Ferrocene-Containing Desmuramylpeptides Adopt A Flexible Conformation and Exhibit NOD2 Agonistic Activity

This study extends our preliminary investigation of ferrocene-containing desmuramylpeptides as potential agonists of nucleotide binding oligomerization domain containing protein 2 (NOD2). We report the rational design, chemical synthesis, conformational characterization and biological evaluation of 13 novel ferrocene conjugates with the desmuramyldipeptide l-Ala–D-isoGln (10c/g, 11a–c, 13d/f) and tripeptide Gly–l-Val–d-Glu–(OEt)2 (12a–f). Detailed spectroscopic analysis revealed the presence of conformationally flexible structures driven by weak intramolecular hydrogen bonds. The ferrocene-carrying desmuramylpeptides were evaluated for their in vitro NOD2 agonist activity on the HEK-Blue NOD2 reporter cell. Of the ferrocene derivatives tested, conjugates 12b and 12c exhibited the strongest NOD2 agonist activity of the series, with the lowest EC50 values of 44.1 and 252.6 nM, respectively. The best performing compound 12b also induced cytokine production in peripheral blood mononuclear cells (PBMCs), both alone and in combination with lipopolysaccharide (LPS). This work thus presents the first potent ferrocene-modified NOD2 agonists and provides a framework for the rational design and further optimization of ferrocene-based NOD2 immunomodulators.