Modular helix stabilization via alkenyl butylcarbamate staples: effects of staple length, stereochemistry, and directionality

Peptide stapling is a widely used approach for stabilizing α-helical peptides, improving their structural integrity, proteolytic resistance, and therapeutic potential. Here, we present a novel stapling strategy employing alkenyl butylcarbamate cross-links formed via ring-closing metathesis (RCM). This platform enables fine control over staple length, stereochemistry, and directionality. Through systematic analysis, the 13-atom hex-2-enyl butylcarbamate staple was identified as optimal, achieving enhanced α-helicity and efficient macrocyclization. We further demonstrate that peptide stereochemistry and staple orientation significantly impact both RCM efficiency and helix stabilization. Notably, the optimized stapled peptides exhibited a 45-fold increase in resistance to trypsin-mediated degradation compared to their unmodified counterparts. In addition, the carbamate linkage provided excellent resistance to non-enzymatic hydrolysis under physiological conditions. Together, these results highlight alkenyl butylcarbamate stapling as a chemically robust, hydrophilic, and conformationally rigid approach for stabilizing α-helical peptides. This strategy offers an attractive alternative to traditional hydrocarbon staples, particularly for therapeutic peptides targeting extracellular or membrane-bound proteins