned at the beginning of the maltodextrin chain
and thus, disproportionation reactions do lead to the
formation of monomers with various saccharide length
rather than saccharides with multiple acrylate groups.
The TLC results (Table 2 and Figure S1, Supporting
Information) discussed above showed only two spots of
enzyme catalyzed transglycosidation products (Rf 0.71 and
0.65). However, the ESI-MS spectrum already revealed that
maltodextrin-acrylates with more anhydroglucose units
were present in the reaction mixture (e.g., (Glc-a)3-Glc-b-
EA). The reaction mixture was therefore subjected to
HPLC analysis using an UV-detector, which has a number
of advantages over refractive index detection for this
specific CGTase catalyzed transglycosidation reaction. First
of all, saccharidic side products (i.e. glucose, maltose, etc.)
in the reaction mixture will not be detected, since
saccharides have no UV absorbance. On the other hand,
the transglycosidation products that contain an acrylate
moiety can be detected due to the double bond that does
absorb UV light. Secondly, the molar absorption coefficient
of all transglycosidation products ((maltooligooxy)-ethyl
acrylates) should be equal, because the double bond
responsible for absorbance does not change upon the
transglycosidation reaction. The molar concentration of
each saccharide-acrylate monomer and the conversion of
the startingglucosideGlc-b-EAcan thereforebedetermined
by integration of the peak areas. Before addition of the
biocatalyst, a peak corresponding to Glc-b-EAwas found at
6.7min (Figure 3, top chromatogram). After the enzymatic
reaction, up to fifteen new peaks were detected in the
reaction mixture (Figure 4, bottom chromatogram). The
new peaks can be assigned to 2-(b-maltooligooxy)-ethyl
acrylates (2-MOEA) with increasing polarity (because
an amino column was used). Since the polarity of the
monomers was mainly determined by the number of
glucose units, the number of glucose units of each
compound can be simply determined by counting the
number of peaks. A similar approach was used by
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Hiroyuki et al.[36] in CGTase catalyzed transglycosidation
of l-menthyl-a-glucopyranoside. We have found that
about 65 mol% of the initial amount of Glc-b-EA was
transferred into 2-MOEA after incubation of the reaction
mixture for 1.5 h. Longer incubation, to 120h, did not
further improve the conversion of the acceptor Glc-b-EA.
The concentration of the 2-MOEAwas found to decrease
upon the number of glucose units in the monomer.
This could be explained by the lower solubility of high
molecular weight 2-MOEA in aqueous MeOH (used for
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Biocatalytic Synthesis of Maltodextrin-Based Acrylates . . .
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preparation of the HPLC sample). This was supported by
the fact that the sum of all peak areas was only 65% of
the area of the starting glucoside and therefore about
35% of acrylic double bonds were lost by precipitation of
the high molecular weight saccharide-acrylates. Neverthe-
less, CGTase from B.macerans catalyzed transglycosidation
appears tobeanextremelyeffective tool for thesynthesisof
saccharide-acrylates with multiple anhydroglucose units
starting from the glucosyl-acrylate Glc-b-EA.
3.2. Conversion of the Acceptor Glc-b-EA toward
2-MOEA
It was reported by various authors that the distribution of
transglycosidation products can be influenced by the
donor:acceptor ratio.[23,37–39] In this research the molar
ratio a-CD: Glc-b-EA was varied between 1:3 and 2:1. The
CGTase catalyzed conversion of the starting glycoside
Glc-b-EA into 2-MOEA was measured by HPLC-UV.
The molar concentration of each maltodextrin acrylate
and the conversion were determined by integration of
the peak areas. The concentration of the major trans-
glycosidation products as a function of the molar ratio
between a-CD and Glc-b-EA was plotted in Figure 4. The
concentration of each transglycosidation product was
taken relative to the amount of the acceptor Glc-b-EA.
The conversion of the acceptor increases by increasing the
molar amount of a-CD. About 40% of the initial amount of
Glc-b-EA was not transferred to higher saccharide mono-
mers at a ratio of 1:2 (a-CD: Glc-b-EA). Increasing the
amount of a-CD to a ratio of 2:1 (a-CD:Glc-b-EA), reduces
the amount of not converted Glc-b-EA to only 15% of
the initial amount. However, further increasing the ratio
to 3:1 does not further increase the conversion of Glc-b-EA.
