Functionalized Celluloses with Regular Substitution Pattern by Glycosynthase-Catalyzed Polymerization

Article abstract of DOI:10.1021/acs.biomac.5b01453

Control of the monomer sequence in polymers is extraordinarily difficult by chemical synthesis, though Nature routinely exerts such control, including in the biosynthesis of polysaccharides. This inability has prevented us from being able to match the exquisite structure-activity control exhibited in biosynthesis of bioactive natural polysaccharides. We here address a powerful approach, whereby enzyme-catalyzed polymerization of properly modified building blocks is introduced as a simple route affording polysaccharides with controlled sequence and functionalization pattern. Targeting cellulose as a versatile scaffold for novel biomaterials, we describe the preparation of a perfectly alternating polysaccharide with repeat unit 6′-azido-6′-deoxycellobiose by a glycosynthase-catalyzed polymerization using the Humicola insolens cellulase Cel7B E197A mutant, and its further functionalization to give novel modified cellulose derivatives with a regular substitution pattern.

Full text of DOI:10.1021/acs.biomac.5b01453

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Article
Functionalized celluloses with regular substitution
pattern by glycosynthase-catalyzed polymerization
Victoria Codera, Kevin J. Edgar, Magda Faijes, and Antoni Planas
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01453 • Publication Date (Web): 16 Feb 2016
Downloaded from http://pubs.acs.org on February 16, 2016
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Functionalized celluloses with regular substitution
pattern by glycosynthase-catalyzed polymerization
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Victoria Codera†, Kevin J. Edgar‡, Magda Faijes†, Antoni Planas*,†
†
Laboratory of Biochemistry, Institut Químic de Sarrià, Universitat Ramon Llull, Via Augusta
390, 08017 Barcelona, Spain and ‡ Department of Sustainable Biomaterials, Macromolecules and
Interfaces Institute, and Institute for Critical Technologies and Applied Science, Virginia Tech,
Blacksburg, Virginia 24061, USA.
KEYWORDS: Cellulose • glycosynthase • functionalization • sequence control • regular pattern
• Humicola insolens cellulase
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ABSTRACT
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Control of the monomer sequence in polymers is extraordinarily difficult by chemical synthesis,
though Nature routinely exerts such control, including in the biosynthesis of polysaccharides.
This inability has prevented us from being able to match the exquisite structureꢀactivity control
exhibited in biosynthesis of bioactive natural polysaccharides. We here address a powerful
approach, whereby enzymeꢀcatalyzed polymerization of properly modified building blocks is
introduced as a simple route affording polysaccharides with controlled sequence and
functionalization pattern. Targeting cellulose as a versatile scaffold for novel biomaterials, we
describe the preparation of a perfectly alternating polysaccharide with repeat unit 6’ꢀazidoꢀ6’ꢀ
deoxycellobiose by a glycosynthaseꢀcatalyzed polymerization using the H. insolens cellulase
Cel7B E197A mutant, and its further functionalization to give novel modified cellulose
derivatives with a regular substitution pattern.
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Figure for the abstract
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1. INTRODUCTION
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Synthetic aminopolysaccharide mimetics of natural chitosans have attracted recent interest
in materials science to modify and improve the physicochemical and biological properties of
chitosanꢀbased biomaterials. Chitosan, a linear heteropolysaccharide of βꢀ1,4ꢀlinked Nꢀ
acetylglucosamine (GlcNAc) and glucosamine (GlcNH2) units, is obtained by chemical or
enzymatic partial deꢀNꢀacetylation of chitin, with variable degrees of acetylation and random
distribution of cationic glucosaminyl units along the polysaccharide chain.1 The cationic nature
of chitosans makes them versatile materials with a broad range of applications.2–6 They are
biocompatible and possess mucoadhesive, bacteriostatic, and hemostatic properties. The
positively charged chitosans are able to bind and encapsulate anionic compounds such as
proteins and DNA, and interact with negatively charged compounds found in mucus and on cell
surfaces, which improves the cell adhesion and proliferation properties of chitosanꢀbased
scaffolds. However, applications in drug and gene delivery systems suffer from low transfection
efficiency.7
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With the aim of mimicking and improving the biomedical properties of chitosans, different
procedures to modify neutral polysaccharides have been reported.8 Modifications of cellulose,
laminarin, curdlan, and other natural polysaccharides are performed by chemical procedures, i.e.
by functionalizing the Cꢀ6 positions with aminoꢀsubstituted side chains or azido groups than can
be reduced to amines or reacted by “Click” chemistry. The properties of these functionalized
polysaccharides will depend on their molecular weight and degree of substitution. However, they
are polydisperse with random distribution of the functional groups, requiring new methodologies
to produce defined and reproducible polymeric materials.
Sequence control is one of the great remaining problems in polysaccharide synthesis.
Nature creates polysaccharides often with a very high degree of control over sequence leading to
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highly specific biological activity, for example the active sequences of glycosaminoglycans
heparin9 and chondroitin sulfate10 that are vital to their interactions with proteins, governing their
ability to prevent thrombosis. Laborious, multiꢀstep, lowꢀyielding chemical syntheses have been
necessary to produce even oligosaccharides of controlled sequence.11
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Enzymatic synthesis opens up access to regular and homogeneous polysaccharides.
