Stereorandomization as a Method to Probe Peptide Bioactivity

Solid-phase peptide synthesis (SPPS) is usually performed with optically pure building blocks to prepare peptides as single enantiomers. Herein we report that SPPS using racemic amino acids provides stereorandomized ( sr ) peptides, containing up to billions of different stereoisomers, as well-defined single HPLC peaks, single mass products with high yield, which can be used to investigate peptide bioactivity. To exemplify our method, we show that stereorandomization abolishes the membrane-disruptive effect of α-helical amphiphilic antimicrobial peptides but preserves their antibiofilm effect, implying different mechanisms involving folded versus disordered conformations. For antimicrobial peptide dendrimers by contrast, stereorandomization preserves antibacterial, membrane-disruptive, and antibiofilm effects but reduces hemolysis and cytotoxicity, thereby increasing their therapeutic index. Finally, we identify partially stereorandomized analogues of the last resort cyclic peptide antibiotic polymyxin B with preserved antibacterial activity but lacking membrane-disruptive and lipopolysaccharide-neutralizing activity, pointing to the existence of additional targets.