Multivalent Carbohydrate-Functionalized Polymer Nanocrystals

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Multivalent Carbohydrate-Functionalized Polymer Nanocrystals Qiong Tong, Magnus S. Schmidt, Valentin Wittmann, and Stefan Mecking Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01460 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 10, 2018

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Biomacromolecules

Multivalent Carbohydrate-Functionalized Polymer Nanocrystals Qiong Tong, Magnus S. Schmidt, Valentin Wittmann*, Stefan Mecking*

University of Konstanz, Department of Chemistry, Universitätsstr. 10, D-78457 Konstanz, Germany

* corresponding author, email: [email protected], [email protected]

Keywords: glyconanoparticles, polyethylene nanocrystals, multivalent binding, catalytic polymerization

Abstract: Nanoparticles with a covalently bound shell of carbohydrate or sulfate groups, respectively, and a polyethylene core were generated by Ni(II)-catalyzed aqueous copolymerization of ethylene with comonomers undec-10-en-1-yl sulfate, undec-10-en-1-yl -Dglucoside or undec-10-en-1-yl -D-mannoside, respectively. Via remote substituents of the

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catalyst, the degree of branching and consequently degree of crystallinity of the polyethylene core could be controlled. This in turn impacts particle shapes, from spherical to anisotropic platelets, as observed by cryo-TEM. Enzyme-linked lectin assays revealed the mannosedecorated nanocrystals to be efficient multivalent ligands for Concavalin A.


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Introduction Carbohydrates, in the form of glycoproteins and glycolipids take part in numerous cellular recognition processes.1,2 Examples include cell-cell and cell-pathogen interactions, as well as immune responses. Furthermore, certain carbohydrate structures have been reported to be markers for diseases such as cancer or diabetes. Interactions between individual carbohydrate epitopes and lectins (carbohydrate-binding proteins) are usually weak. Nature uses multivalent interactions between multiple carbohydrate epitopes and multiple receptors displayed on a protein or cell surface to increase binding affinity (also known as the glycoside cluster effect).3,4 Since the bridging of binding sites is a major factor for increased affinities, the spatial arrangement of the carbohydrates has a decisive impact on achievable binding enhancements.5 However, structural data that prove the chelating binding mode of multivalent ligands is rare.6-11 Glyconanoparticles with their inherent high surface/volume ratio are promising mimics of cell surfaces and can provide a high local concentration of carbohydrates.12,13 With these properties, the application of nanomaterials in drug delivery, as pathogen sensors, toxin inhibitors and as diagnostic platforms can be envisioned. While inorganic nanoparticles offer distinct properties beneficial for e.g. optical detection and localization,14,15 organic polymers as a complimentary concept offer a broader scope of ACS Paragon Plus Environment

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functionality and tailoring of their structure.16,17 By comparison to dynamic blockcopolymer assemblies, polymer particles are more stable and their structure is less dependent on their environment, such as solute or electrolyte concentrations. To this end, dendritic polymers18-21 possessing a unimolecular micelle-like structure with an apolar interior and multiple functional groups on their surface suitable for further reactions such as bioconjugation have been studied.22 Due to its branched nature, their interior is intrinsically amorphous. Crystalline interactions as an ordering principle23 would enable the utilization of different polymer topologies, and the generation of particles with defined non-spherical shapes. Here, we report on nanoscale polymer crystals covalently substituted with multiple carbohydrate moieties on their surface. These very small crystallizable hydrocarbon polymer particles are accessed by aqueous catalytic copolymerization24 with water-soluble catalysts. These glyconanoparticles display an efficient multivalent binding to the plant lectin ConA, as revealed by a drastically increased affinity compared to monovalent analogues.

Experimental Section Materials and General Methods. Complexes 1 and 2 were prepared as reported previously.27,28 Comonomer B1 was synthesized according to a published procedure.32 Thin ACS Paragon Plus Environment

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layer chromatography (TLC) was carried out on aluminum sheets coated with silica gel 60 F254 (Merck, layer thickness 0.2 mm) with detection by UV light ( = 254 nm) and/or by charring with 15% sulphuric acid in ethanol. Flash column chromatography (FC) was performed on Merck silica gel 60 (0.040-0.063 mm) with the solvent systems specified. 1H NMR and 13C NMR spectra were recorded on Bruker Avance 400 and Bruker Avance DRX 600 instruments. Chemical shifts are reported in ppm relative to solvent signals: CDCl3: H = 7.26 ppm, C = 77.0 ppm; DMSO-

d6: H = 2.49 ppm, C = 39.7 ppm. Signals were assigned by two-dimensional 1H,1H and 1H,13C correlation spectroscopy. Electrospray ionization mass spectra (ESI-MS) were recorded on a Bruker Esquire 3000 spectrometer. Elemental analysis was performed on an elementar CHNS vario EL instrument. The molecular weight of isolated polymers was determined by size exclusion chromatography at 160 °C (vs. linear polyethylene standards). Thermal properties of the bulk polymers and dispersions were determined by differential scanning calorimetry (DSC) on a Netzsch Phoenix 204 F1 at a heating and cooling rate of 10 K min-1. Data given refers to second heating cycles. Crystallinities were calculated assuming a melting enthalpy of 293 J g-1 for 100% crystalline polyethylene, crystallinities are correct to refer to the polyethylene portion of the polymer chain (excluding the mass of the comonomer-derived side chain substituents). 1H

and

13C

NMR spectra of the isolated bulk polymers were recorded on a Bruker AC 250 ACS Paragon Plus Environment

