Biohybrid Glycopolymer Capable of Ionotropic Gelation

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Biohybrid glycopolymer capable of ionotropic gelation Ali Ghadban, Luca Albertin, Marguerite Rinaudo, and Alain Heyraud Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm300925j • Publication Date (Web): 28 Aug 2012 Downloaded from http://pubs.acs.org on September 3, 2012

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Biohybrid glycopolymer capable of ionotropic gelation Ali Ghadban 1, Luca Albertin 1,*, Marguerite Rinaudo 2, Alain Heyraud 1 1

Centre de Recherches sur les Macromolécules Végétales (CERMAV-CNRS)†, BP53, 38041 Grenoble, France. 2 European Synchrotron Radiation Facility, BP 220, 6 Rue Jules Horowitz, 38043, Grenoble, France. [email protected]

RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to)

ABSTRACT

Ionotropic gelation is particularly appealing for the formation of hydrogels since it takes place under mild conditions, is not thermoreversible and does not involve toxic chemicals. A well-known example is the gelation of alginate in the presence of calcium ions, which is at the base of numerous applications involving this polymer. In this study, alginate-derived oligosaccharides were converted into acrylamideand methacrylamide-type macromonomers in two steps without resorting to protective group chemistry. They were then copolymerized with 2-hydroxyethylmethacrylamide in aqueous solution to yield high molar mass biohybrid glycopolymers containing between 25% and 52% by mass of oligosaccharide graft

*

Corresponding author, [email protected]. Tel. +33 (0)4 76 03 76 60. Fax +33 (0)4 76 54 72 03. †

Affiliated with Université Joseph Fourier, and member of the Institut de Chimie Moléculaire de 1 Grenoble. ACS Paragon Plus Environment

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chains. A comparative kinetic study showed that both acrylamide- and methacrylamide-type macromonomers reacted since the early stages of the copolymerization, but that the mole fraction in the polymer was smaller than in the feed up to 50-60% conversion and increased markedly afterwards. This effect was slighter for the methacrylamide-type macromonomer though. Copolymers carrying oligosaccharide chains with 16-20 repeating units were synthesized and used for a gelation experiment: When dialyzed against CaCl2 0.5 mol L-1, the polymer carrying (1→4)-α-L-guluronan residues led to a soft isotropic self-standing transparent hydrogel while the polymer carrying (1→4)-β-D-mannuronan residues gave a loose opaque gel. This study demonstrates that alginate-extracted oligosaccharides and aqueous radical polymerization can be combined for the flexible design of biohybrid glycopolymers capable of ionotropic gelation under very mild conditions.

INTRODUCTION Hydrogels are hydrophilic polymer networks swollen with water. Thanks to their high water content (usually a multiple of their dry mass) and soft rubbery consistence they closely resemble soft living tissues and are extensively studied for pharmaceutical and medical applications.

1-10

The attractive

features of hydrogels include their low interfacial tension (which results in a minimal tendency to adsorb proteins from body fluids and in good biocompatibility), the fact that an aqueous environment can protect cells and fragile drugs incorporated into the gel, and the good transport of nutrients to cells and of products from cells throughout the gel pores. 9 Among the various mechanisms leading the formation of hydrogels,

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the complexation of ions with

the creation of extended junction zones (i.e. ionotropic gelation) is particularly appealing 12 since it takes place under mild conditions, is not thermoreversible (as long as an excess of ions is present and the junction zone is sufficiently long) 13, 14 and does not involve toxic chemicals. A well-known example is the gelation of alginate in the presence of calcium ions, which is at the base of numerous applications involving this polymer. Alginate is a heteropolysaccharide extracted from brown algae such as Laminaria hyperborea and Lessonia. At a molecular level it is a linear unbranched copolymer of 2 ACS Paragon Plus Environment

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(1→4)-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues distributed in long homopolymeric blocks (e.g. MMMM or GGGG) and in shorter alternating copolymer blocks (e.g. MGMG). 15, 16 In aqueous solution, alginate forms transparent hydrogels upon addition of some divalent cations (e.g. Ca2+, Ba2+, Sr2+ and Zn2+) mostly due to complexation of said ions by guluronan on adjacent polymer chains and to the formation of junction zones. 17 Depending on the author,18, 19 a minimum of 8 to 18 repeating units in a guluronan block is reported to be necessary to the formation of a stable complex with calcium ions. A major difficulty in the use of alginate for the preparation of hydrogels with well-defined physicochemical properties is the intrinsic variability of the molar mass and composition of the polysaccharide, which depend on the source species, the conditions of growth, the period of harvest and the extraction process.

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Besides, the type of chemical functionality present on any given polysaccharide is

intrinsically limited (only hydroxyl, carboxyl and hemiacetal groups in the case of alginate) and any other residue (e.g. an adhesion peptide or a biodegradable linker) has to be introduced via polymermodification reactions. The latter are often cumbersome, 20-22 rarely efficient 23, 24 and generally limited in scope. 25, 26 An attractive feature of alginate is the possibility to extract its constituting blocks by controlled hydrolysis and selective precipitation.

27-29

Since at pH ≅ 2-3 the rate of hydrolysis of G-M glycosidic

linkages is 3-4 times that of M-M, G-G or M-G linkages, alternating blocks are preferentially degraded and fairly pure homopolymeric sequences are recovered (> 85% in G or M). 30-32 In this way, (1→4)-αL-guluronan and (1→4)-β-D-mannuronan samples with Xn ≅ 20-30 and low mass dispersity can be

obtained in good yield on a multi-gram scale (Xn is the number average degree of polymerization). In our laboratory, we are investigating the use of alginate-derived oligosaccharides as building blocks for the synthesis of biohybrid polymers with original gelation properties. The rationale behind this work is that (1→4)-α-L-guluronan residues with Xn ≥ 18 are ionotropic gelators that can be incorporated into a water soluble synthetic polymer to bestow it with original gelation properties. As a result, the 3 ACS Paragon Plus Environment

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versatility and precision of modern polymer chemistry could be combined with the gelation properties of guluronan to afford well defined glycopolymers (in terms of composition, molar mass, dispersity and functionality) capable of gelling under mild conditions. In this communication we describe the synthesis of (meth)acrylamide-functionalized (1→4)-β-Dmannuronan and (1→4)-α-L-guluronan macromonomers (Scheme 1), their copolymerization with 2hydroxyethylmethacrylamide (HEMAm) in water and the gelation properties of two of the resulting graft copolymers. Scheme

1.

Synthesis

of

(1→4)-β-D-mannuronan-

and

(1→4)-α-L-guluronan-functionalized

macromonomers.

Conditions: i) H2O, 30 °C, 5-18 days. ii) H2O / CH3OH (or DMSO) ≥ 9:1, 0 °C→Tamb, 6-7 ½ hours. EXPERIMENTAL Materials The following chemicals were reagent grade and were used as received: 4,4´- Azobis(cyanopentanoic acid) (ACPA, 98%, Aldrich), CaCl2 (97%, Prolabo), D2O (99.8%, Eurisotop), HCl (37%, Carlo Erba), NaHCO3 (≥ 99%, SDS), DMSO-d6 (99.8%, Eurisotop), ethanol amine (≥ 99%, Fluka), 2,6-di-tert-butyl4-methyl