Glycosyltransferases are the natural enzymes catalyzing glycoside bond formation in the
biosynthesis of oligoꢀ and polysaccharides, but have limited applicability for preparative in vitro
synthesis due to the requirement of expensive glycosyl donors (i.e. sugar nucleotides) and
limited stability of these enzymes. Glycosidases, which catalyze the hydrolysis of oligo and
polysaccharides, have been demonstrated to be effective catalysts for carbohydrate synthesis
under medium engineered conditions and using activated glycosyl donors (kinetically controlled
transglycosylation). In this way, glycosidaseꢀcatalyzed polymerizations of simple glycosyl
donors are regioꢀ and stereospecific for the newlyꢀformed glycosidic bonds. Kobayashi
pioneered the application of cellulaseꢀcatalyzed polymerization of activated disaccharides,
preparing synthetic cellulose for the first time by polymerization of βꢀcellobiosyl fluoride in
acetonitrile/acetate buffer12 and 6ꢀOꢀmethylated celluloses by polymerization of the
corresponding donors.13 Few other polysaccharides (e.g. amylose, xylan, mixedꢀlinked βꢀ
glucans, alternating celluloseꢀchitin, and celluloseꢀxylan hybrids) have been prepared by
kineticallyꢀcontrolled transglycosylation using retaining glycosidases,14,15 with moderate yields
due to the intrinsic hydrolase activity of the enzymes. More recently, glycosynthases have
proved their efficiency for the enzymatic synthesis of oligosaccharides and glycoconjugates.
Glycosynthases are mutated retaining glycoside hydrolases in which the catalytic nucleophile has
been replaced by a smaller and inert residue.16,17 They are hydrolytically inactive but efficiently
catalyze glycoside bond formation when using glycosyl fluoride donors that have the opposite
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anomeric configuration to the normal substrate of the parental wildꢀtype enzyme.18–21 The
concept has been extended to glycosidases acting by substrate assisted catalysis (i.e.
hexosaminidases) using oxazoline donors with engineered hydrolytically inactive mutant
enzymes.19 The glycosynthase technology was expanded to the synthesis of polysaccharides by
promoting the donor selfꢀcondensation reaction.14 We reported the first nonꢀnatural
polysaccharide, an alternating mixedꢀlinked βꢀ1,3 and βꢀ1,4 glucan by polymerization of αꢀ
laminaribiosyl fluoride with a glycosynthase variant of a Bacillus 1,3ꢀ1,4ꢀβꢀglucanase.22 Other
polysaccharides such as cellulose,23 laminarin,24 xylans,25 1,3ꢀ1,4ꢀβꢀglucans,26 and branched
xyloglucans27,28 have also been successfully achieved. Interestingly, Fort et al.23 explored the
ability of the glycosynthase derived from the cellulase Cel7B from Humicola insolens (HiCel7B
E197A) to accept different functionalized cellobiosyl donors. The enzyme was able to accept αꢀ
cellobiosyl fluorides that had functionalities at Cꢀ6’ position such as bromine, amino and
thioglucosyl groups. This particular glycosynthase offers a broad range of synthetic applications
since it has also been employed for the synthesis of xyloglucan oligosaccharides.27
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We envision the glycosynthase technology as a potential tool for the efficient synthesis of
functionalized polysaccharides: simple glycosyl donors prepared with the specific modifications
can be accepted and polymerized by the glycosynthase enzyme to produce modified
polysaccharides. This glycosynthase approach could provide a new generation of
polysaccharides with controlled functionalization patterns and polymerization degrees different
from those obtained by chemical modification routes. We here report the preparation of a
perfectly alternating polysaccharide with repeat 6’ꢀazidoꢀ6’ꢀdeoxycellobiose by a glycosynthase
catalyzed polymerization of a 6’ꢀazidoꢀcellobiosyl donor using the cellulase HiCel7B E197A
mutant, and its further functionalization to give modified cellulose derivatives with a regular
substitution pattern.
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2. EXPERIMENTAL SECTION
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2.1. Structural characterization. H and C NMR, COSY, TOCSY, and HSQC spectra were
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recorded on a Varian Gemini 400 MHz spectrometer operated at 298 K. For H NMR, the TMS
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peak (0 ppm) was used as reference. When using MeOD as solvent, the central peak of the
MeOD multiplet (3.31 ppm) was used as a reference. For 13C NMR, the central peak of the
CDCl3 triplet (77.16 ppm) was used as a reference. When using MeOD as solvent, the central
peak of the MeOD multiplet (49.00 ppm) was used as a reference. Synthetic cellulose and
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functionalized cellulose C NMR spectra were obtained on Varian INOVA and Varian UNITY
400 MHz spectrometers with a minimum of 5,000 scans in CDCl3. Chemical shifts are reported
relative to the solvent peaks.
A Thermo Electron Nicolet 8700 FTIR was used to perform infrared spectroscopy analyses
of the samples as pressed KBr pellets. All samples were dried in a vacuum oven prior to analysis
to avoid moisture. In situ FTIR spectra were obtained using a Mettler Toledo ReactIR 45M with
a SiComp fiber optic ATR probe.