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spectrometer in 1,1,2,2-tetra-chloroethane-d2 at 120 °C. The branching structures were assigned according to [25]. Particle size of the dispersions was measured by dynamic light scattering performed on a Malvern Nano Zeta Sizer ZEN 3600 (173° back scattering) on diluted dispersions by diluting a few drops of as-prepared dispersion in ca. 1 mL of distilled water. The autocorrelation function was analyzed using the Malvern dispersion technology software 5.1 algorithm to obtain volume weighted particle size distributions. Electron microscopy was performed on a Zeiss Libra 120 transmission electron microscope operated at 120 kV acceleration voltage. For cryo-TEM, a Gatan CT3500 cryo-transfer system was used. Specimens for the cryo-TEM investigations were prepared by freezing a thin film of the suspension in liquid ethane. The thin film was created by dripping a small amount of the suspension on a 700 mesh grid. A meniscus, thin enough for use in TEM, forms over the holes in the grid and is rapidly frozen to give a vitrified sample. The sample was then cryo-transferred into the TEM and examined at a temperature around 90 K with minimal electron dose. Synthesis of comonomer A. Comonomer A was synthesized according to [31] with minor modifications. Chlorosulfonic acid (5.2 g, 0.030 mol) was added dropwise into a ice-cooled flask containing 40 ml of pyridine. To the above solution, a solution of undec-10-ene-1-ol (6.5 g, 0.038 mol) in 10 ml pyridine was introduced dropwise via a dropping funnel. The obtained grayish ACS Paragon Plus Environment

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mixture was slowly warmed up to 55 °C, and kept stirring at this temperature overnight. The reaction mixture was then slowly poured into a ice-cooled saturated NaHCO3 aqueous solution under vigorous stirring. The obtained clear solution was kept stirring at room temperature overnight, then extracted with 1-butanol (3×50 ml). The combined organic phase was washed once with brine, dried over MgSO4 and concentrated in vacuo. The crude product was washed repeatedly with diethyl ether and acetone until white solid was obtained, which was redissolved in a solvent mixture of methanol/acetone (1:3), and filtrated through a Nylon filter to remove any undissolved residue. After removing the solvent, 5.3 g (yield: 64 %) comonomer A was obtained as white powder.

Na

8

10

O3SO 11

9

4

6 7

5

2 3

1

Comonomer A: 1H NMR (200 MHz, CD3OD, 298 K):  = 5.80 (m, 1H, H-2), 4.90-5.02 (m, 2H, H-1), 4.00 (t, 2H, 3JHH = 10 Hz, H-11), 2.06 (m, 2H, H-3), 1.69 (m, 2H, H-10), 1.32 (br, 12H, H-4 – H-9); 13C NMR (50 MHz, CD3OD, 298K):  140.1 (C2), 114.7 (C1), 69.3 (C11), 34.9 (C3), 30.730.1, (C4~C8 and C10), 26.9 (C9); Anal. Calcd for C11H21SO4Na: C, 48.51; H, 7.77; Found C, 48.82; H, 8.10.


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General procedure for the deacetylation of glycosides.26 To a solution of the peracetylated glycoside in MeOH is added a solution of sodium methylate (0.5 M in MeOH, 0.15 equiv). The mixture is stirred for 5-24 hours at rt. After neutralization with acidic ion exchange resin (DOWEX 50 W X8, H+ form), the mixture is filtered and the solvent evaporated to yield the deacetylated glycoside in >95 % yield. Synthesis of undec-10-enyl 2,3,4,6-tetra-O-acetyl--D-mannopyranoside (4). Compound 333 (6 g, 12 mmol) and 10-undecenol (1.65 mL, 8.1 mmol) were dissolved in dry CH2Cl2 (100 mL) under N2 atmosphere. BF3 x OEt2 (30 L) was added and the mixture was stirred for 20 h at rt. After addition of NEt3, the solvent was evaporated. Purification by FC (petroleum ether/EtOAc 3:1) yielded 4 (2 g, 50%) as a colorless solid together with the corresponding orthoester (1 g, 24%). Rf = 0.26 (petroleum ether/EtOAc 3:1); 1H NMR (600.1 MHz, CDCl3, 298 K):  = 5.80 (m, 1 H, -CH=CH2), 5.33 (dd, J = 9.9, 3.4 Hz, 1 H, H-3), 5.26 (‘t’, J = 9.9 Hz, 1 H, H-4), 5.22 (dd, J = 3.4, 1.7 Hz, 1 H, H-2), 5.00-4.91 (m, 2 H, -CH=CH2), 4.78 (d, J = 1.7 Hz, 1 H, H-1), 4.27 (dd, J = 12.2, 5.3 Hz, 1 H, H-6a), 4.10 (dd, J = 12.2, 2.3 Hz, 1 H, H-6b), 3.98 (ddd, J = 10.0, 5.3, 2.3 Hz, 1 H, H-5), 3.66 (dt, J = 9.6, 6.8 Hz, 1 H, OCH2), 3.44 (dt, J = 9.6, 6.8 Hz, 1 H, OCH2), 2.15 (s, 3 H, C(O)CH3), 2.09 (s, 3 H, C(O)CH3), 2.03 (s, 3 H, C(O)CH3), 2.02 (m, 2 H, CH2-CH=CH2), 1.98 (s, 3 H, C(O)CH3), 1.62-1.57 (m, 2 H, OCH2CH2), 1.39-1.35 (m, 2 H, OCH2CH2CH2), 1.28 ACS Paragon Plus Environment