phenol

(BHT, ≥ 99%,

Fluka),

methyl

α-D-glucoside

(≥ 99%,

Fluka), 4

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ethylenediaminetetraacetic acid tetrasodium salt EDTA (99%, Acros), methacryloyl chloride (≥97.0% Fluka), NaCl (≥ 99%, Aldrich), NH4OAc (98 %, Aldrich), NaBH3CN (95 %, Aldrich), NaNO3 (≥ 99%, Aldrich), NaN3 (≥ 99%, Merck), sodium hydroxide solutions (pure, Acros). Deionized water was produced in house with a MilliQ apparatus (Millipore) and used for all experiments. Cellulose nitrate membranes were supplied by Sartorius. (1→4)-β-D-Mannuronan and (1→4)-α-L-guluronan oligomers were obtained from Elicityl SA (Crolles, France) and characterized by 1H NMR and SEC-MALLS for their molar mass and, following total acid hydrolysis, by High Performance Anion Exchange Chromatography (HPAEC) for composition. General methods Accurate volumes (10-1000 µL) were measured with calibrated automatic pipettes (Eppendorf Research). Accurate pH and conductivity values were measured with a pH-meter (Cyberscan PC510); alternatively, a special pH indicator paper was used (Macherey-Nagel, ± 0.5 pH units). The density of solutions and liquids was measured with precision hydrometers (Alla France). Dialysis purifications were performed against de-ionized water (100-150 times the volume of the sample) at room temperature over a 48 hours period with Slide-A-Lyzer Dialysis Cassettes (Pierce Biotechnology). During the process the water was changed thrice, typically after 2, 16, and 26 hours. Diafiltrations were carried out using ultrafiltration cells equipped with a cellulose acetate membrane (∅ 63.5 mm, Millipore) and connected to an auxiliary reservoir filled with de-ionized water (p = 2-3 bars; stirring rate ~300 rpm). Purifications were stopped once the conductivity of the eluate had fallen below 5 µS cm-1. TLC analyses were performed on aluminium-backed silica gel plates (60 Å, 15 µm, SDS); following solvent evaporation, the developed plates were exposed to a UV lamp (λ=254 nm) for spots detection. Flash chromatography was carried with a glass column using silica gel from Merck (60 Å, 40-60 µm). Synthesis of N-(2-hydroxyethyl)methacrylamide (HEMAm) In a round bottom flask, ethanol amine (10 mL, 0.160 mol) was dissolved in MeOH (100 mL) and Et3N (21 mL, 0.150 mol) was added. The mixture was cooled in an ice bath and methacryloyl chloride 5 ACS Paragon Plus Environment

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(14.4 mL, 0.150 mol) was introduced drop wise under stirring using a gas tight syringe. After 2 hours the ice bath was removed and the mixture was left reacting for a further 2 hours at ambient temperature. The reaction was stopped by adding HCl 2 N (~8 mL), the volatiles were eliminated at reduced pressure, and 10 g of the resulting solid were re-solubilized in EtOH and adsorbed on silica gel (70 g). The resulting white powder was added at the top a column (∅: 7cm) pre-packed with silica gel (h ≅ 24 cm) and the product was eluted using a gradient of Petroleum Ether/EtOAc/EtOH (from 6:3:1 to 6:2:2). The fractions containing the product were pooled, stabilized with BHT, concentrated at the rotary evaporator and further dried under mechanical vacuum (p ≈ 10-1 mbar) overnight. Viscous oil, Rf 0.45 (Petroleum Ether/EtOAc/EtOH 6:2:2). 1H-NMR (400 MHz, D2O, 298 K) δ (ppm): 1.93 (m, H6, 3H), 3.40 (t, H4, 2H), 3.68 (t, H5, 2H), 5.45 (m, H3b, 1H), 5.71 (m, H3a, 1H).

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C-NMR (100 MHz, D2O, 10 °C) δ

(ppm): 20.41 (C6), 44.34 (C4), 62.60 (C5), 123.95 (C3), 141.61 (C2), 174.83 (C1). ESI-MS: m/z calculated 130.09, found: 130.0 [M.H+]. Note: In the absence of a radical inhibitor the monomer homopolymerized during drying under mechanical vacuum. Synthesis of macromonomers In a typical experiment, (1→4)-β-D-mannuronan (ManA10, Xn = 10, 98 %, 4.50 g, 2.79 mmol) was weighed in a 200 mL Erlenmeyer flask, solubilized in H2O (50 mL) and mixed with an aqueous solution of ammonium acetate (9.08g, 115 mmol in 35 mL H2O) and sodium cyanoborohydride (3.21 g, 48.0 mmol in 15 mL H2O). The final pH was checked with pH paper (pH ≅ 7), a magnetic bar was added and the flask was sealed with a rubber septum and immersed in a water bath preheated at 30 ºC. After stirring at 200 rpm for 7 days, the mixture was transferred to a centrifugation tube and the oligosaccharide precipitated by the addition of ethanol under vigorous stirring (final proportion 80 % v/v). After centrifugation (10 000 rpm for 10 min), the supernatant was discarded and the precipitate was re-dissolved in H2O (~80 mL) and diafiltered (500 Da cut off membrane). The desalted solution was transferred to a round bottom flask, frozen in liquid nitrogen and freeze dried overnight to isolate the 1amino-1-deoxyalditol (ManA10-NH2). Conversion of starting carbohydrate > 90 %. Functionalization 6 ACS Paragon Plus Environment

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rate: 42 %. 1H-NMR (400 MHz, D2O, 323 K, see Scheme 2 for position numbering) δ (ppm): 3.00 (dd, H1a, 1H, J1a,1b 12.9 Hz, J1a,2 9.4 Hz), 3.47 (dd, H1b, 1H, J1b,1a 13.1 Hz, J1b,2 3.0 Hz), 3.61-4.30 (H2-H5, H2´-H5´), 4.62-4.81 (H1´), 5.02 (H1´, residual G unit). Mn (SEC) 1900 Da with dn/dc = 0.165, Ð 1.08. Part of the ManA10-NH2 (1.50 g, 3.57 × 10-4 mol) was dissolved in 34 mL of a 9:1 mixture of sodium carbonate – sodium bicarbonate buffer solution (0.6 mol L-1, pH 9.5) and MeOH. The resulting solution was cooled on an ice bath for 10 minutes and methacryloyl chloride (493 µL, 50.9 × 10-4 mol) was added drop-wise under vigorous stirring (250-300 rpm). From time to time the pH was adjusted to ~9.5 with solid Na2CO3. After 2 hours on ice and 4.5 hours at Tamb the reaction was stopped by precipitating the oligosaccharide with ethanol (final proportion 80 % v/v). The mixture was centrifuged (10,000 rpm, 15 °C, 10 min), the precipitate was re-solubilized in water, diafiltered (500 Da cut off membrane) and freeze dried (ManA10-MAm). Conversion of starting 1-amino-1-deoxyalditol: 100 %. Functionalization rate: 42 %. MS calcd for C40H56NO37 (X=6), 1142.26; found 1142.3 ([M-H]-); calcd for C46H64NO43 (X=7) 1318.29; found 1318.4 ([M-H]-). 1H NMR (400 MHz, D2O, 323 K) δ (ppm): 1.94 (m, H10, 3H), 3.38 (dd, 1H, H1a, J1a,1b 14.0 Hz, J1a,2 7.6 Hz), 3.59-4.34 (H2-H5, H2´-H5), 4.64-4.80 (H1´), 5.00 (H1´, residual G unit), 5.44 (m, 1H, H7 cis to C10), 5.70 (m, 1H, H7 trans to C10).

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Scheme 2. Position numbering used for NMR assignment.

Homopolymerization of 2-hydroxyethyl methacrylamide (HEMAm) (Run no. 1, Table 1) 4,4´Azobis(cyanopentanoic acid) (3.14 × 10-2 g, 1.12 × 10-4 mol) was dissolved in DMSO-d6 (1 mL), cooled to ≅ 8 °C and diluted with an equal volume of D2O. HEMAm (0.350 g, 2.71 × 10-3 mol) was dissolved in D2O (2.4 mL) and filtered through a syringe filter (0.22 µm, cellulose acetate) to remove the suspended inhibitor (BHT). Part of the latter solution (2.0 mL, 1.13 mol L-1), was mixed with a calculated amount of ACPA solution (200 µL, 5.60 × 10-2 mol L-1, 1.12 × 10-5 mol) and transferred to a Schlenk tube. The tube was then sealed with a rubber septum, degassed by 3 freezeevacuate-thaw cycles and transferred to a water bath preheated at 60 °C. After 3.5 hours the polymerization was stopped by plunging the tube in icy water, a sample was withdrawn for 1H NMR and SEC analysis and the remaining polymer was precipitated twice in an excess of acetone. The fiber-like precipitate was dried under mechanical vacuum (10-15 torr) at room temperature (~21°C) for 65 hours. 1

H-NMR (400 MHz, D2O, 298 K) δ (ppm): 0.97 and 1.12 (3H, CH3), 1.75 and 1.86 (2H, CH2

backbone), 3.28 (2H, CH2-NH), 3.66 (2H, CH2-OH).


