Scalable and Chromatography-Free Synthesis of Efflux Pump Inhibitor Phenylalanine Arginine beta-Naphthylamide for Its Validation in Wild-Type Bacterial Strains

Phenylalanine arginine beta-naphthylamine, or PA beta N, is a C-terminus capped dipeptide discovered in 1999 as an RND-type efflux pump inhibitor (EPI). Since then, PA beta N has become a standard tool compound in EPI research and development. Despite this, PA beta N 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 PA beta N was developed using streamlined traditional solution-phase peptide coupling chemistry. With this procedure, gram scale quantities of PA beta N 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-PA beta N itself, we identified that its enantiomer, D,D-PA beta N, also provided 8- to 16-fold potentiation of the antibiotic levofloxacin at 40 to 50 mu g/mL concentrations of EPI in various wild-type Pseudomonas aeruginosa strains. Additionally, D,D-PA beta N was shown to be significantly more hydrolytically stable than L,L-PA beta N, indicating that it may be a useful, and now readily synthesized, tool compound facilitating future EPI research.