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(br. s, 10 H, (CH2)5);

13C-NMR

(150.9 MHz, CDCl3, 298 K):  = 170.6 (C(O)CH3), 170.1

(C(O)CH3), 169.9 (C(O)CH3), 169.7 (C(O)CH3), 139.2 (CH=CH2), 114.1 (CH=CH2), 97.6 (C-1), 69.7 (C-2), 69.2 (C-3), 68.5 (OCH2), 68.4 (C-5), 66.3 (C-4), 62.5 (C-6), 33.8 (CH2-CH=CH2) 29.428.9 (6 x CH2), 26.0 (OCH2CH2CH2) 20.7 (C(O)CH3), 20.6 (C(O)CH3), 20.5 (C(O)CH3), 20.5 (C(O)CH3); ESI-MS: m/z Calcd for C25H40O10: 523.3 [M+Na]+, 539.2 [M+K]+; Found 523.5 [M+Na]+, 539.4 [M+K]+; Anal. Calcd for C25H40O10: C, 59.58; H, 8.05; Found C, 59.93; H, 8.15. Synthesis of undec-10-enyl -D-mannopyranoside (B2). A) Compound 4 was deacetylated according to the general procedure. B) D-Mannose (1 g, 5.56 mmol) was suspended in 10undecenol (5 mL, 25 mmol). TMSOTf (0.25 mL) was added and the mixture was stirred for 5 h at 90 °C. Purification by FC (CH2Cl2/MeOH 8:1) yielded B2 (1.12 g, 60 %) as a colorless solid. 1H

NMR (600.1 MHz, DMSO-d6, 298 K):  = 5.81 (m, 1 H, -CH=CH2), 5.01-4.91 (m, 2 H, -

CH=CH2), 4.81 (d, J = 1.2 Hz, 1 H, H-1), 3.97 (dd, J = 12.2, 1.8 Hz, 1 H, H-6a), 3.94 (‘t’, J = 9.8 Hz, 1 H, H-4), 5.22 (dd, J = 3.0, 1.2 Hz, 1 H, H-2), 3.85 (dd, J = 9.7, 3.0 Hz, 1 H, H-3), 3.76 (dd,

J = 12.2, 1.9 Hz, 1 H, H-6b), 3.61 (dt, J = 9.7, 6.8 Hz, 1 H, OCH2), 3.51 (m, 1 H, H-5), 3.38 (dt, J = 9.7, 6.8 Hz, 1 H, OCH2), 2.04 (q, J = 7.2 Hz, 2 H, CH2-CH=CH2), 1.56-1.52 (m, 2 H, OCH2CH2), 1.40-1.35 (m, 2 H, OCH2CH2CH2), 1.28 (br s, 10 H, (CH2)5); 13C-NMR (150.9 MHz, DMSO-d6, 298 K):  = 139.2 (CH=CH2), 114.1 (CH=CH2), 100.0 (C-1), 72.5 (C-5), 71.7 (C-3), ACS Paragon Plus Environment