HPSEC analyses to determine molecular mass profiles were performed on an Agilent 1100
HPLC system equipped with a refractive index detector using a PSS Gram column (8.0 x 300
mm, 100Å, 10 ꢁm) and a PSS Gram preꢀcolumn (9.0 x 50 mm, 100 Å, 10 ꢁm) maintained at
50ºC, and DMSO with lithium bromide (5 g/L) as eluent at a flow rate of 0.5 mL/min. The
calibration curve was obtained with dextrans as standards (American Polymer Standards
Corporation DXT1 – DXT55 kDa). Freeze dried polymers and standards were dissolved in
DMSO and filtered. From the chromatograms, Mp (molecular mass of the peak maximum), Mw
(weight average molecular mass), Mn (number average molecular mass), DP (degree of
polymerization), and PDI (polydispersity index) were calculated.29
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MALDIꢀTOF spectra were obtained in a Bruker Daltonics spectrometer with 2,5ꢀ
dihydroxybenzoic acid as matrix. Reflectron, positive ion mode, 19 kV acceleration voltage, and
20 kV reflector voltage were used.
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For SEM experiments, the dried product was fixed on a graphite tape, coated with
gold/palladium by ionꢀsputtering, and observed at an accelerating voltage of 10 kV and working
distance of 16 mm using a JEOL JSMꢀ5310 microscope.
Fluorescent polysaccharides were observed with a Stereoscopic microscope Nikon Eclipse
TE 2000ꢀU using a Piston GFP filter, excitation 450ꢀ490 nm, emission: 500ꢀ530 nm, and images
were acquired with a cooled CCD camera.
2.2. Synthesis of donor and acceptor substrates. αꢀCellobiosyl fluoride (CelαF) (1) and 6’ꢀ
azidoꢀαꢀcellobiosyl fluoride (6’ꢀN3ꢀCelαF) (2) were prepared as reported in Fort et al.1 4ꢀ
NitrophenylꢀβꢀDꢀglucopyranoside (GlcpNP) (3) was from Sigma.
4ꢀNitrophenyl 6ꢀazidoꢀ6ꢀdeoxyꢀβꢀDꢀglucopyranoside (6ꢀN3ꢀGlcpNP) (4) was prepared by
tosylation of the 6ꢀOH group of 3, acetylation with Ac2O/py/DMAP, substitution of the tosyl
group by azide, and finally deꢀOꢀacetylation by reaction with sodium methoxide in methanol, as
follows:
4ꢀNitrophenylꢀβꢀDꢀglucopyranoside (3) (2.59 g, 8.59 mmol) was dissolved in dry pyridine
(26 mL) and the solution was cooled in an ice bath. A cold solution of tosyl chloride (4.91 g,
25.78 mmol) in dry pyridine (26 mL) was added and the reaction mixture was stirred for 22
hours under nitrogen atmosphere at room temperature. Acetic anhydride (52 mL) and N,Nꢀ
dimethylaminopyridine (DMAP) (25 mg) were directly added to the solution. The mixture was
stirred for 24 hours at room temperature. Finally, methanol (100 mL) was added to quench the
reaction and, after evaporation until dryness, the product was redissolved with ethyl acetate,
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washed with water, the organic phase dried, and the solvent evaporated under reduced pressure
to yield 9.17 g of 4ꢀnitrophenyl 6ꢀOꢀtosylꢀ2,3,4ꢀtriꢀOꢀacetylꢀβꢀDꢀglucopyranoside (compound I).
Compound I (9.17 g, 15.77 mmol) was dissolved in dry DMF (183 mL). Sodium azide (2.23 g,
34.37 mmol) was added and the solution stirred at 80 ºC for 14 hours. A saturated aqueous
solution of sodium bicarbonate and ethyl acetate was added, and the organic phase was washed
with water, dried, and the solvent evaporated. The product was purified by flash chromatography
(tolueneꢀethyl acetate 20:1). 4ꢀNitrophenyl 6ꢀazidoꢀ2,3,4ꢀtriꢀOꢀacetylꢀβꢀDꢀglucopyranoside
(compound II) (1.94 g, 4.31 mmol) was obtained in 50% overall yield from 3. 13C NMR
(CDCl3, 100 MHz) δ 170.2, 169.6, 169.3 (CO), 126.0, 116.8 (CꢀAr), 98.1 (Cꢀ1), 74.0 (Cꢀ5), 72.4,
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71.0, 69.3 (Cꢀ2, Cꢀ3, Cꢀ4), 51.3 (Cꢀ6), 20.7 (CH3). Η ΝΜR (CDCl3, 400 MHz) δ 8.23 (d, 2H,
JAr = 9.1 Hz, arom), 7.10 (d, 2H, JAr = 9.1 Hz, arom), 5.33 – 5.30 (m, 2H, H2, H3), 5.24 (d, 1H,
J1ꢀ2 = 7.9 Hz, H1), 5.09 (m, 1H, H4), 3.85 (m, 1H, H5), 3.44 (dd, 1H, J5,6b = 7.4 Hz, J6a,6b = 13.4
Hz, Hꢀ6b), 3.35 (dd, 1H, J5,6a = 2.7 Hz, J6a,6b = 13.4 Hꢀ6a), 2.07ꢀ2.05 (m, 9H, CH3). ESIꢀMS
(m/z): 470.1514 [M+NH4]+. Compound II (200 mg, 0.44 mmol) was dissolved in methanol (7
mL) and sodium methoxide (1 mM in methanol). The mixture was stirred for 6 hours at room
temperature. Then, the mixture was neutralized with Amberlite IR 120 resin (H+), filtrated and
freezeꢀdried. 4ꢀNitrophenyl 6ꢀazidoꢀ6ꢀdeoxyꢀβꢀDꢀglucopyranoside (4) was obtained in 98%
yield.
