The 'ins' and 'outs' of amidines in β-sheet folding and fibril disaggregation.

Amidines are a relatively unexplored isostere of the amide bond, offering unique electronic properties and hydrogen-bonding behavior. This study presents the first systematic investigation of amidines within folded β-sheet structures. Using CD, NMR, and aggregation assays, we find that amidines are well tolerated when acting as hydrogen-bond donors but disrupt β-sheet folding when serving as hydrogen-bond acceptors. This donor/acceptor asymmetry contrasts with the behavior of amidines in α-helices, where both roles are accommodated. Importantly, outward-facing amidines disrupt the edge-to-edge hydrogen bonding required for fibril formation, enabling the design of non-aggregating β-hairpin peptidomimetics without reliance on sequence charge. Spectroscopic analysis further reveals that amidines embedded in peptide backbones exist predominantly in their neutral, monoprotonated form even at acidic pH, prompting a reassessment of amidine basicity in structured biomolecules. These findings establish design principles for using amidines in stable, aggregation-resistant peptidomimetic scaffolds.