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Modular Platform of Carbohydrates-modified Supramolecular Polymers Based on Dendritic Peptide Scaffolds

Glycopeptide supramolecular polymers displaying multivalent carbohydrates are particularly suitable for immune-relevant biomaterials, due to the important functions of carbohydrates in mediating cell-cell communication and modulating immune responses. However, the diversity and complexity of carbohydrates limited the generation of glycopeptide supramolecular monomers. Thereby, a modular platform of presenting various carbohydrates, especially more complex oligosaccharides, is highly desirable but remains underexplored. Here, we first prepared the linear amphiphilic glycopeptides that self-assembled into spherical nanoparticles and worm-like nanoparticles. Furthermore, the dendritic glycopeptides that self-assembled into uniform nanorods were designed to generate modular supramolecular polymers with variable functionality, via redesigning the molecular backbone. With various functional oligosaccharide-modified supramolecular polymers, the in vitro studies further indicated that these polymers were not cytotoxic to macrophages, and significantly modulated the production of proinflammatory cytokines. These findings provide a promising platform to develop supramolecular glycopeptide biomaterials with potential applications in immunomodulation and immunotherapy.

Enzyme-linked immunosorbent assay (ELISA): RAW264.7 cells were incubated with carbohydrate-modified peptide supramolecular polymers (10 g/mL, 100g/mL) for 24 h. After collected the supernatant solutions (1500 rpm, 10 min), IL-6 and TNF-α were detected by BioLegend ELISA kit. The experimental steps were as follows: The coated antibody was incubated overnight at 4 °C, then blocked the ELISA dilution for 2 h. After adding the standard or detection sample and incubate for 2 hours, the detection antibody was added and incubated for 1 h. Then the avidin-HRP was added and incubated for 30 minutes, and 3,3',5,5'-tetramethylbenzidine (TMB, catalyzed by HRP to produce a soluble blue product, absorbance at 370 nm) was employed for color reaction. After adding stop solution for TMB substrate (maximum absorbance change to 450 nm), the absorbance at 450 nm was detected by absorption light microplate reader. Figure S1. Hydrodynamic sizes and TEM images of three-branched 3-M3 assemblies. Assembly was performed using a 25 µM aqueous solution (water) of triple-branched 3-M3 monomer at room temperature. Figure S2. TEM images of 3-FITC assemblies. Self-assembly was performed using a 25 µM aqueous solution (water) of 3-FITC monomer at room temperature.   Synthesis of P2: P1 (436 mg, 2.13 mmol) was dissolved in 10 mL of N,N-Dimethylformamide (DMF), and sodium hydride (98 mg, 2.65 mmol) was added. The solution was stirred at room temperature for 20 min. Then methyl iodide (160 μL, 2.57 mmol) was added slowly, with stirring continued for 2 h at room temperature. The solvent was removed by rotary evaporation and chloroform was added. It was washed three times with saturated brine, dried with anhydrous sodium sulfate, filtered and spin-dried and passed through a column. The product was purified by silica gel column chromatography with n-hexane/ethyl acetate (3/1, v/v)  Synthesis of P3-NHS or P0-NHS: 1 eq of P3 or P0 was dissolved in DCM, and added 1.5 eq of NHS. After stirring, dicyclohexylcarbodiimide (DCC) (1.5 eq) was added to the reaction system, and the mixture was stirred at room temperature for 1 h. The product was purified by silica gel column chromatography (gradient elution with ethyl acetate and petroleum ether), and dried through rotary evaporation.

Synthesis of precursor
Synthesis of Fmoc-FFF: Fmoc-FFF was prepared as described in previous work.