2.3. Cel7B E197A protein. The Humicola insolens Cel7B E197A enzyme was kindly provided
by Novozymes (Denmark). The protein was >95% homogeneous as judged by SDSꢀPAGE.
Concentration was determined by UV spectrophotometry (ε280= 6.68 x 105 Mꢀ1·cmꢀ1).
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2.4. Kinetics of donor-acceptor condensations catalyzed by the HiCel7B E197A
glycosynthase. Reactions were done in 96ꢀwell microplates. Donor and acceptor substrates in
100 mM phosphate pH 7.8 were preꢀincubated at 35ºC for 5 min, then the reactions were
initiated by addition of the HiCel7B E197A enzyme, and kept at 35 °C (final reaction volume of
0.3 mL). Aliquots were withdrawn at regular time intervals, diluted 1:10 with formic acid 2%
(v/v) to stop the enzymatic reaction, and analyzed by HPLC (Agilent HPLC equipment,
NovaPak C18 (4 µm, 3.9 × 150 mm) column (Waters), flow rate 1 mL/min, 8.5% MeOH in
water when using cellobiosyl fluoride donor (1) and 12% MeOH in water for 6’ꢀazidoꢀ
cellobiosyl fluoride donor (2), UV detector at 300 nm). Initial rates (vo) were obtained from the
linear progress curves of product formation (normalized area vs. time) at initial reaction times (<
10% conversion) and expressed as vo/[E].
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For the determination of specific activities, reactions were done at 1 mM donor (1 or 2), 7
mM acceptor (3 or 4), and 0.83 µM enzyme. For determination of kinetic parameters, reactions
were done at constant acceptor (3) concentration of 20 mM, varying donor (1 or 2)
concentrations (0.025 to 2 mM), and 0.1 or 0.5 µM enzyme for reactions with donor 1 or 2,
respectively. Kinetic parameters kcat, KM, and kcat/KM were calculated by nonlinear fitting of v0
vs. donor concentration data to the Michaelis Menten model or substrate inhibition model.
2.5. Glycosynthase-catalyzed polymerizations. Reaction mixtures (0.5 mL) consisting of 30 ꢀ
50 mg donor substrate (1 or 2), phosphate buffer (100 mM, pH = 7.8) and HiCel7B E197A
enzyme (4.5 ꢀ 56 µM, concentrations as indicated) were incubated at 40ºC and 250 rpm. A
precipitate was formed during the reaction and no donor was observed by TLC (ACN/H2O 8:2)
after 24 hours. The precipitated product was isolated by centrifugation at 13,000 rpm for 3
minutes, and the precipitate was thoroughly washed with cold water. Finally, the product was
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freezeꢀdried to yield waterꢀinsoluble polymers as white powders. Supernatants were also
lyophilized to recover soluble oligomers. The products were analyzed by HPSEC to determine
the polymer parameters (Mw, Mn, Mp, DP, PDI).
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Synthetic cellulose (5).- Compound 1 (30 mg, 0.087 mmol) was dissolved in phosphate buffer
(0.1M, pH 7.8, 0.5 mL) and incubated with HiCel7B E197A enzyme (56 µM) at 40 °C. After 24
h, the isolated waterꢀinsoluble polymer was freezeꢀdried obtaining a white powder (29 mg, 92%
yield).
Alternating 6-azido-6-deoxycellulose (6).- Compound 2 (30 mg, 0.081 mmol) was dissolved in
phosphate buffer (0.1M, pH 7.8, 0.5 mL) and incubated with HiCel7B E197A enzyme (56 µM)
at 40 °C. After 24 h the isolated waterꢀinsoluble polymer was freezeꢀdried obtaining a white
powder (26 mg, 82% yield). FTIR: 3431 cmꢀ1 (OH), 2112 cmꢀ1 (N3).