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Table 1. Summary of polymerization experiments. run HEMAm no. (mol L-1)

macromonomer

t (min) p (%)

dn/dc

Mn (Da)

[η ]

Ð

(mL g-1) type

c (mmol L-1)

f

F

Fm

(%)

(%)

(%)

1

1.03

-

-

210

80

0.208

395 000

93

1.83

-

-

-

2

0.52

ManA5MAm

26.9

120

68

0.197

208 000

104

2.05

4.9

4.1

25

3

0.58

ManA10MAm 29.6

270

76

0.186

520 000

199

2.32

4.9

6.6a

50a

4b

0.58

ManA10MAm 29.6

270

76

0.187

480 000

193

2.41

4.9

6.6a

49a

5

0.58

ManA10MAm 30.0

300

86 (76)

0.191

743 000

205

2.05

5.0

4.3

39

6

0.58

ManA10Am

29.9

300

85 (51)

0.188

566 000

211

2.59

4.9

3.0

30

7b

0.58

ManA10Am

29.6

300

87 (55)

0.188

611 000

218

2.52

4.9

3.1

31

8

0.58

ManA10Am

31.0

300

84 (68)

0.192

678 000

192

2.40

5.0

4.1

38

9

0.50

ManA16MAm 21.0

270

98 (100) 0.185

482 000

205

2.12

4.0

4.2

52

10

0.39

GulA20MAm

255

82 (76)

612 000

205

1.95

2.7

2.5

44

11.0

0.189

General conditions: D2O / DMSO-d6 (< 2%), pD ≅ 6-7, 60 °C, 4,4´-azobis(cyanopentanoic acid), c0ACPA / mM = 5.1 (run 1), 3.1 (run 2), 1.0 mM (run 3-8) or 1.1 mM (run 9-10). ManAX and GulAX represent (1→4)-β-D-mannuronan and (1→4)-α-L-guluronan, respectively, with number average repeating units “X”. MAm stands for methacrylamide, Am stands for acrylamide. Total conversions (p) for run 1-4 were calculated by 1H NMR. Individual monomer conversions were calculated by 1H NMR for run 6-8 and by SEC for run 5-10 and are reported as pHEMAm (pmacromonomer). Ð is the molar mass dispersity; f and F are the molar fraction of macromonomer in the feed and in the polymer, respectively; Fm is the weight fraction in the polymer; F values were calculated from the 1H NMR spectra of the purified polymer (run 2-4) or from individual monomer conversions. a These values are overestimated due to the presence of residual macromonomer and starting oligosaccharide in the dialyzed / diafiltered polymer sample. b Polymerization carried out in NaCl 0.2 mol L-1. Copolymerization of ManAX-(M)Am with HEMAm (X = 5, 10) (Run no. 2-4 and 6-7, Table 1). In a typical experiment (run no. 2) 4,4´-azobis(cyanopentanoic acid) (ACPA, 1.47 × 10-2 g, 5.24 × 10-5 mol) was dissolved in DMSO-d6 (0.50 mL), cooled to ~ 8 °C and diluted with an equal volume of D2O. HEMAm (0.0880 g, 6.82 × 10-4 mol) was dissolved in D2O (0.40 mL) and filtered through two syringe filters connected in series (1.22 µm glass fiber and 0.22 µm nylon filter) to remove the suspended inhibitor (BHT). ManA5-MAm (0.0940 g, 47% purity, 4.77 × 10-6 mol) was dissolved in D2O (0.62 mL), added to part of the HEMAm solution (0.3 mL, 1.70 mol L-1) and 9 ACS Paragon Plus Environment

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mixed with a calculated amount of initiator (57 µL, 5.24 × 10-2 mol L-1, 2.99 × 10-6 mol). The resulting solution (pD ≅ 6-7) was transferred to a NMR tube equipped with a Young valve, a 1H NMR spectrum at t = 0 was recorded and the tube was sealed, degassed by 4 freeze-evacuate-thaw cycles and transferred to a water bath preheated at 60 °C. After 2 hours the reaction was stopped by plunging the tube in icewater and a 1H NMR spectrum was acquired for global conversion calculation (p). To this end, the 1HNMR spectra of the starting and final polymerization mixture were normalized to the internal anomeric proton of the macromonomer (4.64 ppm) and the ethylenic protons were integrated (5.44 and 5.70 ppm for HEMAm and ManXMAm). The following formula was then applied:

 Aethylenic     Aref t p =1−  Aethylenic    A ref  0

(1)

where Aethylenic and Aref are the area of peak ethylenic and reference peaks, respectively. The polymer was purified by dialysis (20,000 Da cut-off membrane) for 50 hours respectively and freeze dried. 1HNMR (400 MHz, D2O, 323 K) δ (ppm): 0.96 (H7, 2H), 1.11 and 1.74 (H10, 3H), 3.27 (H11, 2H), 3.64 (H12, 2H), 3.6-4.4 (H2-H5, H2´-H5), 4.67 (H1´). The composition of the purified glycopolymer was calculated by normalizing to 2.00 the integral the 1H-NMR CH2-NH signal of HEMAm (3.27 ppm) and by dividing the integral of the internal anomeric protons of the macromonomer (4.67 ppm for mannuronan, 5.04 ppm for guluronan) by (XnNMR-1), where XnNMR is the number of repeating units in the oligosaccharide estimated by 1H NMR. For polymerizations at high ionic strength D2O was replaced with NaCl 0.2 mol L-1 in D2O. When the macromonomer was Man10Am (δethylenic 5.76, 6.18 and 6.32 ppm) individual monomer conversions were calculated from 1H-NMR. Kinetic study of the copolymerization of ManA10-MAm and ManA10Am with HEMAm The above described procedure was modified as follows: The macromonomer was dissolved in D2O together with a calculated amount of methyl α-D-glucoside as internal standard; a t0 sample (~ 120 µL) was drawn from the polymerization mixture, frozen in liquid nitrogen and stored in a freezer until ACS Paragon Plus Environment

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needed. The polymerization mixture (pD ≅ 6-7) was then transferred to a Schlenk tube, sealed with a rubber septum, degassed with 4 freeze-evacuate-thaw cycles and transferred to a water bath preheated at 60 °C. At pre-set intervals, samples (~ 6 mg mL-1) were drawn from the reaction mixture, frozen in liquid nitrogen and stored in the freezer until needed. After 5 hours (end of the kinetic monitoring) more initiator was added and the reaction was left running for another 11.5 hours (final conversion ≅ 100%). Intermediate samples were analyzed by SEC with a column set allowing baseline resolution between monomer, polymer and internal standard peaks (Shodex OH pak SB-(Guard + 802 + 803) HQ columns). Individual monomer conversions (p) were then calculated according to the formula:

