Designed Helical Peptides as Functional Probes for γ-Secretase.

γ-Secretase is a membrane-embedded aspartyl protease complex with presenilin as the catalytic component that cleaves within the transmembrane domain (TMD) of >90 known substrates, including the amyloid precursor protein (APP) of Alzheimer's disease. Processing by γ-secretase of the APP TMD produces the amyloid ß-peptide (Aß), including the 42-residue variant (Aß42) that pathologically deposits in the Alzheimer brain. Complex proteolysis of APP substrate by γ-secretase involves initial endoproteolysis and subsequent carboxypeptidase trimming, resulting in two pathways of Aß production: Aß49 → Aß46 → Aß43 → Aß40 and Aß48 → Aß45 → Aß42 → Aß38. Dominant mutations in APP and presenilin cause early onset familial Alzheimer's disease (FAD). Understanding how γ-secretase processing of APP is altered in FAD is essential for elucidating pathogenic mechanisms in FAD and developing effective therapeutics. To improve our understanding, we designed synthetic APP-based TMD substrates as convenient functional probes for γ-secretase. Installation of the helix-inducing residue α-aminoisobutyric acid provided full TMD helical substrates while also facilitating their synthesis and increasing the solubility of these highly hydrophobic peptides. Through mass spectrometric analysis of proteolytic products, synthetic substrates were identified that were processed in a manner that reproduced physiological processing of APP substrates. Validation of these substrates was accomplished through mutational variants, including the installation of two natural APP FAD mutations. These FAD mutations also resulted in increased levels of formation of Aß-like peptides corresponding to Aß45 and longer, raising the question of whether the levels of such long Aß peptides are indeed increased and might contribute to FAD pathogenesis.

Synthesis of stable C-linked ferrocenyl amino acids and their use in solution-phase peptide synthesis.

Incorporation of ferrocenyl group to peptides is an efficient method to alter their hydrophobicity. Ferrocenyl group can also act as an electrochemical probe when incorporated onto functional peptides. Most often, ferrocene is incorporated onto peptides post-synthesis via amide, ester or triazole linkages. Stable amino acids containing ferrocene as a C-linked side chain are potentially useful building units for the synthesis of ferrocene-containing peptides. We report here an efficient route to synthesize ferrocene-containing amino acids that are stable and can be used in peptide synthesis. Coupling of 2-ferrocenyl-1,3-dithiane and iodides derived from aspartic acid or glutamic acid using n-butyllithium leads to the incorporation of a ferrocenyl unit to the δ-position or ε-position of an α-amino acid. The reduction or hydrolysis of the dithiane group yields an alkyl or an oxo derivative. The usability of the synthesized amino acids is demonstrated by incorporating one of the amino acids in both C-terminus and N-terminus of tripeptides in solution phase.