Acetylated alternating 6-azido-6-deoxycellulose (7).ꢀ Polymer 6 (26 mg) was mixed with
pyridine (118 µL, 20 equiv), acetic anhydride (138 µL, 20 equiv) and DMAP (1 mg). The
reaction solution was stirred for 24 h at 80 ºC. The product was isolated by pouring the reaction
mixture into 2 mL deionized water, followed by filtration. The precipitate was dried overnight in
a vacuum oven at 50 °C. DS = 2.1 determined by 1H NMR spectroscopy. FTIR: 3439 cmꢀ1 (OH),
2109 cmꢀ1 (N3), 1757 cmꢀ1 (C=O). 1Η ΝΜR (DMSO, 400 MHz) δ 5.11ꢀ5.06 (m, 2H, Hꢀ3, Hꢀ
3’), 4.73 (d, 1H, Hꢀ1’), 4.54 (d, 1H, Hꢀ1), 4.56ꢀ4.52 (m, 2H, Hꢀ2, Hꢀ2’), 4.24 (d, 1H, J6a,6b =
11.1 Hz, Hꢀ6a), 4.05ꢀ3.99 (m, 1H, Hꢀ6b), 3.83ꢀ3.74 (m, 2H, Hꢀ5, Hꢀ5’), 3.74ꢀ3.70 (m, 1H, Hꢀ4’),
3.65ꢀ3.60 (m, 1H, Hꢀ4), 3.54 (d, 1H, J6’a,6’b = 12 Hz, Hꢀ6’a), 3.49ꢀ3.39 (m, 1H, Hꢀ6’b), 2.07ꢀ
1.86 (m, 15H, CH3).
13C NMR (DMSO, 100 MHz) δ 170.59 – 169.25 (C=O), 99.62 (Cꢀ1’), 99.27 (Cꢀ1), 76.84 (Cꢀ4),
75.73 (Cꢀ4’), 73.29 (Cꢀ5), 72.64 (Cꢀ3, Cꢀ3’), 72.17 (Cꢀ5’), 71.96 (Cꢀ2, Cꢀ2’), 62.61 (Cꢀ6), 50.37
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(Cꢀ6’), 20.95 – 20.53 (CH3). HSQC, COSY, and TOCSY experiments are given in Supporting
Information.
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2.6. Functionalization of polysaccharides 6 and 7.
Conjugated alternating 6-azido-6-deoxycellulose with Alexa Fluor® 488 alkyne (8).ꢀ A solution
of Alexa Fluor® 488 alkyne (Life Technologies) (2.0 mg, 2.56 µmol) in water (1 mL) and
freshly prepared solution of sodium Lꢀascorbate (1M, 20 mL, SigmaꢀAldrich) were added to a
solution of polymer 6 (1.0 mg, 2.6 µmol/anhydrous cellobiose unit) in DMSO (1 mL). Then,
7.5% aqueous cupric sulfate (24 µL, 7.21 µmol) was added to the reaction mixture, which was
stirred overnight at 24ºC. The unreacted Alexa Fluor reagent was removed by dialysis (PurꢀAꢀ
LyzerTM Mega 1000 Dialysis Kit, SigmaꢀAldrich) against water. The functionalized polymer 8
(1.0 mg, 1.07 µmol) was obtained after lyophilization in 41 % yield. FTIR: 3431 cmꢀ1 (OH,
NH2), 1763 cmꢀ1 (C=O).
Acetylated alternating 6-aminocellulose (9).ꢀ Polymer 7 (20 mg) was dissolved in 2 mL of THF,
followed by the dropwise addition of 13 µL of deionized water. Then, Ph3P (2 eq. per acetylated
glucosyl unit) was added, and the solution allowed to react at room temperature for 12h in a
sealed flask. The solution was transferred to a 3500 MWCO dialysis tubing that was placed in a
beaker containing 600 mL ethanol. As the dialysis proceeded, a precipitate was slowly formed.
After one day of dialysis, the contents inside the tubing were removed, and the precipitate was
isolated by filtration. The precipitate (polymer 9) was dried in a vacuum oven at 50 °C. FTIR:
3431 cmꢀ1 (OH, NH2), 1763 cmꢀ1 (C=O). 13C NMR (TDF, 100 MHz) δ: 171.62 – 170.35 (C=O),
100.26, 100.25 (Cꢀ1, Cꢀ1’), 64.15 (Cꢀ6), 43.25 (Cꢀ6’), 21.74 – 20.37 (CH3).
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3. RESULTS AND DISCUSSION
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3.1. Kinetics of HiCel7B E197A glycosynthase with azido substrates.
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To evaluate the ability of the HiCel7B E197A glycosynthase to accept azidoꢀsubstituted
substrates, different donorꢀacceptor pairs were kinetically analyzed (Figure 1). With CelαF (1) as
donor substrate, specific activities with acceptors GlcpNP (3) and 6N3ꢀGlcpNP (4) were
essentially the same, indicating that the azido group can be accommodated in subsite +1 of the
enzyme’s binding cleft. With 6’N3ꢀCelαF (2) donor and GlcpNP (3) acceptor substrates, activity
was just 2ꢀfold lower than the reference 1+3 reaction, indicating that subsite ꢀ2 also tolerates an
azido substitution. Kinetics with different donor concentrations at saturating GlcpNP acceptor
concentration are plotted in Figure 2, and kinetic parameters given in Table 1. CelαF (1) follows
saturation kinetics with kcat of 17 minꢀ1, whereas 6’N3ꢀCelαF (2) has the same kcat but shows
substrate inhibition at high donor concentration. Therefore, subsites ꢀ2 and +1 accept the 6ꢀazido
substitution, a requirement to evaluate donor selfꢀcondensation to yield alternating 6ꢀazido
polymers.