Svensson et al. obtained similar results in the CGTase
catalyzed synthesis of dodecyl-b-maltooctaoside from
a-CD. They found the reaction rate to level off with
increasing concentration of a-CD. Product inhibition
was the most likely explanation for this effect, but the
increased viscosity or complex formation between the
substrate and the acceptor can be reasons for the reduced
reaction rates as well.[38]
Secondly, the amount of the transglycosidation products
Glc-a-Glc-b-EAand (Glc-a)2-Glc-b-EAwas found todecrease
with increasing amount of donor substrate. The products
Glc-a-Glc-b-EA and (Glc-a)2-Glc-b-EA act as acceptors as
well, leading to the formation of higher maltodextrin
acrylates with seven or more glucose units (secondary
coupling). The increased availability of the donor substrate
clearly stimulates this secondary coupling reaction andas a
result, the amount of the products Glc-a-Glc-b-EA and (Glc-
a)2-Glc-b-EA decreases. Unfortunately, the products of this
secondary coupling reaction could not be quantitatively
determined by HPLC, because the higher molecular weight
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maltodextrin monomers precipitate upon preparation of
the samples.
3.3. CGTase Catalyzed Synthesis of Saccharide
Acrylates by Transglycosidation: a-CD Versus Starch
The coupling of Glc-b-EA and a-CD catalyzed by CGTase
from B.macerans aswe describe above,might be combined
with the known cyclization property of CGTases (the
formation of a-CD from starch). We tested the CGTase
catalyzed synthesis of 2-MOEA from starch and Glc-b-EA.
Reactionswithdifferent amounts of starchwereperformed
and compared to the results obtained when -CD was used
as the glycosyl donor (Table 4). The starch we used was
found to be a suitable glycosyl donor for CGTase catalyzed
transglycosidation, but only 2.6% of the acceptor could
be converted to 2-MOEA with a relatively low molecular
weight (sevenor lessanhydroglucoseunits). Similar toa-CD
as the donor, the conversion of Glc-b-EA was found to be
strongly depended on the ratio starch to Glc-b-EA. When
usinga starch toGlc-b-EA ratioof 7:1w:w, about49mol%of
the initial amount of Glc-b-EA did not react to 2-MOEA.
Secondly, 2-MOEA with more than seven glucose units
could not be detected in the reaction mixture at all. By
decreasing the donor to acceptor ratio (3:1), the Glc-b-EA
conversion was increased to 24 mol%. For synthesis of
2-MOEA with long saccharide residues, a low starch to
Glc-b-EA ratio should be used. Since the conversion of the
acceptor was lower at higher donor to acceptor ratio,
enzymatic reactions other than coupling to the acceptor
must be dominant. It was reported that the turn over
number (kcat) for the disproportionation reaction was the
highest for most CGTases.[23,24] The disproportionation
reaction was found to follow a ping-pong mechanism,
and thus the rate can be described by the following
equation:[24]
14, 14, 1
bH &
n ¼ Vmax�½D��½A�=ðKA
m�½D� þ KDm�½A� þ ½A��½D�Þ ð1Þ
with n is reaction rate; Vmax is maximal reaction rate; [D]
and [A] are the donor and acceptor concentration,
respectively; and KAm and KD
m are affinity constants of the
acceptor and donor, respectively.
According to this equation, both the reaction rate of
the desired transfer to Glc-b-EA and the undesired
disproportionation reaction increase upon increase of the
starch concentration. For the undesired disproportionation
reaction, both [D] and [A] increase linear upon increase
of the starch concentration. On the other hand, only [D]
increases for the desired transfer reaction. Therefore,
increasing the amount of soluble starch promotes the
undesired reaction more than the transfer reaction.
Evidently, the lower the starch:acceptor ratio, the better
the conversion to 2-MOEA.
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Table 4. CGTase catalyzed synthesis of 2-(b-maltooligooxy)-ethyl acrylates from a-CD or starch as donor and Glc-b-EA as acceptor,determined by HPLC-UV.
