Glycopolymers Prepared by Alternating Ring-Opening Metathesis Polymerization Provide Access to Distinct, Multivalent Structures for the Probing of Biological Activity

A myriad of biological processes are facilitated by ligand–receptor interactions. The low affinities of these interactions are typically enhanced by multivalent engagements to promote binding. However, each biological interaction requires a unique display and orientation of ligands. Therefore, the availability and diversity of synthetic multivalent probes are invaluable to the investigation of ligand–receptor binding interactions. Here, we report glycopolymers prepared from bicyclo[4.2.0]oct-6-ene-7-carboxamide and 4,7-dihydro-1,3-dioxepin or cyclohexene. These glycopolymers, synthesized by alternating ring-opening metathesis polymerization, display precise ligand spacing as well as the option of a hydrophobic or acetal-functionalized polymer backbone. Small-angle X-ray scattering (SAXS) data analysis revealed that these [4.2.0] glycopolymers adopted distinct conformations in solution. In aqueous media, [4.2.0]-dioxepin glycopolymers formed swollen polymer chains with rod-like, flexible structures while [4.2.0]-cyclohexene glycopolymers assumed compact, globular structures. To illustrate how these glycopolymers could aid in the exploration of ligand–receptor interactions, we incorporated the [4.2.0] glycopolymers into a biological assay to assess their potential as activators of acrosomal exocytosis (AE) in mouse sperm. The results of the biological assay confirmed that the differing structures of the [4.2.0] glycopolymers would evoke distinct biological responses; [4.2.0]-cyclohexene glycopolymers induced AE in mouse sperm while [4.2.0]-dioxepin glycopolymers did not. Herein, we provide two options for glycopolymers with low to moderate molecular weight dispersities and low cytotoxicity that can be implemented into biological assays based on the desired hydrophobicity, rigidity, and structural conformation of the polymer probe.


Table of Contents
Preparation of SBTI-Alexa 488 Conjugate.A 2 mg/mL solution of soybean trypsin inhibitor (SBTI) was prepared using 0.1 M NaHCO3 (pH: 8.3).500.0µL (1.0 mg) of the protein solution was then transferred into a reaction vial containing Alexa Fluor™ 488 tetrafluorophenyl (TFP) ester that was warmed to 25 °C.
The mixture was allowed to stir gently at 25 °C in the dark for 1 h.The reaction mixture was purified using size exclusion chromatography (Cytiva disposable PD-10 desalting column with Sephadex G-25 resin, 1.0-2.5 mL samples) with 1X Dulbecco's phosphate-buffered saline (DPBS, containing 0.02% sodium azide) as the elution buffer.The bottom yellow-green band in the column corresponding to the protein conjugate was collected.The top green band, which contained unreacted and hydrolyzed dye, was disposed.After purification, the protein conjugate was transferred to a prewetted Amicon Ⓡ Ultra-15 Centrifugal Filter Unit (MWCO 3kD, 15 mL sample) and centrifuged at 5,000 rpm for 5 min to concentrate the protein conjugate to 1.0 mL.Once concentrated, the conjugate was prepared for relabeling.Two reaction vials of Alexa Fluor™ 488 TFP ester were warmed to 25 °C and combined in a single vial using 1.0 mL of protein conjugate.100.0 µL of 0.1 M NaHCO3 was then added to the reaction to increase the pH of the solution.
The reaction mixture was then left to stir at 25 °C in the dark for 1 h before transferring to 4 °C and allowing to stir gently for an additional 24 h in the dark.The relabeled protein conjugate was then purified by size exclusion chromatography for a second time with 1X DPBS buffer (containing 0.02% sodium azide) as the elution buffer and analyzed by UV-vis spectroscopy.