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71.1 (C-2), 68.0 (OCH2), 66.2 (C-4), 61.0 (C-6), 33.8 (CH2-CH=CH2) 29.5-28.9 (6 x CH2), 26.1 (OCH2CH2CH2); ESI-MS: m/z Calcd for C17H32O6: 355.2 [M+Na]+, 371.2 [M+K]+; Found 355.2 [M+Na]+, 371.2 [M+K]+; Anal. Calcd for C17H32O6: C, 61.42; H, 9.70; Found C, 61.45; H, 9.79. Synthesis

of

2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl

2,3,4,6-tetra-O-acetyl--D-

mannopyra-noside (6). Compounds 3 (0.9 g, 1.8 mmol) and 543 (0.2 g, 0.9 mmol) were dissolved at 0 °C in dry CH2Cl2 (10 mL) under N2 atmosphere. BF3 OEt2 (11 L) was added and the mixture was stirred for 4 h at 0 °C. After addition of NEt3, the solvent was evaporated. Purification by FC (petroleum ether/EtOAc 2:1) yielded glycoside 6 (325 mg, 50%) as a colorless solid. Rf = 0.42 (petroleum ether/EtOAc 1:3); 1H NMR (400.1 MHz, CDCl3, 298 K):  = 5.35 (dd, J = 10.0, 3.3 Hz, 1 H, H-3), 5.28 (‘t’, J = 10.0 Hz, 1 H, H-4), 5.26 (dd, J = 3.3, 1.8 Hz, 1 H, H-2), 4.87 (d, J = 1.8 Hz, 1 H, H-1), 4.29 (dd, J = 12.4, 5.0 Hz, 1 H, H-6a), 4.08 (m, 1 H, H-6b), 4.06 (ddd, J = 10.0, 5.0, 2.4 Hz, 1 H, H-5), 3.80 (m, 1 H, Man-O-CH2), 3.69-3.62 (m, 13H, OCH2-CH2O), 3.39 (t, J = 5.2 Hz, 2 H, -CH2N3), 2.15 (s, 3 H, C(O)CH3), 2.00 (s, 3 H, C(O)CH3), 2.04 (s, 3 H, C(O)CH3), 1.98 (s, 3 H, C(O)CH3); 13C-NMR (100.6 MHz, CDCl3, 298 K):  = 170.7 (C(O)CH3), 170.0 (C(O)CH3), 169.9 (C(O)CH3), 169.7 (C(O)CH3), 97.7 (C-1), 70.7-70.6 (6 x OCH2-), 69.5 (C-2), 69.0 (C-3), 68.3 (C-5), 67.3 (Man-O-CH2), 66.1 (C-4), 62.4 (C-6), 50.7 (CH2N3), 20.9 (C(O)CH3), 20.6 (C(O)CH3), 20.6 (C(O)CH3), 20.6 (C(O)CH3); ESI-MS: m/z Calcd for ACS Paragon Plus Environment

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C22H35N3O13: 572.2 [M+Na]+, 588.2 [M+K]+; Found 572.1 [M+Na]+, 588.0 [M+K]+; Anal. Calcd for C22H35N3O13: C, 48.08; H, 6.42; N, 7.65; Found: C, 47.74; H, 6.31; N, 7.17. Synthesis

of

2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl

-D-mannopyranoside

(7).

Mannoside 6 was deacetylated according to the general procedure. 1H NMR (400.1 MHz, CDCl3, 298 K):  = 4.86 (d, J = 1.5 Hz, 1 H, H-1), 4.34-3.98 (m, 4 H, H-2, H-3, H-4, H-6a), 3.81-3.73 (m, 2 H, H-6b, Man-O-CH2), 3.70-3.60 (m, 13 H, OCH2-CH2O), 3.56 (m, 1 H, H-5), 3.40 (t, J = 5.3 Hz, 2 H, -CH2N3); 13C-NMR (100.6 MHz, CDCl3, 298 K):  = 100.2 (C-1), 72.4 (C-5), 71.7 (C-3), 70.9 (C-2), 70.7-70.6 (6 x OCH2-), 66.7 (Man-O-CH2), 66.5 (C-4), 61.1 (C-6), 50.7 (CH2N3); ESIMS: m/z Calcd for C14H27N3O9: 404.2 [M+Na]+, 420.1 [M+K]+; Found 404.0 [M+Na]+, 419.9 [M+K]+; Anal. Calcd for C14H27N3O9: C, 44.09; H, 7.14; N, 11.02; Found: C, 43.94; H, 7.47; N, 10.60. Synthesis

of

2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl

-D-mannopyranoside

(8).

Compound 7 (350 mg, 0.9 mmol) was dissolved in methanol. Pd/C (40 mg, 10% Pd) was added and the mixture was stirred for 4 h at rt under H2 atmosphere. After filtration, the solvent was evaporated to yield 8 (325 mg, 95 %) as a colorless oil. 1H NMR (400.1 MHz, DMSO-d6, 298 K):

 = 4.63 (d, J = 1.5 Hz, 1 H, H-1), 3.69-3.65 (m, 1 H, Man-OCH2), 3.64 (dd, J = 11.6, 1.8 Hz, 1 H, H-6a), 3.58 (dd, J = 3.3, 1.8 Hz, 1 H, H-2), 3.56-3.40 (m, 15 H, H-6b, H-4, OCH2-CH2O), 3.38ACS Paragon Plus Environment

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3.28 8 (m, 2 H, H-3, H-5), 3.35 (t, J = 5.9 Hz, 2 H, OCH2CH2NH2), 2.64 (t, J = 5.9 Hz, 2 H, CH2NH2).