Retention
time [min]
Maltooligooxyethyl
acrylate
a-CD 7:1a)
[%]b)
[%]b)
Blankc)
[%]bStarch 3.5:1a) Starch 7:1a)
6.7 Glc-b-EA 18.4 20.2 49.2 98.7
11.4 Glc-a-Glc-b-EA 12.9 11.5 0.7
20.8 (Glc-a)2-Glc-b-EA 5.5 5.3 0.0
33.3 (Glc-a)3-Glc-b-EA 3.0 2.6 0.2
43.0 (Glc-a-)4Glc-b-EA 2.7 1.8 0.3
50.7 (Glc-a-)5Glc-b-EA 2.2 1.1 0.2
56.7 (Glc-a)6-Glc-b-EA 1.4 0.7 1.2
61.9 (Glc-a)7-Glc-b-EA 1.2 0.4
66.4 (Glc-a)8-Glc-b-EA 1.0 0.2
70.4 (Glc-a)9-Glc-b-EA 1.0 0.2
73.9 (Glc-a)10-Glc-b-EA 0.9 0.1
77.2 (Glc-a)11-Glc-b-EA 0.8 0.1
80.2 (Glc-a)12-Glc-b-EA 0.6 0.0
Total recovered aread) (mol%) 52 44 52 99
a)Ratio donor:acceptor (w:w); b)Concentration in mol%, relative to the initial acceptor concentration; c)Blank reaction was performed
with Glc-b-EA and the biocatalyst without donor substrate; d)Total recovered peak area gives the relative amount of initial double bonds
that are recovered after incubation at 60 8C for 1.5h.
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The results with the starch to Glc-b-EA ratio (3:1) were
comparable with the results using a-CD as the donor
substrate. The amount of unreacted acceptor was in both
cases about 20%. In total, 12 newly formed 2-MOEA were
detected and the conversion of Glc-b-EA to 2-MOEA was
about 29%. However, from a commercial point of view,
starch was much better available and therefore cheaper
than a-CD. This makes the CGTase from B. maceranscatalyzed transglycosidation of Glc-b-EA and starch an
economically attractive synthesis route toward new kinds
of 2-(b-maltooligooxy)-ethyl acrylates.
3.4. Polymerization of Enzymatically Synthesized
2-(b-maltooligooxy)-ethyl acrylates
The polymerization of the 2-MOEA synthesized by CGTase
from B. macerans, was performed by free radical polymeri-
zation (Scheme 1, bottom). Since the 2-MOEA monomers
were highly soluble in water, their polymerization could
be conducted in water, which contributes to an environ-
mentally friendly process.
As mentioned before, the separation and purification of
each individual maltodextrin-acrylate monomer was
extremely difficult due to: (i) the low concentration of
themonomers, (ii) the small difference in polarity between
the monomers, and (iii) the presence of saccharide side
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products that result from the CGTase catalyzed dis-
proportionation and hydrolysis reactions. Therefore,
the concentration of each saccharide-acrylate in the
reaction mixture before and after polymerization was
determined by HPLC using UV detection. The peak data
of fourteen 2-MOEA, before and after polymerization is
listed in Table 5.
Since the acrylic double bonds are responsible for UV
absorption, the polymerization of these acrylic bonds
results in a decrease of UV absorption. As shown in
Table 5, the sumof the peak areas after polymerizationwas
only 15% of the sum of the initial peak areas. Evidently,
the saccharide-acrylates were successfully polymerized
with an overall conversion of 85 mol%, even without
removal of the saccharidic side-products, which originate
from the enzymatic reaction. From the decreased peak
area of each individual saccharide-acrylate monomer, it
appears that the conversion of the smaller and more
abundant monomers Glc-b-EA and Glc-a-Glc-b-EA is
somewhat higher compared to the larger saccharide
monomers, like (Glc-a)13-Glc-b-EA. On the other hand, it
is clear that all saccharide-monomers participate in
the polymerization process and none of the saccharide
monomers is completely excluded. The separation of
the synthesized poly(2-(b-maltooligooxy)-ethyl acrylate)
(P(2-MOEA)) from the reaction mixture was challenging
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Table 5. Peak data of 2-MOEA before and after polymerization determined by HPLC-UV.
Monomer
Retention
time [min]
Peak area
[a.u.]
Peak area after
polymerization [a.u.]
