NMR characterization of Tacrine in aqueous solution and insights into its interaction with amyloid-β1–40 aggregates revealed by STD

The deposition of β-amyloid peptide (Aβ) aggregates in the brain is widely accepted as one of the major factors driving the development of Alzheimer's disease (AD). Tacrine (9-amino-1,2,3,4-tetrahydroacridine) is one of the most potent acetylcholinesterase inhibitors approved for the symptomatic treatment of AD, but its clinical use was discontinued due to hepatotoxicity. Recent studies have reported that tacrine can interact with A β and inhibit its aggregation, although the underlying mechanism is not yet fully elucidated, highlighting the need for detailed spectroscopic characterization under physiologically relevant conditions. Here, we report, to the best of our knowledge, the first complete and unambiguous assignment of the 1 H and 13 C NMR resonances of tacrine in aqueous solution at near-physiological pH using a combination of ROESY, HSQC, and HMBC experiments. pH-dependent 1 HNMR studies revealed that aromatic proton resonances are highly sensitive to protonation state, displaying pronounced but fully reversible chemical-shift variations. The resulting spectral map provides a reliable basis for ligand-observed NMR investigations of tacrine and related compounds in aqueous media. Moreover, saturation transfer difference (STD)-NMR experiments were optimized to investigate the interaction of tacrine with soluble A β 1–40 aggregates. Under the optimized conditions, clear STD effects were observed exclusively in the presence of A β 1–40 , confirming direct ligand–aggregate interaction. Differential saturation transfer among aromatic protons, particularly for H-7, suggests distinct proximity relationships within the tacrine– A β 1–40 complex. Together, these results establish a robust spectroscopic and methodological framework for future NMR studies of tacrine-based ligands and their interactions with amyloid assemblies.