Structural Engineering of Tyrosine-Based Neuroprotective Peptides: A New Strategy for Efficient Blood–Brain Barrier Penetration

The relationship between the structure of walnut-derived peptides and their activity of transport efficiency across the blood-brain barrier (BBB) remains unclear. In this study, a series of walnut-derived peptides were synthesized by substituting leucine (L) with tyrosine (Y), lysine (K), or arginine (R). Three outstanding peptides-EVSGPGYSPN, TWLPYPR, and YVPFPYP-were selected based on their antioxidant capacity and BBB transport efficiency, with EVSGPGYSPN exhibiting the highest activity. Reversed-phase high-performance liquid chromatography (RP-HPLC) and Transwell assay results demonstrated that EVSGPGYSPN can remain stable during gastrointestinal digestion and penetrate the BBB. Pharmacokinetic results revealed that the cumulative concentration of EVSGPGYSPN in the brain reached 1.25 ± 0.91 µg/g at 10 h, while its plasma half-life exceeded 12 h. Furthermore, it significantly reduced reactive oxygen species (ROS) levels to 110.46 ± 15.16%. Nuclear magnetic resonance (NMR) and Fourier-transform infrared spectroscopy (FTIR) results indicated that EVSGPGYSPN is rich in aromatic hydrogen signals and exhibits low methyl signals, which may enhance its antioxidant activity. Circular dichroism (CD) spectroscopy showed that EVSGPGYSPN has the highest random coil content, which facilitates its binding to transporters on the BBB and promotes BBB permeability. This study provides valuable insights into the design of brain-targeted peptide delivery systems.