 Ai   Ai    −  ⋅ (1 − P) AISTD t  AISTD 0  pi = 1 −  Ai    ⋅ P  AISTD 0

(2)

where Ai and AISTD are the area of the monomer and internal standard peak respectively, P is the mass purity of the monomer (assumed to be 1 for HEMAm and determined by 1H-NMR for the monomer). This formula takes into account the fact that macromonomer samples contained an amount (1-P) of the oligosaccharide from which they were derived (or the corresponding alditol) and that the corresponding SEC peaks are perfectly superimposed. Copolymerization of GulA20-MAm and ManA16-MAm with HEMAm The procedure used for kinetic experiments was modified as follows: The polymerization mixture was degassed with 3 freeze-evacuate-thaw cycles; no intermediate samples were drawn; after 4.25 hours the reaction was stopped by plunging the tube in cold water, a tend sample (~ 120 µL) was drawn for SEC analysis and the polymer was recovered by diafiltration (30,000 Da cut off membrane) followed by freeze drying. GulA20-Mam / HEMAm (run no. 10, Table 1), final conversion (SEC): HEMAm 82%, GulA20-MAm 76%. 1H-NMR (400 MHz, D2O, 328 K) δ (ppm): 0.98 and 1.12 (3H, H10), 1.75 and 1.86 (2H, H7), 3.28 (2H, H11), 3.66 (2H, H12), 3.88 (H2, H2´), 4.00 (H4, H4´), 4.11 (H3, H3´), 4.44 (H5, H5´), 5.05 (H1´). ManA16-MAm / HEMAm (run no. 9, Table 1), final conversion (SEC): HEMAm ACS Paragon Plus Environment

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98%, ManA16-MAm ~100%. 1H NMR (400 MHz, D2O, 328 K) δ (ppm): 0.98 and 1.12 (3H, H10), 1.75 and 1.86 (2H, H7), 3.28 (H11, 2H), 3.6-4.4 (H2-H5, H2´-H5´), 4.64 (H1´). Analytical techniques Nuclear Magnetic Resonance. Spectra were acquired on a Bruker DPX400 spectrometer equipped with a Variable Temperature (VT) module (resonance frequency of 400.13 and 100.62 MHz for 1H and 13

C nuclei, respectively). Two different 5 mm detection probes were used: QNP (direct) and BBIZ

(inverse). Unless otherwise specified, for 1H experiments 90° pulses and pulse sequence recycle times of 3 s were used. The probe temperature was calibrated in the range 303-363 K using 80% ethylene glycol in DMSO-d6.

33

1D 1H spectra were obtained with 32-128 scans and 32 K data points, and were re-

processed using MestReNova software (v5.1). Sodium 3-(trimethylsilyl)propanoate (TSP) or sodium 3(trimethylsilyl)propane-1-sulfonate (DSS) were used as an internal reference for samples dissolved in D2O, whereas tetramethylsilane (TMS) was used in all other cases. Chemical shifts (in ppm) were referenced to δTSP = -0.017 ppm (1H) and -0.149 ppm (13C), or to δDSS and δTMS = 0.000 ppm (1H and 13

C). Mass spectrometry. ESI-MS analyses were performed with a Waters ZQ (Altrincham, GB) single

quadrupole atmospheric pressure ionization mass spectrometer fitted with a Z electrospray interface (ESI). The instrument was calibrated with mass spectra generated by ion spray ionization of a 0.1 mol.L1

solution of sodium iodide in aqueous acetonitrile (50%, v/v) in the mass range of 23-1972 amu.

Nitrogen was used as the drying and nebulizing gas. Samples (~1 mg mL-1) were dissolved in deionised water or water/methanol mixtures and infused to the ESI interface at constant flow rate (50 µL min-1). MALDI –Tof analyses were performed on a Voyager DE-Pro (AB Sciex) equipped with a nitrogen laser (λ=337 nm). The spectrometer was operated in negative ion reflector or linear mode and mass spectra were obtained by accumulating 300 laser shots for each analysis. Samples were solubilized in water (20 g L-1) and mixed with an equal volume of 2-(4-hydroxyphenylazo)benzoic acid / 1,1,3,3tetramethylguanidine (1:2 molar ratio) in methanol as described by Przybylski et al.

34

The resulting 12

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Biomacromolecules

solution (1 µL) was then deposited target and allowed to dry at room temperature and atmospheric pressure. Size Exclusion Chromatography (SEC-DV-MALLS). Molar mass, molar mass distributions and intrinsic viscosities were measured with a Size Exclusion Chromatography (SEC) system consisting of an Alliance GPCV 2000 chromatograph (Waters) equipped with a differential refractometer (λ = 880 nm) and a 3 capillary differential viscometer (DV), and interfaced with a multi-angle laser light scattering (MALLS) detector (DAWN HELEOS II, Wyatt Technology Corp., Santa Barbara, California; λ = 658 nm). The system was equipped with a 50×6 mm guard column and two 300×8 mm linear columns (Shodex SB-800 HQ series). An aqueous buffer (NaNO3 0.1 M, NaN3 0.03% w/v, Na-EDTA 0.01 M) was used as eluant at a flow rate of 0.5 mL min-1 while temperature of the columns, DRI and viscometer was maintained at 30 °C. Samples were prepared by diluting the polymerization mixtures or by dissolving pure polymer samples directly in the eluant at concentrations of ~1 g L-1. The resulting solutions were filtered through 0.22 µm sterile syringe filters (Millex GS, Millipore) and injected in 100 µL volumes. Differential refractive index increments dn/dc used for molecular weight calculations were measured at 30 °C with an Optilab rEX differential refractometer (Wyatt Technology Corp., λ = 633 nm). Results were analyzed with ASTRA 5.3 software (Wyatt Technology Corp.). For each slide of the chromatogram, the molar mass was obtained from the intensity of the scattered light at different angles as the reciprocal of the intercept at θ = 0 of a plot: 35

Kc θ  vs. sin2   Rθ 2

(3)

where θ is the scattering angle, K is an optical constant containing the specific refractive index increment of the polymer (dn/dc), Rθ is the Rayleigh ratio and c is mass concentration. To this end, the term 2 A2c in the classic Zimm plot was considered to be negligible. Radii of gyration Rg were obtained from the slope of the same plot and their z-average was computed for the whole sample. Intrinsic viscosities were obtained as mass averages by using the approximation: 13 ACS Paragon Plus Environment

Biomacromolecules

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

[η ] ≅

η sp

Page 14 of 40

(4)

c

where ηsp is the specific viscosity. Eq. 4 is generally valid for dilute solutions of low molar mass polymers of small intrinsic viscosity.

36

In our case c was less than 0.3g L-1 at the peak of the

chromatograms and [η] ≤ 220 mL g-1. Intrinsic viscosities were used to calculate approximated hydrodynamic radii (Rh) for individual polymer chains via the Einstein-Simha equation for inelastic spheres: 35, 36  3M [η ]   Rh ≅   10 πN A 

1/ 3

(5)

where M is the molar mass of the polymer and NA is Avogadro’s number. Differential refractive index increments. The value of dn/dc for poly(HEMAm) (run no. 2) and poly(HEMAm-co-ManA5MAm) (run no. 3) was measured at 30 °C with an offline Optilab® rEX differential refractometer (λ = 633 nm; Wyatt Technology Corp.) operated with ASTRA 5.3 software. To this end, poly(HEMAm) was isolated by re-precipitating it in acetone twice followed by extensive drying under mechanical vacuum, whereas poly(HEMAm-co-ManA5MAm) was purified by diafiltration (30,000 Da molecular weight cut-off) and freeze-dried. In both cases, the residual water content was estimated by TGA (vide infra). Polymer solutions of known concentration were then prepared gravimetrically by dissolving the polymers into the eluant used for SEC analysis (d30= 1.0035 g mL-1) and injected at a flow rate of 0.25 mL min-1. The pure eluant was also injected. The instrument measures the refractive index of (n) for each concentration (c), subtracts the refractive index of the eluant (n0) and calculates dn/dc from the slope of the plot ∆n vs. c according to the following equation: ∆n =

dn ×c dc

(6)

where ∆n = n - n0. The refractive index increments of the copolymers were estimated from the mass fraction (Fm) of each monomer and the dn/dc of the corresponding homopolymers according to the 14 ACS Paragon Plus Environment

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formula: 37 dn/dc = Fm,1(dn/dc)1 + Fm,2(dn/dc)2 To this end, dn/dc values of 0.165 mL g-1 and = 0.208 mL g-1 were used for alginate

