Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, Massachusetts 02155, United States.
Biomacromolecules. 2021 Feb 8;22(2):773-787. doi: 10.1021/acs.biomac.0c01539. Epub 2021 Jan 6.
The oxidation of tyrosine residues of silk fibroin involves the generation of dityrosine and 3,4-dihydroxyphenylalanine (DOPA). However, it remains a challenge to selectively control the reaction pathway to produce dityrosine or DOPA in a selective fashion. Here, silk hydrogels with controllable formation of not only dityrosine and DOPA but also DOPA-Fe complexes within the cross-linked networks were developed. The use of chitosan particles in the Fenton reaction allowed the interaction of Fe ions with silk fibroin to be limited through the adsorption of Fe ions onto chitosan particles by manipulating contact time between the reaction medium and chitosan particles. This led to significant suppression of the premature formation of β-sheet structures that cause steric hindrance to the collisions between tyrosyl radicals and thus enabled higher selectivity toward the formation of dityrosine than DOPA. Remarkably, the addition of ethylenediaminetetraacetic acid (EDTA) to the chitosan particle-assisted Fenton reactions resulted in hydrogels that significantly favored the formation of DOPA over dityrosine due to the increase in the hydroxylation of phenol in the presence of EDTA. Despite the existence of Fe-EDTA complexes, Raman spectra indicated the DOPA-Fe complexation in the hydrogels. Mechanistically, the hydrogel networks with small-sized and uniformly distributed β-sheet structures as well as the abundance of DOPA appear to make non-EDTA-chelated Fe ions more accessible to complexation with DOPA. These findings have important implications for understanding the oxidation of tyrosine residues of silk fibroin by metal-catalyzed oxidation systems with potential benefits for future studies on silk protein-based hydrogels capable of generating intrinsic adhesive features as well as for exploring dual-cross-linked silk hydrogels constructed by chemical cross-linking and metal-coordinate complexation.
丝素蛋白中酪氨酸残基的氧化涉及二酪氨酸和 3,4-二羟基苯丙氨酸（DOPA）的生成。然而，选择性地控制反应途径以有选择地生成二酪氨酸或 DOPA 仍然是一个挑战。在这里，开发了具有可控形成的丝素水凝胶，不仅可以形成二酪氨酸和 DOPA，而且可以在交联网络内形成 DOPA-Fe 配合物。在 Fenton 反应中使用壳聚糖颗粒通过将 Fe 离子吸附到壳聚糖颗粒上，可以限制 Fe 离子与丝素蛋白的相互作用，通过控制反应介质与壳聚糖颗粒之间的接触时间来实现。这导致显著抑制了β-折叠结构的过早形成，β-折叠结构会对酪氨酸自由基之间的碰撞造成空间位阻，从而使二酪氨酸的形成比 DOPA 具有更高的选择性。值得注意的是，向壳聚糖颗粒辅助的 Fenton 反应中添加乙二胺四乙酸（EDTA）会导致水凝胶显著有利于 DOPA 的形成而不是二酪氨酸的形成，这是由于在 EDTA 存在下增加了苯酚的羟基化。尽管存在 Fe-EDTA 配合物，但拉曼光谱表明 DOPA-Fe 配合物存在于水凝胶中。从机制上讲，具有小尺寸和均匀分布的β-折叠结构以及丰富的 DOPA 的水凝胶网络似乎使非 EDTA 螯合的 Fe 离子更容易与 DOPA 配位。这些发现对于理解丝素蛋白中酪氨酸残基的金属催化氧化系统的氧化具有重要意义，这可能对未来研究具有内在粘合特性的丝蛋白水凝胶以及探索通过化学交联和金属配位络合构建的双交联丝水凝胶具有潜在的益处。
