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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: k.u.Loos@rug.nl

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:

DOI: 10.1002/mabi.201400091ary.com

Biocatalytic Synthesis of Maltodextrin-Based Acrylates . . .

www.mbs-journal.de

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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Biocatalytic Synthesis of Maltodextrin-Based Acrylates . . .

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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–

Macromol. Biosci. 20

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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

1272

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.

14, 14, 1268–1279

bH & Co. KGaA, Weinheim 1273

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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W. M. J. Kloosterman, G. Spoelstra-van Dijk, K. Loos

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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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� 2014 WILEY-VCH Verlag GmbH

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

4, 14, 1268–1279

& Co. KGaA, Weinheim www.MaterialsViews.com

Biocatalytic Synthesis of Maltodextrin-Based Acrylates . . .

www.mbs-journal.de

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.

268–1279

Co. KGaA, Weinheim 1275

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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W. M. J. Kloosterman, G. Spoelstra-van Dijk, K. Loos

1276

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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& Co. KGaA, Weinheim www.MaterialsViews.com

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

Biocatalytic Synthesis of Maltodextrin-Based Acrylates . . .

www.mbs-journal.de

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

14, 14, 1268–1279

bH & Co. KGaA, Weinheim 1277

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.

www.mbs-journal.de

W. M. J. Kloosterman, G. Spoelstra-van Dijk, K. Loos

1278

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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� 2014 WILEY-VCH Verlag GmbH

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-

4, 14, 1268–1279

& Co. KGaA, Weinheim www.MaterialsViews.com

Biocatalytic Synthesis of Maltodextrin-Based Acrylates . . .

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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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