(7) 17

and

poly(HEMAm) (this study), respectively. Rheometry. The rheological properties of polymer gels were characterized with an AR2000 rheometer (TA instruments) at 25 °C. To this end, polymers were isolated by diafiltration (30,000 Da molecular weight cut-off), lyophilized, and their residual solvent content was determined by Thermo Gravimetric Analysis. For solution measurements, polymers were dissolved in NaCl 0.1 mol L-1 at twice the critical overlapping concentration (c∗) as estimated from the intrinsic viscosity in NaNO3 0.1 mol L-1 obtained by SEC-IV-MALLS analysis (c∗ ≈ 2.5 / [η]). The resulting solutions were subjected to steadystate and dynamic (oscillatory) experiments using a cone-plate rheometer (diameter = 4 cm, angle = 3°59) with an inter-cone-plate gap of 113 µm. Gels of poly(HEMAm-co-ManA16MAm) and poly(HEMAm-co-GulA20MAm) were prepared by dialyzing a glycopolymer solution in deionized water (c0 ≅ 1.5 c* = 18 mg mL-1, pH ≅ 6-7) against a solution of CaCl2 (0.5 mol L-1) for 28-48 hours. To this end, small dialysis cassettes were used (Slide-A-Lyzer, 0.5-3.0 mL, MWCO 2000 Da, Pierce), from which the gel was recovered by cutting off the dialysis membrane with a scalpel. The resulting material was either punched into a disk (poly(HEMAm-co-GulA20MAm), ∅ = 2.0 cm, h ≅ 0.32 cm) with the same diameter of the rheometer plate, or transferred onto the same plate as a film. In order to prevent slippage effects, a sanded plate was used for gel characterization. For each type of hydrogel, the linear viscoelastic regime was determined by an amplitude sweep to check that the measured moduli were independent of the deformation applied. The rheological properties were then investigated on fresh samples in oscillatory and compression mode using a parallel-plate system. The Young modulus of the hydrogel of poly(HEMAm-co-GulA20MAm) was measured by placing a disk of area S = 3.14 × 10-4 m2 between the two parallel plates of the rheometer and by squeezing it while recording the backward force 15 ACS Paragon Plus Environment

Biomacromolecules

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Page 16 of 40

exerted. The elastic modulus E was calculated by dividing the initial slope of the force vs. deformation curve by the area of the disk. RESULTS AND DISCUSSION Synthesis of macromonomers Glycuronans are polysaccharides containing only uronic acid residues, i.e. monocarboxylic acids formally derived from aldoses by replacement of the hydroxymethyl group -CH2OH with a carboxy group.

38

In order to incorporate a glycuronan into a biohybrid polymer (or other glycoconjugage) it is

advantageous to selectively functionalize its reducing end without resorting to protective group chemistry, the latter being exceedingly time consuming.

39

A common strategy for the derivatization of

mono- and oligosaccharides is their transformation into the corresponding 1-amino-1-deoxyalditols via reductive amination.

40-44

In this reaction, the masked aldehyde group at the reducing end of the

carbohydrate condenses with an amine (or ammonia) molecule to give an amino alcohol, which is then protonated at the oxygen atom and eliminates a water molecule to give an iminium ion. In the presence of a suitable reducing agent, the latter is rapidly reduced to a stable amine.

45, 46

At a laboratory scale,

sodium or lithium cyanoborohydride is the reducing agent of choice for reductive aminations in protic solvent, thanks to its remarkable stability in aqueous solution (at pH > 2.5) and to its pH-dependent chemoselectivity. 47 In particular, if the substrate is a ketone or aldehyde, at pH 6-8 the reduction of the iminium ion is sufficiently faster than that of carbonyl groups to enable its formation and reduction in situ. The reductive amination of oligoalginates with a bifunctional amine in water has already been reported in the literature

48, 49

but the characterization of the resulting materials was basic and

functionalization yields were not determined. Concerning other glycuronans, Ridley et al. reacted analytical quantities of (1→4)-α-D-galacturonan in methanol with a biotin-hydrazide derivative and reduced the resulting hydrazone with NaBH3CN in water.

40

The procedure involved repeated solvent

evaporation steps and is mostly applicable to microscale reactions. Also, the yield of oligogalacturonan–


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Biomacromolecules

hydrazide from a single reaction decreased with the size of the oligosaccharide, from 88% for X = 8 to 39% for X = 16 (where X is the degree of polymerization). In this study (Scheme 1), (1→4)-β-D-mannuronan (ManAX, X = 5, 10 and 16) and (1→4)-α-Lguluronan oligosaccharides (GulA20) were reacted for several days with an excess of ammonium acetate (>40 eq.) in aqueous solution and in the presence of NaBH3CN (~50 eq.). 50 Although conversion of the starting carbohydrate as measured by the disappearance of the reducing end anomeric proton was >90% in all cases, the mole fraction of 1-amino-1-alditol was in the range 37-46 %, thus suggesting the concomitant formation of by-products (most probably alditol). The reaction rate was generally slow and under identical conditions, the reaction of GulA20 was ~3 times slower than that of ManA16, thus indicating a slower formation of the intermediate iminium ion for guluronan in the rate determining step. Optimization of this reaction was beyond the scope of this work and, after elimination of salts by diafiltration, the obtained products were directly used for the following step. The rationale behind this choice was that residual non-aminated oligosaccharides would be inert towards the remaining synthetic steps (acylation and radical polymerization) and could be separated from the final polymer by simple diafiltration or dialysis (the hydrodynamic diameter of the two species differing by two orders of magnitude). In particular, in basic aqueous solution (Schotten–Baumann conditions) an acid chloride will rapidly react with the primary amine and any excess reagent will be hydrolyzed by water (c = 55 mol L-1) before acylating the hydroxyl groups of the carbohydrate (c ≤ 0.6 mol L-1), the two reactions having similar rate constants.

51

Likewise, carbohydrates readily react with oxygen-centered radicals by

hydrogen-atom abstraction at the ring C-H bonds

52‡

but are unaffected by most of the secondary and

tertiary carbon-centered radicals encountered in radical polymerization processes (alkyl ester radicals being a notable exception). 53

‡

For the same reason peroxy initiators should be avoided in the synthesis of glycopolymers. ACS Paragon Plus Environment

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Page 18 of 40

Hence, the purity of the 1-amino-1-alditols obtained by reductive amination was quantified by 1H NMR and the same were directly reacted with (meth)acryloyl chloride (~15 eq.) in aqueous carbonate buffer at pH 9.5. This pH value was found to be the best compromise between the quest for fast amide bond formation (which demands a high pH) and the need to avoid degradation of the glycuronans under basic conditions.

54

When an excess of base was used (> 2 eq.), the acylation step was quantitative and

the limiting step for end-of-chain functionalization was reductive amination. Figure 1 shows the 1H NMR spectra of (1→4)-β-D-mannuronan (Xn = 10; ManA10), of the corresponding 1-amino-1-alditol (ManA10-NH2) and of the final macromonomer ManA10-MAm. Note the almost complete disappearance of the reducing end anomeric protons H1α/β at 4.84-4.89 ppm and 4.20 ppm and the appearance of two double doublets at 3.00 and 3.46 ppm when going from ManA10 to ManA10-NH2. Also, note the deshielding of H1a (from 3.00 ppm to 3.38 ppm) and H1b (no longer visible after acylation) and the appearance of two ethylenic protons at 5.45 ppm (H7 cis to C10) and 5.70 ppm (H7 trans to C10) when going from ManA10-NH2 to ManA10MAm. The fact that the two H1 protons in the latter compounds are not magnetically equivalent is due to the presence of an asymmetric center at C2. The structure of the macromonomer was further confirmed by MALDI-ToF analysis (Figure 2, reflectron mode), in which peaks corresponding to [M-H]- and spaced of 176 Da are visible which correspond to oligosaccharides containing between 5 and 10 repeating units and bearing an end-of-chain methacrylamide group. Higher molar mass species were not visible in the reflectron spectrum but could be seen in spectra acquired in linear mode, albeit with a lower resolution.


