Stapled peptides are regarded as the promising next-generation therapeutics because of their improved secondary structure, membrane permeability and metabolic stability as compared with the prototype linear peptides. Usually, stapled peptides are obtained by a hydrocarbon stapling technique, anchoring from paired olefin-terminated unnatural amino acids and the consequent ring-closing metathesis (RCM). To investigate the adaptability of the rigid cyclobutane structure in RCM and expand the chemical diversity of hydrocarbon peptide stapling, we herein described the rational design and efficient synthesis of cyclobutane-based conformationally constrained amino acids, termed (E)-1-amino-3-(but-3-en-1-yl)cyclobutane-1-carboxylic acid (E7) and (Z)-1-amino-3-(but-3-en-1-yl)cyclobutane-1-carboxylic acid (Z7). All four combinations including E7-E7, E7-Z7, Z7-Z7 and Z7-E7 were proven to be applicable in RCM-mediated peptide stapling to afford the corresponding geometry-specific stapled peptides. With the aid of the combined quantum and molecular mechanics, the E7-E7 combination was proven to be optimal in both the RCM reaction and helical stabilization. With the spike protein of SARS-CoV-2 as the target, a series of cyclobutane-bearing stapled peptides were obtained. Among them, E7-E7 geometry-specific stapled peptides indeed exhibit higher α-helicity and thus stronger biological activity than canonical hydrocarbon stapled peptides. We believe that this methodology possesses great potential to expand the scope of the existing peptide stapling strategy. These cyclobutane-bearing restricted anchoring residues served as effective supplements for the existing olefin-terminated unnatural amino acids and the resultant geometry-specific hydrocarbon peptide stapling provided more potential for peptide therapeutics.
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