A sandcastle worm-inspired strategy to functionalize wet hydrogels

Hydrogels have been extensively used in many fields. Current synthesis of functional hydrogels requires incorporation of functional molecules either before or during gelation via the pre-organized reactive site along the polymer chains within hydrogels, which is tedious for polymer synthesis and not flexible for different types of hydrogels. Inspired by sandcastle worm, we develop a simple one-step strategy to functionalize wet hydrogels using molecules bearing an adhesive dibutylamine-DOPA-lysine-DOPA tripeptide. This tripeptide can be easily modified with various functional groups to initiate diverse types of polymerizations and provide functional polymers with a terminal adhesive tripeptide. Such functional molecules enable direct modification of wet hydrogels to acquire biological functions such as antimicrobial, cell adhesion and wound repair. The strategy has a tunable functionalization degree and a stable attachment of functional molecules, which provides a tool for direct and convenient modification of wet hydrogels to provide them with diverse functions and applications.


Polymer synthesis and characterization
Supplementary Figure 6. Synthesis of Pol-1.
Pol-1 was obtained via atom transfer radical polymerization (ATRP) using 2-(dimethylamino)ethyl methacrylate (DMAEMA) as the monomer and subsequent quaternization. CuCl (Aldrich; >99%) was dissolved in 12 M HCl in a flask, followed by addition of a large amount of water to precipitate out a white solid. After sediment, the supernatant was decanted and more water was added in to the flask with shaking.
The solid was collected from filtration and washed alternately with ethanol and diethyl ether (Et2O). The collected solid was dried under vacuum and stored under nitrogen for direct use as the polymerization catalyst.
The polymer reaction was carried out in a N2 purged glove box at room temperature. DMAEMA (340 μL, 2 mmol), compound 8 (10 mg, 0.01 mmol), 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA, 6.5 µL, 0.024 mmol) was dissolved in anhydrous THF (200 μL) in a reaction vial, followed by addition of CuCl (2.7 mg, 0.02 mmol) and CuCl2 (0.4 mg, 0.004 mmol). After the reaction mixture was stirred at room temperature for 22 h, the mixture was diluted with 500 μL THF and passed through a 4 g neutral alumina column to remove the catalyst. Solvent was removed under reduced pressure and the collected residue was dissolved in THF (1 mL) followed by slow addition of cold PE (45 mL) to precipitate out the crude polymer as white solid. The polymer was collected after centrifugation, removal of the supernatant and dried under N2 flow.
After three cycles of the dissolution/precipitation procedure and drying under vacuum, the purified polymer before quanternization was collected as a white solid (150 mg, 43% yield). The obtained polymer was characterized by GPC.
The above polymer (50 mg) was dissolved in acetonitrile (1 mL) followed by addition of an excess amount 1-bromooctane (550 μL) to the polymer solution. After the reaction mixture was stirred at 55 °C for 24 h, the solvent was removed from the mixture to give an oil. This oil was dissolved in MeOH (0.5 mL), followed by slowly addition of cold Et2O (45 mL) to precipitate out the crude polymer. The acetonide and Boc protecting groups were removed in a mixture of 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIS) and 2.5% H2O for 2 h under gentle shaking. After the solvent was evaporated under a N2 flow, the mixture was dissolved in MeOH (0.5 mL) followed by addition of cold Et2O (45 mL) to precipitate out a white fluffy solid. This precipitate was collected by centrifugation and dried under a N2 flow. After three dissolution-precipitation cycles and drying under vacuum, the final polymer was obtained as a white solid in a form of TFA salt (93 mg, 83% yield). Complete deprotection and purity of Pol-1 was confirmed by 1 H NMR. Figure 7. Synthesis of Pol-2.