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H7 trans

Biomacromolecules

H1

H1a

H7 cis

H1 H1b

H1a

H1(G)

H1

H1α H1β H1(G)

Figure 1. 1H NMR spectra of (a) (1→4)-β-D-mannuronan (Xn = 10), (b) the corresponding 1-amino-1alditol (ManA10-NH2) and (c) the final macromonomer ManA10-MAm. Note that the internal anomeric proton of residual guluronic acid units H1(G) is visible at 5.04 ppm. Conditions: 400.13 MHz, D2O, T = 328 K for (a) and 323 K for (b) and (c).


6 1142.3

1494

1496

1498

9 176

1670.4

10 1846.3

966.3

X=5

1494.4

8

1496.4

1318.4

negative ion mode

1494.4

7

ManA10MAm

Relative Intensisty

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 40

1495.4

Biomacromolecules

1000

1200

1400

1600

1800

m/z

Figure 2. MALDI-ToF spectrum of ManA10-MAm. Reflectron mode, negative ions. For each peak, the degree of polymerization (X) of the corresponding oligosaccharide is also reported. Polymerization experiments and synthesis of graft copolymers The obtained macromonomers were copolymerized with 2-hydroxyethylmethacrylamide (HEMAm) in aqueous solution at 60 °C in the presence of 4,4´-azobis(cyanopentanoic acid) (ACPA; ~1 mmol L-1). HEMAm was chosen as a comonomer since it leads to highly hydrophilic copolymers, been studied for the preparation of soft contact lenses

56

55

has already

and because it closely resembles 2-

hydroxypropylmethacrylamide (HPMAm) (the only difference being a methyl group on the side chain), whose homopolymer is non-toxic and non-immunogenic and whose copolymers have already been used for the preparation of bioconjugates for intravenous administration, some of which are now under 20 ACS Paragon Plus Environment

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clinical trial.

Biomacromolecules 57, 58

Obviously, any other hydrophilic methacrylamide derivative could be used as a

comonomer, but we expect poly(HEMAm) to be as biocompatible as poly(HPMAm) while being even more hydrophilic. The total monomer concentration was 0.5-0.6 mol L-1 in all runs except run no. 10 (Table 1), in which case the lower solubility of GulA20MAm required a slight dilution of the reaction mixture. Non-uniform (dispersity Ð ≅ 2.0-2.6), high molar mass copolymers were obtained in run 3-10 (Mn ≅ 480 000 – 680 000 Da), whereas a somewhat lower molar mass copolymer was obtained with ManA5MAm due to the higher concentration of initiator and, possibly, to the inferior purity of the macromonomer batch. §After purification by diafiltration, the copolymers composition was determined by 1H NMR and it was found that the final materials contained between 2.5 mole % and 4.3 mole % of macromonomer, which correspond to 25% and 52 % by mass of oligosaccharide graft chains. In the case of ManA10MAm and ManA10Am the rate of incorporation of the macromonomer was investigated by adding an internal standard (ISTD, methyl α-D-glucoside) to the polymerization mixture and by taking samples at intermediate reaction times (run 5 and 8 in Table 1). Since the ethylenic protons of HEMAm and ManA10MAm have identical chemical shifts in 1H NMR, individual monomer conversions were calculated by SEC as described in the experimental section. The two experiments were carried out under identical conditions and the resulting conversion vs. time and first-order plots are reported in Figure 3. The rate of polymerization of HEMAm was identical in the two cases and followed pseudo first order kinetics. Both macromonomers were incorporated into the polymer since the early stages of the process but their rate of conversion was slower than that of HEMAm, the difference being more pronounced in the case of ManA10Am. Also, the first order plot (Figure 3b) shows that for the two macromonomers the rate of conversion was slower at the beginning and slightly accelerated with time. The absence of an inhibition period at the beginning of the polymerization confirms that simple filtration of the aqueous

§

ManA5MAm was the only macromonomer not to purified by diafiltration due to its small size. ACS Paragon Plus Environment

21

Biomacromolecules

monomer stock solution through a 0.22 µm nylon filter is an effective way to remove 2,6-di-tert-butyl-4methylphenol (an hydrophobic radical inhibitor). 59

2.0

(b) -ln(1-p)

1.6 1.2 0.8 0.4 0.0 0.8

(a)

0.6

p

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 40

0.4 run 5 ManA10MAm HEMAm run 8 ManA10Am

0.2

HEMAm

0.0 0

40

80

120 160 200 240 280 320

t / min

Figure 3. Conversion vs. time (a) and first-order plot (b) for the radical copolymerization of ManA10MAm (full symbols) and ManA10Am (empty symbols) with 2-hydroxyethylmethacrylamide (D2O, 60 °C; run 5 and 8 in Table 1). The two experiments were carried out under identical conditions and monitored by off-line Size Exclusion Chromatography (p is fractional monomer conversion). Dashed and solid lines indicate linear regression of experimental data.


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Biomacromolecules

From the conversion vs. time data for individual comonomers (Figure 3a), the instantaneous feed (f) and copolymer composition (F) was calculated as:

f macro =

0 f macro ⋅ (1 − pmacro ) 0 0 f macro ⋅ (1 − pmacro ) + f HEMAm ⋅ (1 − pHEMAm )

 dp  0 d[macro] f macro ⋅  macro   dt  dt Fmacro = = d[macro] d[HEMAm]  dp   dp  0 0 + f macro ⋅  macro  + f HEMAm ⋅  HEMAm  dt dt  dt   dt 

(8)

(9)

where [A], f 0A and pA are the concentration, initial feed ratio and conversion, respectively, of species A. This analysis shows that up to 50-60% conversion the mole fraction of macromonomer in the newly formed polymer was smaller than in the feed (Figure 4). ManA10MAm was better incorporated than ManA10Am, with F / f = 0.74 at 10% conversion versus 0.60 for the analogous acrylamide derivative. Also, above 40% conversion F(ManA10Am) started to drift rapidly, by 70% conversion it overtook f and ended up being a multiple of it by the end of the process (F / f ≅ 3 at 83 % conversion). A qualitatively similar behavior was observed for ManA10MAm, but in this case the highest F / f ratio was 1.4 at 76% conversion. Overall, for the methacrylamide-derived macromonomer F increased from 3.9% at p = 0.10 to 10% at p = 0.76 (i.e. a 2.6-fold increase), whereas for the acrylamide analogue F was 3.2% at p = 0.10 but 32% at p = 0.84 (i.e. a 10-fold increase). These findings highlight the importance of the nature of the polymerizable moiety for the copolymerization of glycuronan-derived macromonomers with HEMAm and show that even when both monomers are methacrylamide derivatives (i.e. HEMAm and ManA10MAm), the glycuronan-derived one reacts more slowly. The higher reactivity of ManA10MAm towards poly(2-hydroxyethylmethacrylamid)yl radicals is consistent with the findings of Dainton and Sisley 60 for the copolymerization of methacrylamide and acrylamide, for which the values of r1 = 1.10 ± 0.20 and r2 = 0.74 ± 0.10 were estimated. In our case though, the copolymerization cannot be analysed in the frame of a simple terminal model because of the bulkiness of the macromonomer and the multiple 23 ACS Paragon Plus Environment

Biomacromolecules

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Page 24 of 40

negative charges that it bears. For instance, under the conditions used for copolymerizations neither ManA10MAm nor ManA10Am homopolymerized. To take into account electrostatic repulsion as a cause for the slower incorporation of the macromonomers, parallel experiments were carried out in which either ManA10MAm or ManA10Am were copolymerized with HEMAm in deionized water (run 3 and 6 in Table 1) or in NaCl 0.2 mol L-1 (run 4 and 7). Here, the role of the salt was to screen the electrostatic repulsion and facilitate the incorporation of the macromonomer. In the case of ManA10MAm, the copolymer composition was not determined since the purification step did not eliminate all unreacted macromonomer and since the individual monomer conversion was not available (the ethylenic protons of the two monomers have identical chemical shift in 1H NMR). Nevertheless, for an identical total conversion (p = 0.76) polymers with similar molar masses were obtained in deionized water and in NaCl 0.2 mol L-1, suggesting that electrostatic screening did not significantly increase the mole fraction of incorporated macromonomer. This conclusion is supported by the results obtained with ManA10Am: The addition of salt resulted in an increase from 51% to 55% of the final macromonomer conversion, from 570 000 Da to 610 000 Da of the molar mass of the copolymer and from 3.0% to 3.1% of the mole fraction of macromonomer in the polymer; i.e. differences comparable with the error inherent to the experimental procedures used.


