Enzymatic Halogenation of Silk Fibroin from

Abstract

Silk fibroin from the silkworm, , is a unique biomaterial that has been extensively studied for a variety of applications that utilize robust mechanical properties, biological compatibility, and controlled self-assembly properties. This study tested carbon-halogen (C-X) bond halogenation to alter the chemical composition of silk fibroin with the intention to generate novel functional materials. In brief, silk fibroin side-chain modification used halogen salts (NaX, X = Cl, Br, and I), hydrogen peroxide (H₂O₂), and the vanadium-dependent haloperoxidase from to produce primarily halogenated tyrosine residues along the amorphous regions of the silk fibroin protein. Halogenation was confirmed with various methods, including 1D ¹H NMR, X-ray photoelectron spectroscopy, and analysis of chymotrypsin peptide digests by Q-TOF Liquid Chromatography-Mass Spectrometry. Secondary structure analysis by FTIR-ATR, circular dichroism, and Raman spectroscopy revealed increase in helical conformation of solubilized halogenated silk fibroin, while dried film functionality demonstrated higher abundance of β-sheet structures by maintenance of random coil content. Evaluation by contact angle measurement demonstrated increased hydrophilicity on silk fibroin films following addition of halogens by supporting the formation of water insoluble hydrogels after treatment with various organic and salt solvents. This study is the first to characterize the effects of enzymatic halogenations on the properties of silk fibroin, and this post-translational modification will be useful for the addition of non-natural small molecules or ligands, introducing new material types afforded by the silk fibroin structure.

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(A) Proposed halogenation pathway for functionalization of ortho-positioned H on tyrosine rings. ¹H NMR spectra of unmodified (black) and halogenated samples (blue = chlorinated, red = brominated, and purple = iodinated) of silk fibroin. (B) LC-QTOF FASTA Sequence Alignment with silk fibroin light chain. Sequences of silk fibroin light chain given in one-letter amino acid code. Halogenated amino acids are printed in bold. The detected halogenation of Cl, DiCl, Br, DiBr, I, and DiI is marked above the identified halogenated amino acids. Numbers on side mark amino acid positions.

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XPS analysis of unmodified and halogenated silk fibroin films. Unmodified = unmodified silk, Cl = chlorinated silk, Br = brominated silk, and I = iodinated silk.

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High-resolution XPS plots of individual halogen peaks.

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(A) CD spectra of unmodified silk fibroin without methanol (black), unmodified with methanol (pink), chlorinated (red), brominated (blue), and iodinated silk fibroin (green). (B) FTIR spectra of unmodified and halogenated silk fibroin.

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Deconvoluted Amide I peaks from unmodified and halogenated samples. T = beta turns, R = random coil, B = beta-sheet, and SC = side chain mostly from tyrosine.