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3.2. Glycosynthase-catalyzed polymerizations.
Glycosynthaseꢀcatalyzed polymerizations were performed at high donor concentration, pH
7.8 and 40 ºC (Figure 3, Table 2). Polymeric products precipitated as a white powder after 24 h
reactions. TLC analysis showed that the polymerizations proceeded quantitatively and neither
fluoride donors nor their hydrolysis products were detected. The waterꢀinsoluble polymers were
isolated in high yield (> 90%) after centrifugation, water washes, and lyophilization, and then
structurally characterized.
Analysis of the degree of polymerization and molecular mass distribution of the insoluble
polysaccharides by HPSEC showed that cellulose (5) (Table 2) presents a monomodal profile
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with a Mw value of 4.3 kDa which corresponds to (4Glcβ4Glcβ)n, n = 13, and the alternating 6ꢀ
azidoꢀ6ꢀdeoxycellulose (6) has a Mw value of 5.8 kDa ((4[6N3Glc]β4Glcβ)n, n = 17) (Figure 4).
Similar results were obtained after acetylation of the 6ꢀazido polysaccharide with Ac2O and
pyridine (polymer 7). MALDIꢀTOF MS shows a series of peaks up to 5.5 kDa corresponding to
the low molecular mass oligomers, peaks separated by ∆m/z of 324 and 349 for polymer 5 and 6,
respectively, which correspond to the theoretical mass of the repeating (Glcβ4Glc) unit for
polymer 5 and the (6N3Glcβ4Glc) unit for polymer 6, and ∆m/z of 560 for the acetylated
polymer 7 Figure S1, Supporting Information). Analysis of the acetylated polymer 7 by 1H NMR
confirmed the presence of the azide after acetylation with a degree of acetylation (DA) of 2.2
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(maximum theoretical DA = 2.56) (Figure 5A). C NMR (Figure 5B) showed the formation of
the new βꢀ1,4 glycosidic linkage (δC 76 ppm) and disappearance of the signal at δC 69 ppm
assignable to the unsubstituted C4 of the nonꢀreducing end of the cellobiosyl donor. The signal at
δC 62 ppm corresponds to the acetylated C6 positions while the signal at δC 51 ppm was assigned
to the azidated Cꢀ6’ positions consistent with the presence of azide groups on alternating
glucosyl units. 2D NMR experiments (COSY, TOCSY, and HSQC) allowed the assignment of
the two different spin systems for 6ꢀazidoꢀglucosyl and glucosyl units (Figure S2, Supporting
Information). The FTIR spectrum of the alternating 6ꢀazidoꢀ6ꢀdeoxycellulose (6) (Figure 6A)
shows an intense stretching band of the azido group at 2112 cmꢀ1, and the absorption band of the
sugar OH groups at 3431 cmꢀ1, whereas the acetylated polymer 7 (Figure 6B) retains the azide
band at 2112 cmꢀ1, a new band at 1753 cmꢀ1 for the C=O of the acetyl groups, but still contains a
small absorption band at 3431 cmꢀ1 due to free hydroxyl groups resulting from the incomplete
acetylation reaction.
Polysaccharide morphology was analyzed by SEM revealing that the 6ꢀazidoꢀ6ꢀ
deoxycellulose (6) formed porous spherulites like the nonꢀmodified cellulose (5), although they
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seem to be larger, with 5ꢀ7 µm versus 2ꢀ4 µm average diameter, respectively (Figure 7). These
artificial polysaccharides notably differ from natural cellulose such as MCC (microcrystalline
cellulose), whose particles of 10ꢀ50 µm are composed of aggregate bundles of multiꢀsized
cellulose microfibrils.30 The spherulite morphology with variable porosity seems to be common
for artificial polysaccharides produced by in vitro enzymatic syntheses, as previously seen in the
first artificial cellulose obtained by transglycosylation with T. viridiae cellulase31 or alternating
βꢀ1,3/1,4 glucan obtained by a glycosynthaseꢀcatalyzed polymerization.22,26
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Substituted celluloses at C6 have been achieved by chemical modification of MCC, where
PPh3/NBS bromination of cellulose is completely selective at C6,32 but further SN2 displacement
of the bromide is limited due to the poor solubility of the 6ꢀbromoꢀ6ꢀdeoxycellulose. Edgar and
coworkers33 showed that esterification of brominated cellulose broadens the potential for further
modifications. In this way, 6ꢀazidoꢀ6ꢀdeoxyꢀ2,3ꢀdiꢀOꢀacylꢀcellulose was obtained from MCC
resulting in a polysaccharide of DP 50 and DS (azide) of 0.92.34 The enzymatic protocol here
reported provides access to novel structures, where azide substitutions are regularly distributed at
C6 of alternating glucosyl units on a cellulose chain with free hydroxyl groups, with DP of about
40 and DS (azide) of 0.5. It is notable that nature employs this very architectural motif of amine
substitution on alternating monosaccharides in all of the active glycosaminoglycans. By
extension, the same strategy with homologous azidoꢀcellooligosaccharide donors (i.e. 6’’ꢀazidoꢀ
cellotriosyl and 6’’’ꢀazidoꢀcellotetraosyl fluoride donors) should afford other regularly
functionalized celluloses with different substitution patterns.
