Enzymatic Post-Translational Halogenation for Adding Functionality to Biomaterials

Silk fibroin from the silkworm, Bombyx mori, is a unique biomaterial that has been extensively studied for a variety of applications due to its promising properties such as controllable self-assembly, robust mechanical properties, and biological compatibility. Previously, there have been numerous methods describing the chemical modification of silk fibroin that utilize synthetic or enzymatic means that do not use halogens as a means of functionalization. Herein, a halogenation strategy is presented to modify silk fibroin with the aim of developing a novel functional material through the carbon-halogen (C-X) bond. Modification with NaX (X = Cl, Br, and I) salts, hydrogen peroxide (H2O2), and a vanadium dependent haloperoxidase (VHPO) from Curvularia inaequalis produced halogenated tyrosine residues along the protein’s amorphous regions. Halogenation was confirmed using various methods including 1D and 2D 1H NMR and a chymotrypsin digest with LCMS. Secondary structure was analyzed by FTIR-ATR, circular dichroism (CD), and Raman spectroscopy which revealed that halogenated silk fibroin prefers helical conformations in solution and beta sheet structures when made into dried films but still has random coil content. Addition of halogens increased hydrophilicity on silk fibroin films evaluated by contact angle measurements. Finally, to showcase the C-X bond as a route for functionalization, Suzuki-Miyaura coupling was employed to add a fluorescent molecule, fluorescein, through a palladium catalyzed reaction scheme. Although coupling efficiency was observed to vary for chlorine, bromine, and iodine, the results demonstrate that this strategy can be used to add new functional groups on to silk fibroin which can potentially modulate the material’s characteristics and enhance function