Sialic Acid-Responsive Polymeric Interface Material: From Molecular Recognition to Macroscopic Property Switching

Biological systems that utilize multiple weak non-covalent interactions and hierarchical assemblies to achieve various bio-functions bring much inspiration for the design of artificial biomaterials. However, it remains a big challenge to correlate underlying biomolecule interactions with macroscopic level of materials, for example, recognizing such weak interaction, further transforming it into regulating material’s macroscopic property and contributing to some new bio-applications. Here we designed a novel smart polymer based on polyacrylamide (PAM) grafted with lactose units (PAM-g-lactose0.11), and reported carbohydrate-carbohydrate interaction (CCI)-promoted macroscopic properties switching on this smart polymer surface. Detailed investigations indicated that the binding of sialic acid molecules with the grafted lactose units via the CCIs induced conformational transformation of the polymer chains, further resulted in remarkable and reversible switching in surface topography, wettability and stiffness. With these excellent recognition and response capacities towards sialic acid, the PAM-g-lactose0.11 further facilitated good selectivity, strong anti-interference and high adsorption capacity in the capture of sialylated glycopeptides (important biomarkers for cancers). This work provides some enlightenment for the development of biointerface materials with tunable property, as well as high-performance glycopeptide enrichment materials.

(6.0 mmol), 5.0 g ammonium carbonate and 15 mL anhydrous DMSO. The tube was sealed and placed in an automated microwave synthesizer (Biotage Initiator 8 EX), the reaction was proceeded at 40 °C in 10 watts power for 4 hours. Then the reaction mixture was freeze-dried to remove the excess of ammonia and DMSO to obtain β-glycosyl amine (1-Amino-lactose). 1 Then 0.34 g β-glycosyl amine (1-Amino-lactose 1.0 mmol), 0.39 g fluorescein isothiocyanate (FITC, 1.0 mmol), and 5 mL anhydrous DMF were added to a 25 mL round-bottom flask. The reaction mixture was stirred at room temperature overnight. After that the solvent was removed under reduced pressure. The crude product was purified through a Shimadzu UFLC 20A purity system using a C18 reversed-phase analytic chromatographic column (10 mm×250 mm, Boston Analytics, Corp. China) to afford fluorescein-labelled lactose as yellow power 0.527 g, yield: 72%. Hydrogen nuclear magnetic resonance ( 1 H NMR, 300 MHz, CD3OD) ppm: 3.45-3.51 The above fluorescein-labelled lactose (0.366 g, 1 mmol) was dissolved in 5 mL dry pyridine, then acetic anhydride (0.714 g, 7 mmol) was added to the above solution and kept stirring for 12 hours at ambient temperature. Subsequently, the reaction solution was poured into an ice water (50 mL), the precipitate was washed with ice water and petroleum ether for three times.
After that the crude product was purified through Shimadzu UFLC 20A purity system using a C18 reversed-phase chromatographic column, to afford fluorescein-labelled acetylated lactose as yellow power 0.369 g, yield: 36%. 1

Synthesis of the polymer PAM-g-lactose
Scheme S2. Synthetic procedure of PAM-g-lactose.
Lactose 1 (34 g, 0.1 mol) was added into a 500 mL three necked RB flask containing Ac2O (160 mL) and HClO4 (1 mL) at 20 °C. The reaction mixture was stirred for 2.5 hours while maintaining reaction temperature below 30 °C. After that, the reaction mixture was placed in an ice bath and red phosphorus (12.0 g), Br2 (20 mL) and distilled H2O (14 mL) were added in sequence while maintaining reaction temperature below 20 °C. The ice bath was removed and the reaction was continued for another 4 hours at room temperature. The mixture was diluted with chloroform (120 mL), poured into ice water, neutralized with saturated aqueous Na2CO3.
The combined organic layer was dried over CaCl2 and concentrated under vacuum to afford 40 g crude yellow oil product 2 (75%) which could be used without further purification.

S5
After stirring for 48 hours at 60 °C, the crude product was obtained, and purified by dialysis in water and methanol for 3 days, using a piece of dialysis membrane (2 cm×10 cm, molecule weight cut-off: 10000). White powder of PAM-g-lactose was obtained after freeze-drying. 1