3.3. Functionalization of polysaccharides 6 and 7
The azido cellulose 6 and the acetylated derivative 7 are reactive intermediates for further
modifications (Figure 3). The accessibility and reactivity of the 6ꢀazido groups was first tested
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by “click” chemistry.35 The alternating 6ꢀazidoꢀ6ꢀdeoxycellulose (6) was subjected to a copperꢀ
catalyzed azide/alkyne cycloaddition (Huisgen reaction)36 with the alkyneꢀfunctionalized Alexa
Fluor dye. After isolation of the water soluble product (8) by dialysis and lyophilization, FTIR
spectroscopy (Figure 6C) showed the appearance of a new absorption band at 1621 cmꢀ1
corresponding to the carbonyl group of the Alexa Fluor amide function, and the disappearance of
the stretching band of the azide groups at 2112 cmꢀ1, although some residual band was apparent
due to incomplete reaction. The dyeꢀlabeled polymer 8 was observed as a green fluorescent solid
by fluorescence microscopy. Next, reduction of the azido groups to amines was studied.
Staudinger reduction has been recently applied to acylated 6ꢀazidoꢀ6ꢀdeoxycelluloses with DS
(azido) of 0.9ꢀ0.95 to selectively reduce the azido groups to amines in the presence of easily
reducible ester groups.34 The same protocol was here applied to reduce the acetylated alternating
6ꢀazido polymer 7 with the aim of obtaining a novel 6ꢀaminoꢀ6ꢀdeoxy cellulose derivative with a
regular pattern of one amino group every two glucosyl units in the polysaccharide chain. The
reaction was performed by reacting the acetylated polymer 7 dissolved in THF with PPh3 to form
a phosphazide which evolves to an iminophosphorane. It is then hydrolyzed by water to produce
the polymer 9 and triphenylphosphine oxide. Unfortunately, this new polysaccharide has poor
solubility in water and common organic solvents, as did the reported acylated 6ꢀaminocelluloses
obtained from MCC.34 Due to the solubility issues, the reaction was performed in deuterated
THF and the 13C NMR spectrum of the crude reaction mixture was directly recorded (Figure 5C).
The spectrum showed the formation of the amine (δC 43 ppm assigned to C6’ꢀNH2). FTIR
analysis confirms the presence of the free amine (broad N−H stretching band at 3390 cm−1 and
the N−H bend at 1570 cm−1) but shows a residual signal of the azide at 2112 cmꢀ1, while the
carbonyl signal of the acetyl groups remains at 1753 cmꢀ1 (Figure 6D).
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4. CONCLUSIONS
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As opposed to most chemically modified celluloses where the substitution pattern is
intrinsically random, the glycosynthaseꢀcatalyzed polymerization of properly modified glycosyl
donors gives access to novel functionalized celluloses with defined and regular substitution
patterns. We have shown that a 6’ꢀazidoꢀcellobiosyl donor is readily polymerized to give
alternating 6ꢀazidoꢀ6ꢀdeoxycellulose with a regular pattern of one azido group every two
glucosyl units, which can be further reacted to install different functional groups or substituents.
The strategy can be extended to other functionalized donors, i.e. triꢀ or tetrasaccharide donors, to
afford other regularly functionalized glycopolymers with different substitution patterns.
Likewise, other functional groups may be introduced in the glycosyl donor (i.e. –COOH, ꢀ
CONH2, ꢀSH) provided that the donor is accepted as a substrate by the glycosynthase, which
otherwise can be engineered for the desired specificity by protein engineering and directed
evolution approaches. This strategy provides access to novel polysaccharide structures with
promising applications.
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AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: antoni.planas@iqs.edu
Notes
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The authors declare no competing financial interest
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
ACKNOWLEDGMENTS. This work was supported by Grant BIO2013ꢀ49022ꢀC2ꢀ1ꢀR from
MINECO, Spain (to AP). KJE thanks the USDA for financial support (grant 2011ꢀ67009ꢀ
20090). The HiCel7B E197A enzyme was kindly provided by Novozymes (Denmark). V.C.
acknowledges a starting predoctoral grant from Obra Social La Caixa, and a predoctoral
fellowship from the Institut Químic de Sarrià. We acknowledge Shu Liu (Department of
Chemistry, Virginia Tech), and Dr. Xavier Batllori (Department of Organic Chemistry, Institut
Químic de Sarrià) for the NMR spectra.
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Table 1. Kinetic parameters of HiCel7B E197A glycosynthase for donors CelαF (1) and 6’ꢀN3ꢀ
CelαF (2) at saturating (20 mM) GlcpNP (3) acceptor in 100 mM phosphate buffer pH 7.8, 35ºC.
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Donor
kcat
KM
KI
kcat/KM
(minꢀ1)
(mM)
(mM)
(mMꢀ1minꢀ1)
17.1 ± 0.9 0.10 ± 0.03
16.4 ± 2.4 0.15 ± 0.04 1.5 ± 0.5
ꢀꢀ
173
113
1
2
Table 2. Enzymatic polymerizations by the HiCel7B E197A glycosynthase. Polysaccharide
parameters determined by HPSEC.