Conversion
[%]
Fraction in
polymer
Glc-b-EA 6.73 5 239 323 489 279 90.7 0.375
Glc-a-Glc-b-EA 11.44 3 691 350 493 814 86.6 0.252
(Glc-a)2-Glc-b-EA 21.26 1 559 000 299 384 80.8 0.099
(Glc-a)3-Glc-b-EA 33.82 868 286 137 930 84.1 0.058
(Glc-a-)4Glc-b-EA 43.48 761 608 131 155 82.8 0.050
(Glc-a-)5Glc-b-EA 51.08 634 689 112 885 82.2 0.041
(Glc-a)6-Glc-b-EA 57.09 405 688 117 884 70.9 0.023
(Glc-a)7-Glc-b-EA 62.20 344 800 108 631 68.5 0.019
(Glc-a)8-Glc-b-EA 66.61 298 868 84 028 71.9 0.017
(Glc-a)9-Glc-b-EA 70.52 273 521 55 336 79.8 0.017
(Glc-a)10-Glc-b-EA 74.02 258 114 59 277 77.0 0.016
(Glc-a)11-Glc-b-EA 77.53 234 942 47 871 79.6 0.015
(Glc-a)12-Glc-b-EA 80.45 171 012 50 681 70.4 0.009
(Glc-a)13-Glc-b-EA 82.99 105 824 36 376 65.6 0.005
(Glc-a)14-Glc-b-EA 85.26 76 835 17 251 77.5 0.005
Overall conversion 14 923 860 2 241 782 85.0
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since the crude reaction mixture contains a mixture of
(oligo)saccharides, unreacted monomers and the polymer
P(2-MOEA).
Themost commonly used approach in polymer purifica-
tion is precipitation of the polymer in a non-solvent.
The polymer P(2-MOEA) was successfully precipitated in
methanol and the precipitate was analyzed with 13C NMR
(see Figure S4, Supporting Information). Using 13C NMR,
the saccharidic impurities in the product could easily be
detected by the signal corresponding to carbons of
the reducing ends at 92 (C1-a) and 96 (C1-b) ppm. The
precipitated polymer contained a mixture of polymer
P(2-MOEA) and a large quantity of maltodextrins. Dialysis
with a 25 kD membrane could not separate the polymer
P(2-MOEA) from the saccharidic impurities. These saccha-
ride side products with a molecular weight higher than
25 kD, should be removed from the reaction mixture
before polymerization of the 2-(b-maltooligooxy)-ethyl
acrylate monomers in order to obtain the pure polymer
P(2-MOEA).
Therefore, an alternative synthesis routewas developed.
In this second synthesis route the high molecular weight
saccharide side products resulting from the enzymatic
reaction were precipitated by addition of 50 vol% MeOH
before the free radical polymerization. Subsequently, the
reactionmixturewas polymerized by free radical polymer-
ization and the polymer was further purified by dialysis
using a 15 kD membrane. According to 1H NMR (Figure 5),
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the obtained polymer P(2-MOEA) is free of saccharidic
impurities.
3.5. Characterization of Poly(2-(b-maltooligooxy)-
ethyl acrylate)
The 1H NMR spectrum (Figure 5) of the pure polymer
P(2-MOEA) clearly shows the characteristic protons of the
CH2-CHR backbone at 1.4–2.6 ppm (see Scheme 1 for proton
numbering). We already showed that the 2-MOEA mono-
mers contain Glc-b-EA as the primary unit resulting of the
enzyme catalyzed transglycosidation reaction. Therefore,
the average number of glucopyranosyl units could be
determined from the ratio the C1-Hbsignal at 4.50 ppm
(Glc-b-EA), with the signal at 5.24 ppm (C1–Ha) correspond-
ing to a-(1,4)-linkages in the 2-MOEA monomers. The
integration of both peaks results in an a to a linkages ratio
of 1.7:1.0. Thus, the side chains of the polymer consist of on
average three anhydroglucose units, the first being Glc-b-
EA, the rest being a-1,4-anhydroglucopyranosyl units.
Similarly, the composition of the polymer P(2-MOEA)
was derived from the HPLC data (Table 5). The a to b ratio
canbe calculated from theHPLCdata if assumed that: a) the
detector response (extinctioncoefficient) foreachmonomer
is equal and b) the loss of double bonds (reduction in peak
area) can only be the result of polymerization. Taking the
fraction of each 2-MOEAmonomer in the polymer (Table 5),
last column, the average a to b ratio in a polymer chain can
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Figure 5. 1H NMR spectrum of poly(2-(b-maltooligooxy)-ethyl acrylate). The peak indicated by ‘‘br’’ corresponds to C1-protons of a-(1,6)-branches.
