Toward Clickable Protein Networks: Orthogonal Amidation of Self-Assembled Lysozyme and Bovine Serum Albumin Nanofibers

Self-assembled protein nanofibers (PNFs) are promising building blocks for the development of sustainable materials due to their functional versatility, inherent biodegradability, and thermodynamic stability. In order to broaden the applications of PNFs, chemical modification offers a simple way to incorporate specific functionalization throughout the PNF fiber backbone. To this end, we demonstrate a highly efficient amidation of self-assembled PNFs from bovine serum albumin (BSA) and hen egg white lysozyme (HEWL), using adipic acid dihydrazide (ADH) and aminoacetaldehyde dimethyl acetal (AADA) as bioorthogonal modifiers, offering the possibility to form covalent networks via click chemistry. Critically, we compare (dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM)-mediated amidations with the widely used N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride and N-hydroxysuccimine (EDC/NHS) mediation system, showcasing superior reaction efficiency and reduced pH dependency in the case of DMTMM. Importantly, the PNF backbone remained largely intact, albeit some shortening of the fibers was evidenced. As a proof of concept, aldehyde-functionalized HEWL PNFs and hydrazide-functionalized BSA PNFs were mixed together to demonstrate kinetically bioorthogonal hydrazone cross-linking, relevant for many biomedical applications. Overall, this work provides an efficient and simple approach for modifying PNFs, which could find use in applications ranging from biomedicine (drug delivery or tissue engineering) to cosmetics, food production. and agriculture.