Silk fibroin is a highly versatile and biocompatible protein biomaterial widely used in tissue engineering, drug delivery systems, and optical applications. Its unique mechanical properties and biodegradability make it an ideal candidate for biomedical scaffolds. However, to expand its functionality, covalent modification with bioactive molecules such as enzymes, sensors, or fluorescent tags is essential. Traditional methods often rely on non-specific chemical conjugation targeting amino acid side chains like serine and tyrosine. In this study, we introduce a novel chemoenzymatic strategy to specifically functionalize silk fibroin with azide groups using O-GalNAc-transferases and azide-modified UDP-GalNAc substrates. This approach enables site-specific incorporation of azide moieties onto serine residues, which are abundant in the fibroin sequence, thereby providing a highly selective platform for subsequent “click” chemistry ligation.SCG2 Antibody Purity & Documentation
The method leverages the enzymatic activity of recombinant O-GalNAc-transferases (GalNAc-T1, T10, and T13) to transfer azido-functionalized GalNAc from UDP-GalNAc to hydroxyl groups of serine residues in fibroin. The azide-labeled fibroin was then reacted with DBCO-Sulfo-Cy5, a strained cycloalkyne-bearing fluorophore, enabling efficient and specific conjugation via copper-free click chemistry. SDS-PAGE analysis under fluorescence confirmed successful glycosylation, with strong fluorescent smears observed only in samples treated with GalNAc-T1 and T13—indicating successful azide incorporation.55721-31-8 IUPAC Name No signal was detected with GalNAc-T10, which requires pre-existing O-glycans for activity, thus serving as a negative control.PMID:34974482
To assess functional impact, larger-scale reactions were conducted to form a hydrogel. After conjugation with DBCO-Sulfo-Cy5, the modified fibroin was concentrated and induced to gel by incubation at 37 °C. A solid, stable hydrogel formed within one hour, confirming that the enzymatic modification did not impair the natural self-assembly of fibroin. Excess unreacted DBCO-Sulfo-Cy5 was effectively removed using a centrifugal filter and further purified by ethanol-salt extraction. Agarose gel electrophoresis confirmed the presence of high-molecular-weight conjugated species, and the resulting hydrogel retained its bright blue fluorescence, demonstrating covalent attachment of the fluorophore.
This work presents a robust, biocompatible, and site-selective method for modifying silk fibroin without altering its structural integrity. By combining enzymatic specificity with click chemistry efficiency, this strategy opens new avenues for creating advanced functional biomaterials with precise molecular architecture. The ability to attach any cycloalkyne-containing molecule offers broad potential for applications in targeted therapeutics, biosensing, and regenerative medicine.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com