Potent Racemic Antimicrobial Polypeptides Uncovered by a Stereochemical Series of Cationic d/l Backbones

Antimicrobial polypeptides (AMPs) are often engineered to adopt a secondary structure (e.g., α-helix, β-sheet, or turns), reinforcing the prevailing view that ordered conformations are preferred for enhanced activity. Here, we report a first-of-its-kind stereochemical series of cationic polypeptides with defined d/L ratios (100%L, 100%d, 80%d/20%L, 50%d/50%L, 20%d/80%L) via N-carboxyanhydride (NCA) polymerization and thiol-ene postfunctionalization. Circular dichroism spectroscopy confirms helix-like signatures for all compositions, except the racemic 50d/50L, which lacks a defined structure under membrane-mimicking conditions. Unexpectedly, the racemic polymer is strongly antimicrobial against Gram-positive pathogens (minimum inhibitory concentration, MIC = 4-16 μg/mL) and matches the proteolytic stability of the d-enantiomer analogues. As an adjuvant, it sensitizes multidrug-resistant Gram-negative bacteria, enhancing the efficacy of a wide variety of antibiotics by up to 128-fold across multiple classes and showing highly synergistic effects (fractional inhibitory concentration index, FICI≤0.5). Mechanistic assays reveal comparable outer-membrane perturbation, increased intracellular antibiotic accumulation, membrane depolarization, and reactive oxygen species (ROS) elevation across helical and nonhelical members, indicating that an ordered secondary structure is not required for either direct antimicrobial action or potentiation. By establishing enantiomeric-ratio control as a stereochemical design and identifying racemic composition as a practical, scalable alternative, this study provides guidance for developing robust, low-hemolytic AMP mimics and a universal adjuvant for antibiotics, particularly suited to low-resource settings where access to advanced therapeutics is limited.