We have previously reported the incorporation of an azide-bearing unnatural amino acid, 4-azidophenylalanine (AzPhe), into silk fibroin, and showed that the azide group of AzPhe can be used as a selective handle for chemical modification by click chemistry. In this study, we investigated the intermolecular crosslinking between AzPhe-incorporated silk fibroin molecules by click chemistry, because crosslinking could be an effective way to alter the mechanical properties of silk fibroin materials. A bifunctional crosslinking agent, DBCO-PEG4-DBCO, which bears azide-reactive DBCO groups on both ends of the PEG chain, was used for crosslinking, and molecular-weight changes after the crosslinking reaction were investigated by electrophoresis. The results showed that the crosslinking reaction with DBCO-PEG4-DBCO proceeded with just a small fraction of silk fibroin. It was assumed that the steric constraint between silk fibroin molecules inhibited the reaction. In order to create some distance between the reactive sites for crosslinking and the main chain of silk fibroin, we first reacted DBCO-PEG4-DBCO with AzPhe-incorporated silk fibroin to introduce a DBCO-functionalized PEG linker and then reacted the resulting DBCO-PEG4-modified silk fibroin with another bifunctional crosslinking agent, PEG7-bis-azide, which bears DBCO-reactive azide groups on both ends of the PEG chain. As a result, increased amounts of crosslinked silk fibroin were observed by electrophoresis, indicating that the distance between reactive sites and silk fibroin main chain is important for efficient intermolecular crosslinking.
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