Supplementary
The reaction was carried out in a N2 purged glove box at room temperature using a similar condition as mentioned above for Pol-1 synthesis. 2-hydroxyethyl acrylate (HEA, 104 μL, 1 mmol), compound 8 (5.0 mg, 0.005 mmol), 2,2'-bipyridine (bpy, 2.3 mg, 0.015 mmol) were dissovled in anhydrous ethanol (100 μL) in a vial. Then CuBr (0.7 mg, 0.005 mmol) was added to the reaction and the reaction mixture was stirred at 70 °C for 48 h. The reaction mixture was then diluted with 500 μL MeOH and passed through a 4 g neutral alumina column to remove the catalyst. The collected solution was concentrated and the resulting oil was dissolved in MeOH (0.5 mL), followed by slowly addition of cold methyl tert-butyl ether (MTBE, 45 mL) to the mixture to precipitate out the crude polymer as white solid. The polymer was collected after centrifugation and removal of the supernatant, and then the polymer was dried under a N2 flow. After three cycles of the dissolution/precipitation procedure, the white solid was dried under vacuum. The above polymer was characterized by GPC. The protecting groups were removed by following aforementioned protocol in Pol-1 synthesis to give the final product of Pol-2 as a white solid (41 mg, 34% yield). Complete deprotection and purity of Pol-2 was confirmed by 1 H NMR.

Supplementary Figure 8. Synthesis of Pol-3.
Pol-3 was obtained by reversible addition-fragmentation chain transfer polymerization (RAFT). 2,2'-Azobis(2-methylpropionitrile) (AIBN) was recrystallized from MeOH before use. The polymerization was conducted in a N2 purged glove box to control the moisture level less than 5 ppm. DMAEMA (340 μL, 1 mmol), compound 9 (10.5 mg, 0.01 mmol), AIBN (0.82 mg, 0.005 mmol) were dissolved in anhydrous DMF (600 μL) in a reaction vial. The reaction was stirred at 60 °C for 24 h and stopped by cooling the reaction bottle in an ice-water bath and exposing to air. The crude polymer was dissolved in THF (1 mL) and precipitated out with PE (45 mL). This dissolution-precipitation process was repeated for another four cycles, followed by drying under vacuum to give the purified polymer at the protected stage as a pale pink solid (170 mg, 49% yield). The polymer at protected stage was characterized by GPC. The quaternization process of the polymer is similar to that described above for Pol-1. The polymer (50 mg) and 1-bromooctane (550 μL) was added in 1 mL acetonitrile and the mixture was stirred at 80 °C for 24 h. Quaternized PDMAEMA polymer was purified by dissolving the crude polymer in MeOH (0.5 mL) and precipitated in Et2O (45 mL) for three times. The collected final product was dried under vacuum. The deprotection process was the same as the protocol aforementioned for Pol-1 to give final polymer of Pol-3 as a pale pink solid (95 mg, 85% yield).
Complete deprotection and purity of Pol-3 was confirmed by 1 H NMR. The polymerization was conducted in a N2 purged glove box to control the moisture level less than 5 ppm. Nε-tert-butyloxycarbonyl-L-lysine N-carboxyanhydride (Boc-L-Lys-NCA) was prepared by following previous literature 4 . To a solution of Boc-L-Lys-NCA (68.1 mg, 0.25 mmol) in anhydrous THF (1 mL) was added a solution of compound 7 (2.1 mg, 0.0025 mmol) in THF (1 mL) and the reaction mixture was stirred at room temperature for 2 days. Polymer purification by precipitation and further removal of the protecting groups were conducted by followed the aforementioned protocol in Pol-2 synthesis to give final fully deprotected Pol-5 as a white solid in the form of TFA salt (60 mg, 86% yield). The side-chain protected polymer was characterized by GPC. Complete deprotection and purity of Pol-5 was confirmed by 1 H NMR. Figure 11. Synthesis of Pol-6.

Supplementary
The polymerization was conducted in a N2 purged glove box to control the moisture level less than 5 ppm. N-ε-tert-butyloxycarbonyl-L-lysine N-carboxyanhydride (Boc-L-Lys-NCA) and γ-benzyl-L-glutamate NCA (BLG-NCA) were prepared by following previous literature 4  The polymerization was conducted in a N2 purged glove box to control the moisture level less than 5 ppm. The racemic mixture of β-lactam monomers DMβ and CPβ were obtained by following previous The polymerization was conducted in a N2 purged glove box to control the moisture level less than 5 ppm. The racemic mixture of β-lactam monomers COβ was obtained by the following a previous method 5 .