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Abstract Phenylalanine arginine β‐naphthylamine, or PAβN, is a C‐terminus capped dipeptide discovered in 1999 as an RND‐type efflux pump inhibitor (EPI). Since then, PAβN has become a standard tool compound in EPI research and development. Despite this, PAβN lacks a detailed or efficient synthesis, and standard parameters for its use in wild‐type bacterial strains are inconsistent or non‐existent. Therefore, a scalable and chromatography‐free synthesis of PAβN was developed using streamlined traditional solution‐phase peptide coupling chemistry. With this procedure, gram scale quantities of PAβN were synthesized alongside analogues and stereoisomers to build a focused library to evaluate simple structure activity relationships. While most analogues were less active than the broadly utilized L,L‐PAβN itself, we identified that its enantiomer, D,D‐PAβN, also provided 8‐ to 16‐fold potentiation of the antibiotic levofloxacin at 40 to 50 μg/mL concentrations of EPI in various wild‐typePseudomonas aeruginosastrains. Additionally, D,D‐PAβN was shown to be significantly more hydrolytically stable than L,L‐PAβN, indicating that it may be a useful, and now readily synthesized, tool compound facilitating future EPI research.