13C-NMR

(100.6 MHz, DMSO-d6, 298 K):  = 100.0 (C-1), 74.0 (C-5), 72.8

(OCH2CH2NH2), 70.9 (C-4), 70.3 (C-2), 69.8-69.5 (5 x OCH2-), 67.0 (C-3), 65.7 (Man-O-CH2), 61.3 (C-6), 41.2 (CH2NH2); ESI-MS: m/z Calcd for C14H29NO9: 356.2 [M+H]+, 378.2 [M+Na]+; Found 356.0 [M+H]+, 378.0 [M+Na]+. Copolymerization in homogeneous aqueous phase was carried out in a 300 mL stainless steel mechanically stirred (1000 rpm) pressure reactor equipped with a heating/cooling jacket supplied by a thermostat controlled by a thermocouple dipping into the polymerization mixture. To a mixture of the desired amount of the comonomer and the respective catalyst precursor complex in a 250 mL Schlenk-flask were added 60 ml of distilled and degassed water at room temperature. When necessary, the sample was briefly sonicated to facilitate complete dissolution of the comonomer. The resulting homogeneous solution was then cannulatransferred to the argon-flushed reactor cooled to 12 °C. The reactor was pressurized to a constant pressure of 40 bar ethylene while the temperature was adjusted to 15° C. After 60 min reaction time ethylene feeding was interrupted, the reactor was carefully vented, and the obtained dispersion was filtrated through a plug of glass-wool. To determine the solids content, an aliquot of about 20 g dispersion was precipitated with 200 ml of MeOH. The obtained bulk ACS Paragon Plus Environment

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polymer was filtrated and thoroughly washed with methanol and water, and dried under vacuum at 50 °C overnight. Covalent immobilization of mannoside 8 on microtiter plates. 1,4-Phenylene diisothiocyanate (PDC) (125 mg, 0,65 mmol) was dissolved in DMSO and EtN(i-Pr)2 (100 L) was added. To the solution, DMSO was added to give a final volume of 25 mL. The freshly prepared solution was added to microtiter plates (Nunc, CovaLink NH modules, Cat. No. 478042) (100 µL per well) and incubated for 3 h at room temperature. The plates were emptied and rinsed with isopropanol (2x), water (2x), and carbonate/bicarbonate buffer (100 mM, pH 10.1) (2x). Then the microtiter plates were incubated with a freshly prepared solution of mannoside 8 (1.5 mM) in sodium carbonate/bicarbonate buffer (100 mM, pH 10.1) over night at room temperature (100 µL per well). The plates were emptied and rinsed with isopropanol (1x) and phosphate-buffered saline (10 mM sodium phosphate, 150 mM NaCl, pH 7.3) containing 0.05% Tween-20 (PBST) (4x). Enzyme-linked lectin assay (ELLA). The coated microtiter plates were blocked with 1% bovine serum albumin (BSA) in HEPES buffer (10 mM, pH 7.3) for 90 min at 37 °C (150 µL per well) and then washed with HEPES buffer containing 0.05% Tween-20 (HEPEST) (3x). Solutions of ConA-HRP (final concentration of 10 µg mL–1) and nanoparticles or mannoside 8 in serial dilutions in HEPES buffer containing CaCl2, MnCl2, and MgCl2 (each 1 mM) were pre-incubated ACS Paragon Plus Environment

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for 60 min at 37 °C. Then the ConA-HRP/inhibitor solutions were transferred to the blocked microtiter plates (100 µL per well). After incubation at 37 °C for 60 min, the wells were emptied and rinsed with HEPEST (3x). A solution of 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (12.5 mg per 25 mL) in citrate/phosphate buffer (200 mM, pH 4.0) containing 0.015% of H2O2 was added (100 µL per well). The plates were incubated for 20 min at room temperature in the dark. The reaction then was stopped by addition of 1 M H2SO4 (50 µL per well). After 5 min in darkness, the absorption was measured at 405 nm using a plate reader (BMG Labtech, FLUOstar OPTIMA). Percent inhibition was calculated according to: % inhibition = ([A(no inhibitor) - A(with inhibitor)]/A(no inhibitor)) x 100% IC50 values are reported as the concentration of soluble ligand required for inhibition of 50% of the binding of ConA-HRP to coated microtiter plates. For nanoparticles, the concentration refers to the concentration of mannose residues in the solution. All tests were performed in triplicate.


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Results and Discussion Nanoparticle synthesis. Copolymerization of ethylene with two different types of comonomers containing non-ionic saccharide and ionic sulfate groups were carried out based on considerations of colloidal stabilizing ability of different functional groups (Scheme 1). Two water-soluble neutral Ni(II) catalyst precursors (1, 2)27,28 with different remote substituents29 (R in Scheme 1) on the bidentate salicylaldiminato ligand were employed, with the aim of controlling the microstructure and crystallinity of the obtained copolymer particles, as already observed in ethylene homopolymerization in both toluene and aqueous media. The length of the alkyl linker between the olefinic double bond and the functional groups in the comonomers was chosen to be sufficiently long to prevent possible deactivation of the catalyst via chain-walking to the carbon of the functional group and further conceivable -X elimination.30


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Scheme 1. Copolymerization of ethylene with amphiphilic comonomers. TPPTS = tris(sodium m-sulfonatophenyl) phosphine. TPPDS = bis(sodium m-sulfonatophenyl) phenyl phosphine.