Synthesis of two-branched oligosaccharides-tripeptide.
Synthesis of A3: Fmoc-FFF (200 mg, 0.293 mmol) was dissolved in DMF, Ethyl cyanoglyoxylate-2-oxime (42 mg, 0.293 mmol) was added. Then N,N'-Diisopropylcarbodiimide (DIC) was slowly added in an ice-water bath. After stirring for 1 h in an ice-water bath, the DMF solution of PL1 was slowly added. Stirring in ice-water bath for 1 h, then the mixture was slowly warmed to room temperature overnight. The product was roto-evaporated to remove the solvent, and directly added Trifluoroacetic acid (TFA) / Dichloromethane (DCM) (1/1, v/v) for deprotection, then dried through rotary evaporation.   Synthesis of 2-M3: A6 (5 mg, 6.79 μmol) was dissolved in DMF, and 40 μmol of maltotriose was added. After adjusted the pH of the mixture to 3.7 with acetic acid, the mixture was stirred at room temperature for 48 h. The excess maltotriose was removed by dialysis, then the solution was dried by lyophilization. 1

Synthesis of three-branched oligosaccharides-tripeptide.
Synthesis of C2: C2 was prepared as described in previous work. 1 Synthesis of D1: D1 was prepared as described in previous work. 3 Synthesis of D2: Fmoc-FFF (168 mg, 0.248 mmol) was dissolved in DMF, and Ethyl cyanoglyoxylate-2-oxime (42 mg, 0.293 mmol) was added, and then DIC was slowly added in an ice-water bath. After stirring for 1 h, D1 (100 mg, 0.124 mmol) in DMF solution was slowly added. After stirred in ice-water bath for 1 h, the mixture was slowly warmed to room temperature overnight. After roto-evaporated to remove the solvent, the product was deprotected by piperidine, and separated by semi-preparative HPLC with methanol/water (2/1, v/v). The product was acidified with 10 mM HCl, then the solution was dried by lyophilization. Synthesis of B4: B4 was prepared as described in previous work. 1 B4 was synthesized by solid phase peptide synthesis on a CS136XT by CS Bio (Menlo Park, USA). A TentaGel R Trityl resin loaded with Bis-(2-aminoethyl)-ether (0.5 g, 0.5 mmol/g loading capacity, 0.25 mmol), purchased from Rapp Polymere (Tübingen, Germany), was used as solid support. The chain was elongated using the Fmoc-protected amino acids, Fmoc-aminohexanoic acid (Fmoc-Ahx-OH) and 3 × Fmoc-L-Phe-OH. For the coupling reactions, amino acid (1.0 mmol, 4.0 eq. relative to resin loading capacity) were pre-activated in DMF with HBTU (1.0 mmol, 4.0 eq.), HOBt (1.0 mmol, 4.0 eq.) and DIPEA (1.5 mmol, 6.0 eq.). The solution was then transferred to the reactor and shook during 40 min for the actual coupling. After several washing steps, the cleavage of the terminal Fmoc-group was achieved by treatment with a solution of piperidine in DMF (20 v%) for 5 min and another 20 min. After the attachment of the third phenylalanine, the free N-terminus was reacted with a mixture of C2 (1.0 mmol, 4.0 eq.), HATU (1.0 mmol, 4.0 eq.) and HOAt (1.0 mmol, 4.0 eq.) in DMF. Finally, B4 could be cleaved from the resin using a mixture of TFA/TIPS/H2O (9.5/0.25/0.25). The filtrate was codistilled three times with toluene, the remainder suspended in water and subjected to lyophilization.  Synthesis of B6: In the mixed solution of MeOH and THF (1:1), 19 mg of B5 was added. Then 33 eq of 0.1 M LiOH was slowly added, and the mixture was stirred at room temperature for 1 h. After neutralizing with an equal amount of HCl (1 M), the solvent was removed by rotary evaporation, and the mixture was separated by SEC with the DMF as eluent, then the solution was dried by lyophilization.
Synthesis of 3-FITC: B4 (25 mg, 24.5 μmol) and FITC (95 mg, 245 μmol) was added to a 10 mL round-bottomed flask and dissolved in DMF. Then a catalytic amount of TEA was added. The reaction was stirred overnight at room temperature. After the reaction was completed, the mixture was dialyzed for 2 d. After lyophilization and demethylation, the product was reacted with D2 (Similar with B13). And the final product was purified by semi-preparative HPLC with methanol/water (2/1, v/v), then dried by lyophilization.