Hyaluronan (HA)-inspired glycopolymers as molecular tools for studying HA functions

Hyaluronic acid (HA), the only non-sulphated glycosaminoglycan, serves numerous structural and biological functions in the human body, from providing viscoelasticity in tissues to creating hydrated environments for cell migration and proliferation. HA is also involved in the regulation of morphogenesis, inflammation and tumorigenesis through interactions with specific HA-binding proteins. Whilst the physicochemical and biological properties of HA have been widely studied for decades, the exact mechanisms by which HA exerts its multiple functions are not completely understood. Glycopolymers offer a simple and precise synthetic platform for the preparation of glycan analogues, being an alternative to the demanding synthetic chemical glycosylation. A library of homo, statistical and alternating HA glycopolymers were synthesised by reversible addition–fragmentation chain transfer polymerisation and post-modification utilising copper alkyne–azide cycloaddition to graft orthogonal pendant HA monosaccharides (N-acetyl glucosamine: GlcNAc and glucuronic acid: GlcA) onto the polymer. Using surface plasmon resonance, the binding of the glycopolymers to known HA-binding peptides and proteins (CD44, hyaluronidase) was assessed and compared to carbohydrate-binding proteins (lectins). These studies revealed potential structure-binding relationships between HA monosaccharides and HA receptors and novel HA binders, such as Dectin-1 and DEC-205 lectins. The inhibitory effect of HA glycopolymers on hyaluronidase (HAase) activity was also investigated suggesting GlcNAc- and GlcA-based glycopolymers as potential HAase inhibitors.

Gel Permeation Chromatography (GPC) was performed on an Agilent 1260 infinity system operating in DMF with 5 mM NH 4 BF 4 and equipped with refractive index detector and variable wavelength detector, 2 PLgel 5 μm mixed-C columns (300 × 7.5 mm), a PLgel 5 mm guard column (50 × 7.5 mm), a differential refractive index (DRI) and a variable wavelength detector (VWD). The system was eluted with DMF at a flow rate of 1 mL min -1 and the DRI was calibrated with linear narrow polystyrene standards. All the samples for GPC were previously filtered through a 0.22 μm filter.
Infrared spectra were collected on a Tensor 27 (Bruker) FTIR spectrometer cooled by N 2 (l) coupled with a platinum attenuated total reflectance (ATR) for both solid and liquid samples.
The polymers mass was measured on a Bruker Daltonics AuToFlex MALDI-ToF mass spectrometer, with a nitrogen laser at 337 nm and equipped with positive ion ToF detection. A 9:1:1 solution of: matrix (30 mg/mL), potassium trifluoroacetate (10 mg/mL) and the sample (10 mg/mL) dissolved in THF, with 2 μL being deposited onto the stainless-steel target plate. Spectra were recorded in reflectron mode with the laser set at 20-30% power and masses determined against 200-6000 Da calibration.
Small angle x-ray scattering patterns of glycopolymer solutions (200 µM) were obtained with a SAXSLAB GANESHA 300-XL. Cu  radiation was generated by a Genix 3D Cu-source with an integrated monochromator, 3-pinhole collimation and a two-dimensional Pilatus 300K detector. The scattering intensity q was recorded at intervals of 0.012 < q < 0.3 Å -1 (corresponding to lengths of 10-800 Å). Measurements were performed under vacuum at the ambient temperature. The scattering curves were corrected for counting time and sample absorption. The solution under study was sealed in thinwalled quartz capillaries about 1.5 mm in diameter and 0.01 mm wall thickness. The scattering spectra of the solvent were subtracted from the corresponding solution data using the Irena package for analysis of small-angle scattering data 1 Data analysis was based on fitting the scattering curve to an appropriate model by software provided by NIST (NIST SANS analysis version 7.0 on IGOR). 2 Model fitting of the small-angle scattering pattern The form factor of a semiflexible chain with excluded volume effects is expressed by 3 This describes a chain comprised of a series of locally stiff segments by three parameters b -Kuhn's length, a measure of the chain flexibility, L -the chain's contour length, and R -radius of the chain's cross-section.
The mass of the peptide was determined using an LC-MS Agilent '1100 LC/MSD Trap' in methanol.
Circular dichroism (CD) was used to measure the secondary structure of the peptide on a Chirascan CD spectrometer (Applied Photophysics) from 190 to 300 nm at 25 °C in H 2 O at 110 μM in a 3 mm quartz cuvette, with a blank run in pure water.
TEM analysis was carried out on glycopolymer solutions at 1 mM at pH 7 after being aged for 48 hours. The glycopolymer solutions were loaded onto the carbon film-coated copper grids (400 mesh, Agar Scientific, UK) and negatively stained by 2 wt% uranyl acetate (Agar Scientific, UK). The excess staining solution on the grids was removed with filter paper and the grids were allowed to dry at room temperature for at least 3 hours. Bright-field TEM imaging was performed on a JEOL 1230 TEM operated at an acceleration voltage of 100 kV and the TEM images were recorded by an SIS Megaview III wide-angle CCD camera Dynamic Light Scattering (DLS) on the glycopolymers was performed in a Nano-ZS Zetasizer (Malvern Instruments). Glycopolymer solutions were prepared above the CAC point at 1 mM at pH 7 and allowed to stand for 2 days to ensure s formation of aggregates. The sample was diluted 10-fold and passed through a 0.2 μm filter. The cell and the DLS machine were cleaned using compressed air to ensure removal of large dust particles, before reading.

