Optimization of the Linker Length in the Dimer Model of E22P-Aβ40 Tethered at Position 38

Since amyloid β (Aβ) oligomers are more cytotoxic than fibrils, various dimer models have been synthesized. We focused on the C-terminal region that could form a hydrophobic core in the aggregation process and identified a toxic conformer-restricted dimer model (E22P,G38DAP-Aβ40 dimer) with an l,l-2,6-diaminopimelic acid linker (n = 3) at position 38, which exhibited moderate cytotoxicity. We synthesized four additional linkers (n = 2, 4, 5, 7) to determine the most appropriate distance between the two Aβ40 monomers for a toxic dimer model. Each di-Fmoc-protected two-valent amino acid was synthesized from a corresponding dialdehyde or cycloalkene followed by ozonolysis, using a Horner-Wadsworth-Emmons reaction and asymmetric hydrogenation. Then, the corresponding Aβ40 dimer models with these linkers at position 38 were synthesized using the solid-phase Fmoc strategy. Their cytotoxicity toward SH-SY5Y cells suggested that the shorter the linker length, the stronger the cytotoxicity. Particularly, the E22P,G38DAA-Aβ40 dimer (n = 2) formed protofibrillar aggregates and exhibited the highest cytotoxicity, equivalent to E22P-Aβ42, the most cytotoxic analogue of Aβ42. Ion mobility-mass spectrometry (IM-MS) measurement indicated that all dimer models except the E22P,G38DAA-Aβ40 dimer existed as stable oligomers (12-24-mer). NativePAGE analysis supported the IM-MS data, but larger oligomers (30-150-mer) were also detected after a 24 h incubation. Moreover, E22P,G38DAA-Aβ40, E22P,G38DAP-Aβ40, and E22P,G38DAZ-Aβ40 (n = 5) dimers suppressed long-term potentiation (LTP). Overall, the ability to form fibrils with cross β-sheet structures was key to achieving cytotoxicity, and forming stable oligomers less than 150-mer did not correlate with cytotoxicity and LTP suppression.