The catalyst precursors were observed to be stable and inert towards the substrate up to 48 hours in the presence of a large excess of the saccharide containing monomer. 1H NMR spectra of precatalyst 1a recorded in the presence of 30 eq. of the monomer containing a –(OCH2CH2)3glucose moiety (i.e. 120 eq. of –OH groups) after 48 h at room temperature show that the chemical shift of the nickel-bound methyl proton signal (Ni(II)-CH3) remained at -1.50 ppm (d, 3J

PH

= 7.2 Hz), and the intensity did not change significantly, by reference to the integrations of

the proton signals resonating at 2.86 and 2.99 ppm assigned to DMF (the precatalyst was


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obtained as the DMF adduct) (Figure S1). This also suggests that at room temperature precatalyst 1a does not polymerize the saccharide monomer in the absence of ethylene. The minor signal resonating at -1.32 ppm is due to the partial dissociation of the labile ligand TPPTS in CD3OD.28 A AcO AcO AcO

OAc O + O 3

RO 4 RO RO

6

BF3 OEt2, CH2Cl2, rt

HO 9

NH

50 %

Cl OR O

5 3

1

2

O

NaOMe, MeOH quant.

4: R = Ac B2: R = H

B HO HO HO

OH O OH +

D-mannose

TMS-OTf, 90 °C

HO 9

60 %

B2

Scheme 2. Synthesis of mannose-derived comonomer B2.

Comonomer A31 and the glucose-derived comonomer B132 were synthesized according to published procedure with slight modifications. For the synthesis of mannose-derived comonomer B2 trichloroacetimidate 333 was reacted with 10-undecenol in a Lewis acidcatalyzed Schmidt glycosylation followed by Zemplén deacetylation as shown in Scheme 2, A.


17


Page 18 of 49

Alternatively, B2 could be obtained in a single step from D-mannose by Fischer glycosylation (Scheme 2, B).


18

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Biomacromolecules

Table 1. Copolymerization of ethylene with amphiphilic comonomers a

en try

cat .

como.

(mM)

branching / 1000 C h

results

Ccomo yield (g)

TON g

Xcomo (mol. %)i

conv. como.j

Me

Et

long br. (≥C4)

bulk polymer properties Tm / Tc (°C)k

%cryst.l

Mn (∙ 103 g mol1) m

size (nm)

Mw / Mn

A%

o

n

m

nx per nm2 q

1

1 b

A

42

0.70

2500

2.0

20%

4

0

10

112 / 92

34%

n.a.

n.a.

12

50%

0.8

2

2a

A

43

0.48

1700

0.6

4%

45

2

6

68 / 46

17%

8.0

2.1

12

15%

0.3

3b

1 b

B1

5.8

0.14

500

1.4

20%

2

0

7

115 / 85

35%

n.a.

n.a.

n.a.

n.a.

n.a.

4c

2a

B1

8.5

0.20

730

0.4

6%

45

1

5

79 / 56

15%

1.6

3.3

90

75%

1.2

5d

1a

B2

9.0

0.49

1700

1.2

36%

4

0

6

116 / 93

34%

n.a.

n.a.

15

35%

0.6

6d

2a

B2

6.5

0.68

1200

0.2

11%

40

2

5

77 / 66

14%

n.a.

n.a.

40 p

15%

0.3

7e

1 b

-

-

2.6

9300

-

-

3

0

0

133 / 107

50%

204

1.4

10

60%

1.0

8e

2a

-

-

1.8

6400

-

-

55

3

3

75 / 63

18%

10

2.2

10

80%

1.4

9f

1a

-

-

2.0

7100

-

-

5

0

0

130 / 109

60%

120

1.8

80

60%

1.0

a

Reaction conditions: 10 mol catalyst precursor, 30 bar ethylene pressure, 15 °C, 60 mL of

water, stirring speed of 1000 rpm, polymerization time of 60 min. partially dissolved,

c

b

the monomer was only

5 ml MeOH were added in addition to dissolve the comonomer,

d

in the

presence of 0.1 g SDS. e ethylene homopolymerization in the presence of 0.75g SDS, f ethylene homopolymerization in the presence of 0.075 g SDS, and at an ethylene pressure of 40 bar,

g

mol (ethylene converted) mol (Ni)-1. h determined from 13C NMR, i determined from the integration of +B4+ carbon, j calculated from the incorporated vs. initially added comonomer, k determined by DSC l crystallinity corrected for the weight fraction of polyethylene segments in the copolymer chain,

m

determined by GPC,

n

volume average size determined by DLS,

o

surface area

coverage, calculated by (head group area of incorporated surfactant) / (surface area of all particles) by assuming the specific surface area per surfactant head group to be 0.62 nm2 and taking into account the particle size determined by DLS, p bimodal distribution, also contains about 40 vol.-% of >200 nm particles. q number of functional groups or surfactant head groups per nm2 particle surface, calculated by (number of incorporated surfactant molecules) / (particle surface area)