UV-vis Analysis of SBTI-Alexa 488 Conjugate.
UV-vis analysis of the SBTI-Alexa 488 conjugate was performed on a Shimadzu UV-vis spectrophotometer (UV-2550).1X DPBS (containing 0.02% of sodium azide) was used as a reference and all of the readings were taken at 25 °C.Appropriate dilutions of the SBTI-Alexa 488 conjugate were made with 1X DPBS to ensure that the absorbance values were less than 1.0.Absorbance was measured in a black quartz cuvette with a 1 cm path length at 280 nm (A280) and 494 nm (A494) to account for the absorbance maximum of the Alexa Fluor™ 488 dye (Figure S1).The concentration of the labeled protein and the degree of labeling were calculated.The molar extinction coefficient of Alexa Fluor™ 488, correction factor of Alexa Fluor™ 488 emission at 280 nm, and the molecular weight of SBTI were all incorporated into the calculations (Equation S1 and Equation S2).The degree of labeling for various batches of protein conjugates ranged between 1 and 3 moles of dye per mole of protein, which was sufficient for our experiments (Table S1 Guinier-Porod Model and Power Law Equations.A Guinier-Porod model 5 was used for fitting data where the intensity is calculated as a piecewise function based on the scattering vector q1 (Equation S3).
Below q1, scattering is dominated by Guinier scattering (Equation S4), and above q1, scattering is defined by Porod scattering (Equation S5).The fitting parameters are the radius of gyration (Rg), the Porod exponent (m), a dimensionality parameter (s), and two scale factors: Guinier (G) and Porod (D) (Equation S6).S3) S4) S6) (Equation S7) The radius of gyration, Rg, for cylinders is provided in Equation S7.This model was combined in some cases with an additional power law model which contains parameters for scale and power (Equation S8).
The scale is not directly the volume fraction, and these additional parameters were added to account for any corrections to the background subtraction.The combined model also contains parameters for scale and background which multiply or add to all scattering intensity by a number.
General Methods.Air and moisture-sensitive reactions were performed in a glovebox under nitrogen atmosphere or using a standard Schlenk vacuum line under nitrogen.Analytical thin-layer chromatography (TLC) was performed on aluminum-backed sheets coated with silica gel 60F254.Non-UV active compounds were detected on the plates by staining with 10% (w/v) phosphomolybdic acid in ethanol.Flash column chromatography was performed using a CombiFlash system with RediSep normal phase silica columns (Teledyne ISCO, silica gel 60, 230−400 mesh).HRMS(ESI) was performed with the Agilent LC-UV-TOF consisting of a 1260 UPLC, a UV-Vis diode-array detector (DAD) and a TOF mass analyzer.
General preparation of poly(1′)100 and poly(1)100.Grubbs' third generation catalyst 4 was prepared as described by the literature. 2 D-Mannose (1a′), L-fucose (1b′), poly(1a)100, poly(1b)100, and acetylated precursors were prepared as described by the literature with matching spectra. 3,4neral Preparation of poly(1′)50.To a septum-sealed vial containing nitrogen and a stir bar, a solution of 4 (1.8 mM, 1 equiv) in dichloromethane (600 µL) was added and chilled to 0 °C for 10 min.A solution of 1a′ or 1b′ (90.2 mM, 50 equiv) in dichloromethane (400 µL) was then added to the vial and the reaction was initiated at 0 °C for 10 min.The reaction mixture was stirred at 25 °C for an additional 90 min.An excess of ethyl vinyl ether (0.1 mL) was used to terminate the reaction and left to stir for 30 min at 25 °C.
Ethyl ether chilled to -20 °C was then used to precipitate the polymer, resulting in an off-white solid.After drying in vacuo to remove residual solvents, the polymer was analyzed by GPC and NMR to determine dispersity and purity.

General
Scheme S1.Synthesis of poly(1′)50 and poly(1)50   The Kratky plot shows q vs. q 2 *I(q).This can give insight into the arrangement or folding of scatterers in solution.The Kratky plots for poly(1)100 and poly(1)50 show a broad peak at low-q, followed by a steady increase at higher q.This is usually correlated to partially unfolded proteins or slightly swollen polymer chains. 6This is consistent with the flexible cylinder model used to fit the data as it suggests that the polymers may be coiling or folding due to more favorable interactions between polymer chains rather than the solvent.

Preparation of poly( 1 ) 50 .
To a reaction vial containing poly(1′)50 (50 mg), K2CO3 was added in excess.4.0 mL of an anhydrous mixture of MeOH:THF (2:1 v/v) was added to the vial and the reaction was left to stir at 25 °C for 90 min.The reaction mixture was concentrated in vacuo, neutralized using 5.0 mL of 1 N HCl in H2O:THF (1:1 v/v), and left to stir for 90 min.The reaction mixture was transferred to a prewetted cellulose ester Spectra/Por Ⓡ Float-A-Lyzer Ⓡ G2 dialysis device (MWCO 3.5-5kD, 5 mL) and dialyzed against DI water for at least 3 d.The mixture was then lyophilized for 2 d to afford an off-white solid.

Figure S3 .
Figure S3.Comparison of the average percent of live mouse sperm after treatment with glycopolymers or the negative control (DPBS).The average percentage of live sperm treated with DPBS was 13%.Data represents mean ± standard error of the mean of the cell viability at each of the five polymer concentrations.One-way ANOVA was used to compare the average cell viability of glycopolymers to the average cell viability of the negative control where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.There was no statistically significant difference between the cell viability of any of the samples treated with glycopolymers or the negative control.