Synthesis of maleimide-modified gelatin: Avoiding the peculiar case of maleimide polymerization

Gelatin, a widely used protein biopolymer in the food industry, also holds great potential for biomedical applications because of its unique characteristics. To fully harness its potential, modification is often required, such as the introduction of maleimide groups, which enable conjugation with thiols via very fast thiol-maleimide Michael addition. However, due to the reactivity of maleimides, care must be taken to minimize side reactions. While reactions with amines can be suppressed by lowering the pH, a commonly overlooked issue is maleimide polymerization, which can occur via radical or, more critically, nucleophile/base-initiated anionic mechanisms. This problem was observed during the synthesis of maleimide-modified gelatin using a heterobifunctional N-hydroxysuccinimide/maleimide linker, resulting in insoluble, crosslinked products. In this work, we show that this maleimide polymerization can be initiated by the amine (lysine/hydroxylysine) and imidazole (histidine) moieties of the gelatin. To address this issue, a Diels-Alder/retro Diels-Alder protection strategy using furans was employed, enabling the synthesis of soluble maleimide-modified gelatin (GelMal) with improved degree of modification, controlled molecular weight, and shorter reaction time. The best result was obtained when a 2,5-dimethylfuran-protected NHS/maleimide linker was reacted with gelatin at 50 °C for 1 h in a 40 % ethanol solution in buffer at pH 9 and the subsequent deprotection of the maleimide was conducted in a 0.1 % methanesulfonic acid solution in water at 50 °C for 24 h under a reduced pressure of 300 mbar. This resulted in a degree of functionalization of 77 %. The functionality of GelMal synthesized with this procedure was validated through reactions with 6-monodeoxy-6-monothio-β-cyclodextrin, thiophenol, and dithiothreitol.