Cell Viability
Cell viability was assessed using the LIVE/DEAD™ viability/cytotoxicity kit for mammalian cells (Invitrogen) and following the manufacturer's instructions. Briefly, LuC4 cells (expressed for a human oral squamous cell carcinoma cell line) were seeded at 5,000 cells per well into a 96 well plate and grown in a humidified atmosphere at 37 °C, 5% CO 2 for 48 hours. Cells were treated with glycopolymers at a range of concentrations, 0.1, 1, 10, 100, µg/mL, and incubated for 24 hours. Media was removed, and live/dead dyes (green-fluorescent calcein-AM/red-fluorescent ethidium homodimer-1) were added in PBS and incubated for 1 hour. Fluorescence was read using Synergy HT (BioTek) at 530 nm for calcein-AM (live cells) and at 645 nm for ethidium homodimer-1 (dead cells). The number of live and dead cells was calculated as a percentage of the fluorescent output compared to a control containing no glycopolymer for the live control and methanol fixed cells for the dead assay.

Self-Assembled Monolayers (SAMs) of HA-binding peptide (Pep-1)
The formation of the Pep-1 SAMs was performed on gold-coated coverslips, cleaned using basic piranha mixture before use. The clean gold surfaces were placed in an ethanoic solution of thiolated Pep-1 (1% w/v) for 24 hours at 37 ºC, after which the surface was washed with water and dried under a nitrogen stream. The contact angle was measured using a Drop Shape Analyzer -DSA100 (Kurss). The incubation process was repeated in an aqueous solution of 1.5 MDa HA (0.5 % w/v). The contact angle was again measured and compared to a control of a bare gold surface which had been treated in a similar way but with no peptide or HA in the incubation solutions.