19


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Copolymerizations were studied in the presence of comonomers exclusively, without additional surfactant (Table 1, entry 1 to 3). Compared to ethylene homopolymerizations (in the presence of SDS surfactant) under otherwise identical conditions (Table 1, entry 7 and 8), both activity and productivity of the catalyst in copolymerizations are significantly lower. Ethylene mass flow traces recorded during copolymerizations showed the catalyst life time to be similar as observed in the homopolymerization, and lower catalyst activities were observed from the early stages of polymerizations on (Figure S2). 1H NMR spectra of the isolated copolymers (vide infra) show that the amount of internal olefinic protons, resulting from chain transfer by ß-hydride elimination and isomerization, is nearly the same as in the homopolymers. Available GPC traces and 1H NMR spectra show that the molecular weights of the obtained copolymers are slightly lower than, but still in the same order of magnitude as the homopolymers. The number-average molecular weights determined from the total double bond content from 1H NMR is in good agreement with available GPC data. GPC also shows that molecular weight distributions remained around Mw/Mn ca. 2. These observations indicate that the incorporation of the bulky comonomer slowed down chain growth,24 as expected, but did not significantly enhance deactivation of the catalyst by presence of the polar functional groups. A less efficient colloidal stabilization of primary particles may contribute additionally to decreased catalyst productivity. ACS Paragon Plus Environment

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Biomacromolecules

Also, a conceivable deactivation of the catalytically active species by the OH-moieties of the comonomer may occur to a minor extent (in NMR studies in CD3OD, it was observed that in the presence of ethylene, the deactivation rate of salicylaldiminato-Ni(II)Me(pyridine) complex was accelerated, despite the observation of polymer formation, which indicates a higher propensity of the polymerization active species to undergo alcoholysis34). Note that the copolymerizations were carried out at comonomer concentrations below or rather close to their respective c.m.c. (the c.m.c. of comonomer A was determined to be ca. 45 mM by measuring the concentration dependence of the surface tension of their neat aqueous solutions. By comparison, the c.m.c. of SDS was determined to be ca. 7 mM, which agrees with a previously reported value of 8.9 mM); however, a possible deactivation of the catalyst by an increased exposure to the aqueous phase in the absence of micelle formation can be excluded, as comparative ethylene homopolymerization carried out far below the c.m.c. of SDS showed no significant decrease of activity (Table 1, entry 9). In the absence of SDS, the copolymerization with glycoside-containing comonomer B1 occurs with a significantly decreased productivity, probably due to its very poor solubility in water. This hypothesis is supported by the observation of increased productivity upon addition of a small amount of MeOH (ca. 8 vol.-% vs. water), a very good solvent for this type of monomer (Table ACS Paragon Plus Environment

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1, entry 4). However, particle sizes of 90 nm were obtained, probably due to the insufficient particle stabilization in presence of MeOH. Copolymerizations with comonomer B2 were therefore carried out in the presence of 0.1 g SDS (Table 1, entry 5 and 6). Improved catalyst productivity and monomer incorporation ratio were obtained. However, a drawback of this approach is that physically adsorbed SDS can be only partially removed via dialysis (vide infra). Copolymer composition and microstructure. Covalent incorporation of the comonomers into the polymer backbone was confirmed by

13C

NMR and DEPT spectra of the isolated bulk

copolymers. In copolymers obtained with catalyst 1, the characteristic long chain branch tertiary carbon signal was observed at 38.1 – 38.3 ppm in the copolymers,

which

is

absent

in

ethylene

13C

NMR and DEPT spectra of the

homopolymers

(Figure

1).

In

ethylene

homopolymerization with catalyst 2, a small amount of long chain branches were also observed owing to its higher ‘chain running’ ability, however, the integration of the B4+ carbon signal in the

13C

NMR spectra of copolymers increased to an extent corresponding to the other

resonances arising from incorporated comonomers, as calculated from the increased amount of long alkyl chain branch tertiary carbon signals (Figure 2, corresponding DEPT, 13C and 1H NMR spectra of the isolated copolymers with comonomer B1 and B2 are given in Figure S3-S7,


22

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Biomacromolecules

comparative 13C and 1H NMR spectra of the isolated homopolymers obtained with precatalyst 1 and 2 are given in Figure S8 and S9, respectively).


23


Page 24 of 49

Figure 1 13C NMR and 1H NMR spectra of isolated copolymer from entry 1 (Table 1) in C2D2Cl4 at 130 °C. The broad proton resonance at 4.33 ppm is presumably due to partial hydrolysis of –SO4-Na+ to –OH.