Page 25 of 40

fmacro

Fmacro

F cummacro

fHEMAm

FHEMAm

F cumHEMAm

0.90 0.80

Mole fraction

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

(b) ManA10Am / HEMAm

0.70

0.08

0.30

0.04

0.20

0.00 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0.10

0.95 0.90 (a) ManA10MAm / HEMAm

0.10 0.08 0.06 0.04 0.02 0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

p

Figure 4. Variations in feed (f) and copolymer composition (F) with conversion for the copolymerization of ManA10MAm (a) and ManA10Am (b) with HEMAm (run no. 5 and 8 in Table 1). The cumulative mole fraction in the copolymer (F cum) is also shown. As explained in the introduction, between 8 and 18 repeating units in a guluronan block are thought to be necessary to the formation of a stable complex with calcium ions. 18, 19 Based on the results reported above, two macromonomers (ManA16MAm and GulA20MAm) possessing a suitably long glycuronan 25 ACS Paragon Plus Environment

Biomacromolecules

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Page 26 of 40

chain were copolymerized with HEMAm and the resulting graft copolymers were purified by diafiltration and freeze drying (run 9 and 10 in Table 1). In the two cases, high molar mass polymers were obtained (Mn ≅ 550 000 Da, Ð = 2.0-2.1) whose structure was investigated by 1H NMR (Figure 5; see Scheme 2 for position numbering): The absence of ethylenic proton signals at 5.45 and 5.70 ppm confirms the elimination of all unreacted monomer. The broad peaks in the 0.90 – 2.00 ppm region are due to the polymethacrylamide main chain (H7 and H10) whereas the side chain methylene protons of HEMAm are visible at 3.28 ppm (H11) and 3.66 ppm (H12). The internal anomeric proton peak H1 is found at 4.64 ppm for (1→4)-β-D-mannuronan and at 5.05 ppm for (1→4)-α-L-guluronan, whereas all other methine protons from the carbohydrates resonate in the region 3.7-4.3 ppm. From the integration of H11 and H1 the mole faction of oligosaccharide in each polymer was calculated and the corresponding mass fraction was deduced by taking into account the relative molar mass of the comonomers. It was thus found that poly(HEMAm-g-ManA16) and poly(HEMAm-g-GulA20) contained 52% and 44% by mass of graft chains, respectively.


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H12 H11

H7

H2-H4 H1

H10

H12 H2-H5 H1 H11

H7 H10

Figure 5. 1H NMR spectra of (a) poly(HEMAm-g-ManA16) and (b) poly(HEMAm-g-GulA20). See Scheme 2 for peak assignment. The integral of CH2-NHR (H11) was normalized to 2 in both spectra. Conditions: 400.13 MHz, D2O, 328 K. Ionotropic gelation Poly(HEMAm-g-GulA20) is a high molar mass biohybrid copolymer carrying (1→4)-α-L-guluronan residues that are long enough to form stable complexes with suitable divalent cations. Such complexes were expected to act as cross links between polymer chains and to induce ionotropic gelation of the polymer. In this respect, poly(HEMAm-g-ManA16) was designed as a “negative control”, i.e. an alginate-derived biohybrid polymer incapable of ionotropic gelation. The two polymers had similar molecular size and architecture and the main difference was the degree of branching: Fmacromonomer = 4.0 % for poly(HEMAm-g-ManA16) vs. 2.7 % for poly(HEMAm-g-GulA20). 27 ACS Paragon Plus Environment

Biomacromolecules

Initially, the presence of loose inter-chain interactions in the absence of gelling ions was checked by preparing semi-dilute solutions of both polymers in NaCl 0.1 mol L-1. To this aim, samples were dissolved at twice the critical overlapping concentration c∗ (estimated by the intrinsic viscosity measured in NaNO3 0.1 mol L-1) and their rheological behaviour was investigated. Figure 6 shows the variation of the steady state shear viscosity (η) with shear rate ( γ& ) for the two polymers: Clearly, in the narrow shear rate window tested ( 0.5 s −1 ≤ γ& ≤ 10 s −1 ) both solutions behaved as Newtonian fluids (η ≈ constant). Dynamic (oscillatory) experiments confirmed that in the frequency range 0.5 – 10 Hz the value of the loss modulus G´´ was the same for poly(HEMAm-g-ManA16) and poly(HEMAm-g-GulA20) (Figure 7) and that G´´ was systematically higher than the corresponding storage modulus G´. Indeed, in the case of poly(HEMAm-g-GulA20) G´ was too small to be accurately measured.

-1

-1

10 -2 9x10 -2 8x10 -2 7x10

η / (Pa.s)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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6x10

-2

5x10

-2

4x10

10 -2 9x10 -2 8x10 -2 7x10

poly(HEMAm-g-GulA20)

6x10

-2

5x10

-2

-2

4x10

-2

3x10

-2

3x10

-2

2x10

-2

2x10

-2

10

-2

10

poly(HEMAm-g-ManA16)

1

. -1 γ/s

-2

10

Figure 6. Viscosity η as a function of the shear rate γ& for poly(HEMAm-g-ManA16) (blue triangles) and poly(HEMAm-g-GulA20) solutions (red squares) in NaCl 0.1 mol L-1 at 25 °C.


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gel

solution


,

,


,

3

10

3

2

10

1

10

0

10

-1

10

-2

10

10

1

10

0

10

G'' (Pa)

2

10

G' (Pa)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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-1

10

-2

10

1

Frequency (Hz)

10

Figure 7. Variation of G´ and G´´ with frequency for the gels obtained from poly(HEMAm-g-ManA16) and poly(HEMAm-co-GulA20) at 25 °C in the linear domain. Note that in the case of poly(HEMAm-gGulA20) the value of G´ is missing because it was too small to be accurately measured. A gelation experiment was then realized starting from polymer solutions in deionized water (c0 ≅ 1.5 c∗). The latter were transferred to small dialysis cassettes possessing two parallel membranes and dialyzed against a CaCl2 0.5 mol L-1 for 28-48 hours. The resulting gels were recovered by cutting off the dialysis membrane with a scalpel: As shown in Figure 8, poly(HEMAm-g-ManA16) formed a loose opaque gel with the consistency of an after shave balm that was peeled off the dialysis membrane; whereas poly(HEMAm-co-GulA20) gave a soft self-standing transparent hydrogel from which a disk was punched out for rheological characterization (vide infra).


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Figure 8. Hydrogels of poly(HEMAm-g-ManA16) (left, laying on the dialysis membrane) and poly(HEMAm-g-GulA20) (right) obtained by dialysis against with CaCl2. The rheological properties of both gels were investigated in oscillatory and compression mode using a parallel-plate rheometer. For the two samples, G´ and G´´ were found to be essentially independent of frequency and the storage modulus was ~11 times the loss modulus, thus confirming a rubbery behaviour (Figure 7). Furthermore, at 0.5 Hz the viscoelastic moduli were one to three orders of magnitude higher than those observed in NaCl 0.1 mol L-1. As expected, the guluronan-derived gel had G´, G´´ values ~300 times higher than those of the mannuronan-derived one. Indeed, the mechanism leading to the formation of a gel of poly(HEMAm-gManA16) is unclear: The current understanding of the structure of alginate gels is that mannuronan sequences do not take part in formation of junction zones but rather act as flexible chains between them. 15

In our case, we speculate that at high Ca2+ concentration mannuronan graft chains precipitate and

form aggregates that are big enough to scatter visible light and render the system opaque. Previous work by Donati et al.