[donor] yielda Mw
Mn
(kDa)
Mp
(kDa)
b
d
f
P donor
n (DP)c
PDIe
(mM)
174
162
162
(%) (kDa)
92
90
88
4.3 13 (26) 2.9 1.5 3.7
5.8 17 (34) 4.9 1.2 4.7
9.0 16 (32)
5
6
1
2
2
7 g
7
1.2
8
a
b
Conditions: [enzyme]= 56 µM, 100 mM phosphate buffer, pH 7.8, 40ºC. Average yield from 4 repetitions; Mw,
weight average molecular mass; c n, number of condensations, (DP) degree of polymerization for Mw. d Mn, number
e
f
average molecular mass. PDI, polydispersity index; Mp, molecular mass of the peak maximum in the
chromatograms. g Glycosynthase reaction followed by acetylation, parameters of the acetylated polymer.
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FIGURE LEGENDS
Figure 1. Donorꢀacceptor condensations catalyzed by the HiCel7B E197A glycosynthase.
1
8
9
Specific activities (s.a.) at 1 mM donor (1 or 2), 7 mM acceptor (3 or 4), 0.83 µM enzyme in 100
10
11
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47
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49
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51
52
53
54
55
56
57
58
59
60
mM phosphate buffer pH 7.8, 35ºC.
Figure 2. Kinetics of HiCel7B E197A glycosynthase for donors CelαF (1) and 6’ꢀN3ꢀCelαF (2)
at saturating (20 mM) GlcpNP (3) acceptor in 100 mM phosphate buffer pH 7.8, 35ºC.
Figure 3. Glycosynthaseꢀcatalyzed polymerizations of cellobiosyl donors 1 and 2, and further
derivatization of the alternating 6ꢀazidoꢀ6ꢀdeoxycellulose polymers.
Figure 4. Size exclusion chromatography (HPSEC) of artificial polysaccharides cellulose (5) and
6ꢀazido 6ꢀdeoxycellulose (6).
Figure 5. NMR spectra of acetylated alternating 6ꢀazidoꢀ6ꢀdeoxycellulose (7) and acetylated
alternating 6ꢀaminoꢀ6ꢀdeoxycellulose (9). (A) 1H NMR (CDCl3) of 7. (B) 13C NMR (CDCl3) of
7. (C) 13C NMR (TDF) of 9. Monodimensional and 2D spectra (COSY, TOCSY, and HSQC) of
7 in DMSO are given in Figure S2, Supporting Information, and assignments of proton and
carbon chemical shifts are detailed in the Experimental Section.
Figure 6. FTIR spectra of alternating 6ꢀazido 6ꢀdeoxycellulose and their derivatives. A) 6ꢀAzido
6ꢀdeoxycellulose (6). B) Acetylated 6ꢀazido 6ꢀdeoxycellulose (7). C) Conjugated 6ꢀazido 6ꢀ
deoxycellulose with Alexa Fluor (8). D) Acetylated 6ꢀamino 6ꢀdeoxycellulose (9).
Figure 7. SEM of the polysaccharides 5 (left) and 6 (right).
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FIGURE 1
5
6
7
8
9
F
R1
O
R1
R2
O
R2
O
OH
O
OH
O
O
HO
HO
HO
HiCel7B E197A
HO
O
HO
HO
O
HO
O
HO
HO
+
O
O
OH
OH
OH
OH
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NO2
NO2
OH
OH
s.a. (%)1
100
102
48
1
R1 = OH
R1 = N3
3
4
R2 = OH
R 2= N3
Reaction
1 + 3
1 + 4
R1
OH
OH
N3
R2
OH
N3
2
2 + 3
OH
FIGURE 2
Glc4Glc
F
α
16
12
8
6'N3Glc4GlcαF
4
0
0
0,5
1,0
1,5
2
[donor] (mM)
FIGURE 3
OH
O
OAc
O
OH
O
OH
OAc
OH
O
Glycosynthase
Acetylation
O
HO
HO
HO
O
O
HO
AcO
O
HO
OAc
OH
AcO
O
O
O
HiCel 7B E197A
40ºC, 24 h, pH = 7.8
H
Ac2O / py / DMAP
80ºC, 24 h
Ac
OH
F
OAc
OH
n
R
R
R
n
7
R = N3
1
2
R = OH
R = N3
5
6
R = OH
R = N3
PPh3, THF, H2O
RT
CuSO4
Staudinger
reduction
R = N3
'Click'
Sodium ascorbate
Alexa Fluor® 488 alkyne
DMSO/H2O
OH
O
OAc
O
OH
OAc
O
HO
HO
O
O
AcO
O
OH
OAc
AcO
O
N
H
O
NH2
NH2
Ac
OH
-
OAc
SO3
n
n
9
N
N
O
-
SO3
HN
+
O
R' =
NH2
O
O
8
R'
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FIGURE 4
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FIGURE 5
A
B
C
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FIGURE 6
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A
C
B
D
FIGURE 7
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