Surface Plasmon Resonance (SPR)
A BIAcore 2000 system SPR (Cytiva) was used for interaction analysis. The CD44 protein (Recombinant Human CD44 Fc Chimera Protein, 48.6 kDa, 0.025 mg/ml) in 10 mM sodium acetate buffer solution at pH 5.5 was immobilized via a standard amino coupling protocol onto a CM5 sensor chip that was activated by flowing a 1:1 mixture of 0.1 M N-hydroxysuccinimide and 0.1 M N-ethyl-N'-(dimethylaminopropyl) carbodiimide over the chip for 6 min at 25 °C at a flow rate of 20 µL/min. Subsequently, active channels including 1 (blank) were blocked with a solution of ethanolamine (1 M, pH 8.5) for 10 min at 5 µL/min. Polymer solutions were prepared at varying concentrations (2000 nM-125 nM) in PBS buffer plus 0.005% Tween20 at pH 7.4. Sensorgrams for each glycopolymer concentration were recorded with a 300 seconds injection of polymer solution (association) followed by 150 seconds of buffer alone (dissociation). Regeneration of the sensor chip surfaces was performed using 100 mM glycine HCl at pH 2.0. Kinetic data were evaluated using a single set of sites (1:1 Langmuir Binding) model and also a bivalent model in BIAevaluation 3.1 software.
All lectins (Recombinant Human Proteins, 0.025 mg/ml) were immobilized as described above and the system was equilibrated with filtered HEPES buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 5 mM CaCl 2 , 0.01% P20 surfactant solution). For the immobilization of Pep-1, a gold surface without any modification was used and thiolated peptide (1 mg/ml) was run at a flow rate of 2 µL/min for 15 min at 25 °C to provide high attachment of peptide on the gold surface. Regeneration of the sensor chip surfaces was performed using 10 mM HEPES pH 7.4, 150 mM NaCl, 10 mM EDTA, 0.01% P20 surfactant solution for the lectins. For regeneration of peptides immobilized surface, 50 mM NaOH solution was used. Hyaluronidase (0.025 mg/ml) was immobilized as described above for lectins. A 1-min pulse of a mixture of 50 mM NaOH and 1 M NaCl was used to regenerate the surface after each binding cycle.

1,2,3,4-tetra-O-acetyl-α-D-glucopyranuronate
The synthesis has been modified from previously reported method 6 . Briefly, glucuronic acid (5.14 g) was added to a stirring solution of Ac 2 O (25 mL, 264 mmol) and H 2 SO 4 (5 drops) which was then heated to 65 ºC. Once the added sugar was dissolved, more glucuronic acid (5.09 g) was added (Total glucuronic acid; 10.23 g, 52.7 mmol). The solution was stirred for 1 hour and then cooled to 25 ºC. H 2 O (75 mL) was added and then the product was extracted into DCM (3 x 50 mL). The organic layers were combined, dried over MgSO 4 and reduced under negative pressure and azeotrope against toluene. The product was used without further purification.

2-Azidoethanol
The synthesis was based in a previously reported method 7 . Briefly, 2-bromoethanol (10 g, 80 mmol) was suspended in H 2 O (20 mL) followed by the addition of NaN 3 (9.53 g, 144 mmol) and NBu 4 Br (590 mg). The reaction was stirred for 16 hours at 80 ºC. The reaction was cooled and extracted into Et 2 O (10 x 50 mL). The organic layers were combined and reduced to give the product as a clear oil. General procedure for the glycosidation by Lewis acid Methyl 1,2,3,4-tetra-O-acetyl-α-D-glucopyranuronate (1 equiv) and propargyl alcohol (4 equiv) were suspended in anhydrous DCM (25 mL). The reaction was cooled to 0 ºC at which point BF 3 .OEt 2 (2 equiv) was added. The reaction was stirred at 0 ºC for 15 minutes and then allowed to warm to 25 ºC and stirred for 16 hours. The reaction was diluted with DCM and then washed with water (2 x 50 mL) and brine (50 mL). The organic layers were combined, dried over MgSO 4 and reduced under negative pressure. The oil was purified by flash chromatography, EtOAc: hexane (1:1).

General procedure for polymerisations
The synthesis has been modified from previously reported method 9 . The polymers were synthesised by RAFT polymerisation, using BDTMP as the RAFT agent and V601 as the initiator. Briefly, all the components were combined and purged in a Schlenk tube under nitrogen flow for 30 minutes, after which point the reaction was placed into an oil bath at 70 ºC to initiate the reaction. Once complete, the reaction was quenched in an ice bath followed by exposure to the atmosphere. The solution was precipitated into cold methanol or isopropanol and the product collected by filtration.

