Expanding the Chemical Space of Mechanically Interlocked Peptides (MIPs) via Rational Design and Metabolite Profiling

Mechanically interlocked peptides (MIPs) possess exceptional biological stability, making them promising scaffolds for therapeutic development. However, despite advances in biological and chemical synthesis, their accessible chemical space remains limited. In particular, practical methodologies and rational design principles for mechanically interlocking and stabilizing conventional linear peptides are still underdeveloped. Here, we present a robust strategy for peptide interlocking and stabilization using active-template Cu-(I)-catalyzed azide-alkyne cycloaddition (AT-CuAAC). Through systematic exploration of amino acid side chains, macrocycle size, peptide length, and reaction conditions, we established design guidelines and constructed a diverse library of interlocked short peptides. This approach was further applied to longer peptide sequence, [Y]⁶-AngII, using both convergent and iterative assembly routes, significantly broadening its applicability. Further biological experiment demonstrated that mechanical interlocking confers exceptional stability, with interlocked peptides maintaining >95% integrity in plasma after 48 h and 20% in whole blood after 24 h, far outperforming noninterlocked counterparts. Metabolite profiling reveals the mechanical bond protects ≥8 contiguous residues, with terminal hydrolysis dominating degradation while core regions remain shielded. Collectively, this work provides a robust and broadly applicable approach for MIP construction, offering key insights into the mechanistic basis of mechanical stabilization, and expands the toolkit for designing robust peptide therapeutics.