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be determined by the following equation:
a
b¼ ðf
¼P
Glc�a�Glc�b�EAþ 2f ðGlc�aÞ2�Glc�b�EAþ 3f ðGlc�aÞ3�Glc�b�EAþ . . .Þðf Glc�b�EA þ fGlc�a�Glc�b�EA þ f ðGlc�aÞ2�Glc�b�EA þ . . .Þ
xf ðGlc�aÞx�Glc�b�EA
1
with f ðGlc�aÞx�Glc�b�EA being the molar fraction of a residue
in the polymer
Using the HPLC data given in Table 5 and Equation 2, the
ratio a to b in P((Glc-a)x-Glc-b-EA) was calculated to be
2.15:1.0. This is in fair agreement with the polymer
composition derived with 1H NMR spectroscopy. Thus,
both the HPLC results and the 1HNMR spectrum prove that
the 2-MOEA monomers that were synthesized by CGTase
catalyzed transglycosidation of Glc-b-EA and starch, can
be polymerized successfully by aqueous free radical
polymerization.
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4. Conclusion
The CGTase from B. macerans catalyzed synthesis of
2-(b-maltooligooxy)-ethyl acrylate and methacrylate
monomers from starch and a-CD was demonstrated.
The aimed 2-(maltooligooxy)-ethyl acrylate and meth-
acrylate monomers could not directly be synthesized
by alcoholysis of the alcohols 2-HEA and 2-HEMA with
a-CD. Nevertheless, CGTase from B. macerans catalyzed
transglycosidation between a-CD and 2-(b-glucosyloxy)-
ethyl methacrylate (Glc-b-EMA) and 2-(b-glucosyloxy)-
ethyl acrylate (Glc-b-EA) results in novel maltooligo-
saccharide acrylate monomer. 1-Methacrylamido-2-D-
gluconoylaminoethane (MEGA) appeared to be unsuitable
as acceptor in the CGTase catalyzed transglycosidation
reaction. During the transglycosidation of Glc-b-EMA and
Glc-b-EA the b-linkage was preserved and the newly
attached glucopyranose units were linked by a-(1,4)-
glycosidic bonds. The b-linkages in the acceptors Glc-b-
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EA and Glc-b-EMA have two big advantages: (i) the b-
linkage is not hydrolysable by the enzyme, and (ii) the
acrylate moiety is always positioned at the beginning of
the a-(1,4)-glucan and thus, disproportionation reactions
do never lead to saccharide monomers with multiple
acrylate groups. Therefore, the biocatalytic pathway
results in unique 2-(b-maltooligooxy)-ethyl acrylatemono-
mers. At least fourteen 2-(b-maltooligooxy)-ethyl acrylates
with various length were detected by HPLC analysis of
the reaction mixtures. The acceptor conversion appeared
strongly depended on the ratio a-CD:Glc-b-EA. The conver-
sion was improved to 85 mol% by a donor:acceptor ratio
of 2:1w:w.
Alternatively to a-CD, starch can be used as the donor
substrate as well. The enzymatically synthesized 2-(b-
maltooligooxy)-ethyl acrylatemonomerswere successfully
polymerized by aqueous free radical polymerization.
The conversion of the monomers and the average
composition of the polymer could be easily derived from
HPLC analysis. A simple purification method is provided
to obtain P(2-(b-maltooligooxy)-ethyl acrylate) that is
free of saccharide side products.
We have shown an environmentally friendly synthesis
route toward a new kind of saccharide-acrylate monomers
using CGTase from B. macerans as the catalyst. The 2-(b-
maltooligooxy)-ethyl acrylates could be polymerized by
cheap and environmentally friendly free radical polymeri-
zation in water. This green synthesis approach yields a
novel type of saccharide-acrylate polymer that contains
pending saccharide chains with various lengths ranging
from 1 up to 15 glucopyranosyl units.
Acknowledgements: This research was financed by BASF, Lud-wigshafen, Germany. The authors kindly acknowledge TheodoraTiemersma-Wegman for her assistance with HPLC and ESI-MSanalysis.
Received: February 17, 2014; Revised: April 14, 2014; Publishedonline: May 26, 2014; DOI: 10.1002/mabi.201400091
Keywords: acrylates; biocatalytic; cyclodextrin; glucosyl transfer-ase; polyglucosides
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