61

showed that in the presence of Ca2+ 0.025 mol L-1 pure (1→4)-β-D-mannuronan

remains soluble in water and does not undergo gelation. Nevertheless, when Smidsroed et al. 62 dialyzed an alginate sample with M / G = 9 against Ca2+ 0.34 mol L-1 they obtained a “voluminous slurry of aggregates” characterized by “very high turbidity and virtually no stiffness”, pretty much similar to what observed here. By contrast, we postulate that in the case of poly(HEMAm-g-GulA20) gelation occurs 30 ACS Paragon Plus Environment

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Biomacromolecules

via the intermolecular complexation of Ca2+ ions involving two or more guluronan graft-chains per junction zone and that the latter are connected by flexible poly(HEMAm) chains (Scheme 3). Scheme 3. Mechanism of ionotropic gelation of poly(HEMAm-g-GulA20).

= Ca2+

junction zone

polymer chains in solution

polymer network

The Young modulus (E) of the gel obtained from poly(HEMAm-g-GulA20) was determined by the initial slope of the force vs. deformation curve for a disk of known area and roughly constant thickness (Figure 9). A value of E = 5400 Pa was found which is much smaller than that obtained for an alginate gel under similar conditions (E = 28,000 Pa; FGulA = 0.45, Mw = 490 000 Da, [η] = 1280 mL g-1. c0 = 10 g L-1, dialysis against CaCl2 0.1 mol L-1):

63

Clearly, when compared to natural alginates, the use of a

biohybrid polymer enables the handling of less viscous solutions and leads to softer hydrogels.


Biomacromolecules

0.40 0.35 0.30 0.25

F/N

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.20 2

R = 0.998

0.15 0.10 0.05 0.00 0.00

1.69 N 0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

|∆y| / y0 Figure 9. Force (F) applied by the gel of poly(HEMAm-co-GulA20) vs. deformation under compression at 25 °C. The corresponding spring constant was obtained by a linear fit at small deformation (< 4%). The inset shows the hydrogel disk used for this experiment. Finally, gel beads of poly(HEMAm-co-GulA20) were obtained by simply dripping an aqueous solution of the polymer into CaCl2 0.5 mol L-1, akin to what normally observed with alginates.

64

This result

demonstrates that the biohybrid glycopolymer strategy can be applied to the preparation of functional hydrogels for cell encapsulation. CONCLUSION Acrylamide and methacrylamide macromonomers were synthesized in two steps starting from (1→4)β-D-mannuronan and (1→4)-α-L-guluronan oligosaccharides without resorting to protective group chemistry. Overall yields varied from poor (34%) to fair (46%) and were limited by the yield of the reductive amination step. The final materials consisted of a mixture of macromonomer and unfunctionalized oligosaccharides, the latter being inert towards the tertiary carbon-centered radicals involved in the ensuing radical polymerizations. Hence, the purity of the macromonomers was 32 ACS Paragon Plus Environment

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quantified by 1H NMR and they were used without further purification. Copolymerization with 2hydroxyethylmethacrylamide in aqueous solution afforded high molar mass biohybrid glycopolymers containing between 25% and 52 % by mass of oligosaccharide graft chains. A comparative kinetic study showed that both acrylamide- and methacrylamide-type macromonomers reacted since the early stages of the copolymerization, but that the mole fraction of macromonomer in the polymer was smaller than in the feed up to 50-60% conversion and increased markedly afterwards. This effect was slighter for the methacrylamide-type macromonomer though and methacrylamide derivatives were preferred for the synthesis of copolymers carrying longer oligosaccharide chains (Xn =16-20). After purification, the latter were used for a gelation experiment in the presence of CaCl2: Whereas the polymer carrying (1→4)-α-Lguluronan residues led to a soft isotropic self-standing transparent hydrogel, the polymer carrying (1→4)-β-D-mannuronan residues gave a loose opaque gel. Our study demonstrates that alginate-extracted oligosaccharides and aqueous radical polymerization can be brought together for the design of biohybrid glycopolymers capable of ionotropic gelation under mild conditions. This way, the versatility and precision of modern polymer chemistry can be combined with the gelation properties of (1→4)-α-L-guluronan to afford well defined glycopolymers (in terms of composition, molar mass, dispersity, architecture and functionality) with alginate-like gelation properties. For instance, this strategy may be used to prepare functional hydrogels for cell encapsulation and in-vitro 3-D cell culture simply by incorporating comonomers carrying adhesion peptides (or) biodegradable linkers

67, 68

65, 66

and

in the formulation of the staring polymer. This approach is now being

extended to the synthesis of well-defined biohybrid polymers with more complex architectures by aqueous reversible activation-deactivation radical polymerization. AKNOWLEDGMENTS

We thank the French Ministère de l'Enseignement supérieur et de la Recherche (A.G.), the Réseau de Recherche Chimie pour le développement durable CNRS-INRA (A.G. and A.H.), the Cluster de 33 ACS Paragon Plus Environment

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Recherche Chimie Durable et Chimie pour la Santé of the Rhône-Alpes region (L.A.), the competitiveness cluster Axelera (Lyon, France) and the Agence Nationale de la Recherche (ANR-09CP2D-02 ALGIMAT) for financial support. We also thank L. Buon, Ph. Colin-Morel, I. Jeacomine, F. Delolme (IBCP Lyon) and E. Bayma-Pecit for technical assistance with chromatographic, NMR, mass spectrometry and rheological measurements. SUPPORTING INFORMATION AVAILABLE Procedures for the compositional analysis of oligoglycuronans, Thermo Gravimetric analyses, the synthesis of ManA5MAm and ManA10Am and for copolymerizations; characterization of the oligosaccharides used in this study, summary of macromonomers’ synthesis; MALDI-ToF spectrum of ManA10-Am; 1H NMR spectra of ManA10-Am and GulA20-MAm. This information is available free of charge via the Internet at http://pubs.acs.org/. REFERENCES 1.

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64. Videos

Biomacromolecules

of

the

gel

beads

prepared

this

way

are

available

from

http://www.cermav.cnrs.fr/pages_perso/albertin_luca.htm. 65. Burke, R. S.; Pun, S. H. Bioconjugate Chem. 2009, 21, 140-150. 66. Jackson, D. C.; O'Brien-Simpson, N.; Ede, N. J.; Brown, L. E. Vaccine 1997, 15, 1697-1705. 67. Agarwal, S. Polym. Chem. 2010, 1, 953-964. 68. Chung, I. S.; Matyjaszewski, K. Macromolecules 2003, 36, 2995-2998.


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For Table of Contents use only

Biohybrid glycopolymer capable of ionotropic gelation Ali Ghadban 1, Luca Albertin 1,** , Marguerite Rinaudo 2, Alain Heyraud 1 1

Centre de Recherches sur les Macromolécules Végétales (CERMAV-CNRS)††, BP53, 38041

Grenoble, France. 2 European Synchrotron Radiation Facility, BP 220, 6 Rue Jules Horowitz, 38043, Grenoble, France.

junction zone

= Ca2+

**

Corresponding author, [email protected]. Tel. +33 (0)4 76 03 76 60. Fax +33 (0)4 76 54 72 03. ††

Affiliated with Université Joseph Fourier, and member of the Institut de Chimie Moléculaire de Grenoble. 40 ACS Paragon Plus Environment

Biohybrid Glycopolymer Capable of Ionotropic Gelation

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