24

Page 25 of 49

x

2.64

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

65.0

64.5

64.0

63.5 f1 (ppm)

63.0

62.5

Figure 2. 13C NMR and 1H NMR of isolated copolymer from entry 2 (Table 1) in C2D2Cl4 at 130 °C.


25


Page 26 of 49

The integration of the +B4+ carbon signal was used to determine the molar incorporation ratio of the comonomer. The obtained values agree within experimental errors with the integration of the characteristic proton signal arising from the methylene group in -position to the functional group of the comonomer observed at 3.7 ppm (the minor signal at 4.3 ppm was also observed in a 1H NMR spectrum of SDS taken in C2D2Cl4 under the same conditions. It is presumably due to partial hydrolysis of the sulfate group to alcohol, which has been reported previously35), to which any conceivable contribution from unreacted comonomers can be excluded, since the characteristic allylic carbon signals at 138 and 113 ppm and the proton (CH2=CH-) multiplet at 5.77~5.87 ppm of the free comonomers were not detected. The characteristic carbon signals of the methylene group in -position to the functional group (designated as x) in the isolated copolymers were observed at nearly the same chemical shift as in the free comonomers (at the resonance 63.4~63.5 ppm for sulfate containing copolymers), as expected, due to the presence of the long alkyl linker, except for the copolymers obtained with comonomer type B, for which partial decomposition of the functional groups during the overnight

13C

NMR measurements at 130 °C is suspected. This is suggested by different chemical

shifts of the methylene carbon signal of the -position to the saccharide groups at 63.2 ppm by


26

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Biomacromolecules

comparison to 68.7 ppm for the neat comonomer, as well as other –OH attached methine carbon signals in the region of 66~73 ppm (Figure S6). The integration of characteristic carbon signals of the -position to the functional groups were observed to be lower than the expected value calculated from the incorporation ratio determined from B4+ integration, probably due to its longer relaxation time. NMR analysis (1H-,

13C

NMR, 1H,1H-gCOSY and 1H,13C-gHSQC) of the free comonomers in

C2D2Cl4 show that the x carbon resonates at a chemical shift of 29.5 ± 0.3 ppm, depending on the individual polar functional groups. In the copolymers, this signal is obscured by the intense + peak at 30 ppm (cf. Experimental section and Figure S11 for detailed NMR characterization of the free comonomer A and B2. Presumably, in the polymers the chemical shifts of the proton and carbon atoms remote from the long alkyl chain attached branching points do not differ from the free comonomers). The specific carbon resonance at 26.2 ppm (assigned as x) supports the presence of the functional group, and the integration of this signal agrees with the incorporation ratio determined from the B4+ signal, except in the copolymers obtained with comonomer A where a lower value was observed. The reason for the latter remains unclear. In general, ethylene incorporation dominates due to the selectivity of the catalysts applied for ethylene. About 0.2 to 2.0 mol.-% of comonomer were incorporated into the polyethylene ACS Paragon Plus Environment

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Page 28 of 49

backbone. This observation qualitatively agrees with the previous studies on the copolymerization of ethylene with nonpolar 1-olefins, such as 1-butene and norbornene, with lipophilic salicylaldiminato Ni(II) complexes in both toluene and miniemulsion, in which ethylene incorporation was also observed to be strongly favored.24,36 Interestingly, in copolymerization with all three types of comonomers, both higher productivity and a higher comonomer rincorporation were observed for precatalyst 1 by comparison to 2. Decreased productivity in copolymerization with precatalyst 2 could originate from its stronger propensity for ‘chain walking’ along the alkyl chain towards the functional group, which could deactivate the catalyst. A tentative explanation for higher incorporation ratios with precatalyst 1 is that possibly rapid crystallization occurred in the higher crystalline particles formed by catalyst 1 (vide infra), which could expel the lipophilic catalyst active site towards the particle surface and hereby facilitate its accessibility for the amphiphilic comonomers located at the particle/water interface. During the polymerization with precatalyst 2, due to the more pronounced amorphous nature of the formed particles, the lipophilic catalyst active site is probably located more at the interior of the particle, thus its accessibility for the amphiphilic comonomer is more restricted.


28

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Biomacromolecules 13C

NMR analysis also shows that microstructure control via the remote substituents on the

catalyst ligand backbone occured in a similar fashion as in ethylene homopolymerization. Homopolyethylene obtained with catalyst precursor 1 has a highly linear structure with only 3 methyl branches/1000 carbon atoms. In contrast, a degree of branching of 55 methyl branches/1000 carbon atoms and a small extent of ethyl and long alkyl chain branches are observed in homopolyethylene obtained with precatalyst 2, which results in significant differences in crystallinity of the two homopolymers (ca. 50% and

Multivalent Carbohydrate-Functionalized Polymer Nanocrystals

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