General procedure for microwave-assisted solid-phase peptide synthesis (SPSS)
The peptides were synthesised in an automated microwave peptide synthesizer (Liberty Blue, CEM) by solid-phase method on a Rink amide 4-methylbenzhydrylamine resin following standard 9-fluorenyl methoxycarbonyl (Fmoc) protocol. Couplings were done with the Fmoc-amino acids, HOBt and DIC in 4 times excess dissolved in DMF. The Fmoc group was deprotected with 20% piperidine in DMF (v/v). After each cycle, the resin was washed with DMF before the next cycle.

N-terminal thiol capping
After synthesis has been completed, the resin with bound peptide was removed from the synthesiser and 3-(((4-methoxyphenyl) diphenylmethyl)thio)propanoic acid was coupled to the peptide Nterminal using HOBt and DIC in 4 times excess. The mixture was shaken for 2 hours. After the capping cycle, the resin was subsequently washed with DMF followed by DCM and Kaiser test performed to confirm the coupling.

Thiol peptide cleavage
The peptide and acid-labile side groups were cleaved by incubating the resin with TFA/thioanisole/anisole/EDT (90:5:2.5:2.5) for 3 hours. The solution was collected, concentrated by reduced pressure and precipitated in cold ether. The resulting suspension was collected by centrifugation and decanting the supernatant, followed by lyophilisation of the resulting in the crude peptide.

Peptide characterization and purification
The crude peptides were passed through a 0.2 µm filter and then purified on an AutoPurification System (Waters) using a preparative reverse-phase C18 column (XBridge, 130 Å, 5 μm, 30×150 mm, Waters) with a gradient running from 98:2 to 0:100 of water/acetonitrile supplemented with 0.1% TFA over 30 minutes at 20 mL/min. The fractions were detected by the SQ Mass Detector (Waters). These fractions were combined, reduced under negative pressure and then lyophilised to produce a white powder. Figure S1. Low-resolution MALDI-ToF MS for P 9 between (a) 3000 -6000 m/z showing multiple distributions with (b) a zoom between 4900 -5400 m/z to look at a single repeat and the smaller distributions assigned (Table S1) between the major distribution highlighted by the arrow, with those polymers initiated with the initiator rather than the RAFT agent highlighted in blue.  Figure S2. Offset FT-IR spectra to track the formation of the pendant azide at 2100 cm -1 of P 2 (light blue) from the chloride of P 1 (blue) followed by the consumption of the azide in the CuAAC to form the glycopolymer (pVB-GlcNAc, P 6 )   Figure S4. Example of GPC traces for the formation of a glycopolymer (pVB-GlcNAc, P 6 ) from the polymerisation of VBC to form P 1 (blue) through the conversion of the chloride to azide, P 2 (light blue) to the glycopolymer, P 6 (grey) by CuAAc. Figure S5. Example of NMR spectra (offset) to trace the formation of a glycopolymer (pVB-GlcNAc, P 6 ) the pendant group from pVBC (P 7 ) via PVBAz (P 2 ) showing the shift of the CH 2 at 4.6 ppm (P 1 ) to 4.3 ppm (P 2 ) as well as the introduction of the triazole peak at 8.1 ppm and sugar peaks between 3.0 -5.0 ppm (P 6 ).          Figure S12. Glycopolymer mean size distribution of the aggregates obtained by ImageJ analysis of TEM images (left) and DLS on the glycopolymers at 0.1 mM at pH 7. Analysed by 2one-way ANOVA analysis, where * significant at P < 0.0332, ** significant at P < 0.0021, *** significant at P < 0.0002 and **** significant at P < 0.0001.   Figure S17. Circular dichroism spectra of acetylated (Ac-GAHWQFNALTVR-CONH 2 , non-thiolated) Pep-1 at 110 μM in water at various pHs, showing a random coil. Figure S18. Contact angle of gold-coated coverslips, before and after immersion is an ethanoic solution of thiolated Pep-1 followed by the incubation with 1.5 MDa HA. Analysed by 2one-way ANOVA analysis, where * significant at P < 0.0332, ** significant at P < 0.0021, *** significant at P < 0.0002 and **** significant at P < 0.0001.