Synthesis of PAM-g-lactose@SiO2 enrichment material
(a) Silica gels (5.0 g, average particle size: 5 μm and average pore diameter: 300 Å) were suspended in 25 mL of hydrochloric acid (HCl, 0.1 mol·L -1 ) for 48 hours at ambient temperature to generate sufficient hydroxyl groups on the silica surface. The hydroxyl activated silica gels were separated by centrifugation at 7000 rpm for 5 minutes. Then, the silica gels were washed three times with ultrapure water and then ethanol by repetitive dispersion/precipitation cycles, and then the silica gels were dried under vacuum.
(b) 3-triethoxysilylpropyl isothiocyanate (3.0 mL) was dissolved in anhydrous toluene (40 mL), and the aforementioned silica gels (5.0 g) were added. The mixture was stirred and refluxed for 6 hours. The product was separated by centrifugation at 7000 rpm for 5 minutes.
Then, the isothiocyanate-modified silica gels (denoted as NCS@SiO2) were washed three times with toluene and then ethanol by repetitive dispersion/precipitation cycles to remove the unreacted materials, and then the silica gels were dried under vacuum.
(c) 0.20 g PAM-g-lactose was allowed to react with 0.50 g 3-triethoxysilylpropyl isothiocyanate-modified SiO2 microspheres (denoted as NCS@SiO2) 5 in distilled water for 2 days, via a coupling reaction between the amide residues in PAM and the active NCS sites on the silica gels. Pure PAM-g-lactose@SiO2 was obtained through alternate dialysis (2 cm×10 cm) with methanol and distilled water for three times.
Reference material PAM@SiO2 was also obtained by using the same method.

Fluorescent titration experiment details
To investigate the binding properties (Ka) of lactose towards various saccharides, we used the fluorescein isothiocyanate (FITC) to label the lactose molecule to perform the fluorescent titration experiment, which is a typical and rapid method for measuring affinity constant between host and guest molecules in supramolecular chemistry. 6 Here, fluorescent titration experiments between fluorescein-labelled lactose and different saccharides, including galactose, fucose, glucose, mannose, GalNAc, GlcNAc, and Neu5Ac, were conducted in buffer solutions with pH 7.4 and 3.8, respectively. The host fluorescein-labelled lactose was prepared as stock S6 solution in Tris-buffer solution (10 mM, pH 7.4) and formate-buffer solution (10 mM, pH 3.8) for 5.0×10 -6 mol· L -1 , respectively. Guest monosaccharides were prepared to 1.75×10 -3 and 1.75×10 -2 mol· L -1 of stock solutions in H2O. The work solutions were prepared by adding different volumes of guest solutions to a series of test tubes, and then same amount of stock solution of host fluorescein-labelled lactose was added into each test tube, followed by dilution to 3.00 mL by Tris-buffer solution or formate-buffer solution. After being shaken for 1 minute, the work solutions were measured immediately at 20 °C using a spectrometry. Association constant (Ka) were obtained according to intensity changes at the maximum emission peak.
Detailed Ka values are listed in Table S1.
Similar method was adopted to investigate the binding capacities of fluorescein-labelled maltose and fluorescein-labelled cellobiose to Neu5Ac and glucose in formate-buffer solution (10 mM, pH 3.8).
Similar method was adopted to investigate the binding capacities of fluorescein-labelled acetylated lactose to diverse monosaccharides in Tris-buffer solution, respectively. The detailed Ka values are shown in Table S2.
Moreover, the binding capacities of the fluorescein-labelled lactose towards three other typical acidic analogues (gluconic acid, ascorbic acid and tartaric acid) were also investigated in formate-buffer solution (10 mM, pH 3.8).

AFM measurement details
The general characterizations (film morphology and thickness) of the polymer film were performed using AFM in the ScanAsyst mode with a Nanoscope V controller and the software Nanoscope v8.12. The resulting images were processed using Nanoscope Analysis v1.40.
The surface stiffness of copolymer film was investigated by AFM in PeakForce QNM mode at ambient atmosphere and a constant temperature of 25 °C. AFM with PeakForce QNM mode allows quantitative nanomechanical mapping of material surface properties, including Young's modulus, while simultaneously imaging the topography of sample at a high resolution.
In the PeakForce QNM mode, the reduced modulus E* is obtained by fitting the retraction curve using the Derjaguin-Muller-Toporov (DMT) model: 7 Where F is the force on the tip, FAdh is the adhesion force, R is the tip end radius, d-d0 is the distance between tip and sample. For the cycling experiment, a cycle includes the alternative measurement after the polymer film was immersed in sugar solution of 2.0 × 10 -2 mol·L -1 for 20 minutes and pure water for 20 minutes, respectively.

H NMR experiments
In order to investigate the binding details between lactose and Neu5Ac, 1

Cell culture and protein extraction
Cell culture and protein extraction were carried out as reported. 8

Part S2 Supporting Tables and Figures
Where F represents the fluorescent intensity, and CH and CG are the corresponding concentrations of host and guest.         values and glycopeptides are marked with red pentacles. As the best commercial materials applied in glycoproteomics, the enrichment selectivity of Sepharose is not satisfied, which could not work well when the BSA interference level is higher than 50-fold fetuin.