Synthesis and Fluorescence Profiling of Naphthylamine Analogs

N-phenyl-1-naphthylamine (NPN) has been widely used to probe the integrity of the outer membrane of gram-negative bacteria. NPN fluoresces in the hydrophobic environment of lipid bilayers, whereas its fluorescence is quenched in the aqueous extracellular milieu. Because NPN does not intrinsically accumulate in intact gram-negative bacteria, it has been broadly applied as a reporter of outer membrane permeabilization. Despite its widespread utility, systematic derivatization of this probe has remained largely unexplored. Here, we investigated the structure-fluorescence relationships of NPN through the synthesis of a diverse library featuring varied linkages to the fluorogenic naphthylamine core. Subsequent library expansion was informed by density functional theory and time-dependent density functional theory studies, which suggested possible origins of fluorescence quenching in aqueous environments. The final resulting library comprised 95 compounds, of which 49 exhibited a fluorogenic profile similar to that of NPN. A selected subset of analogs was then evaluated in a fluorescence-based accumulation assay using varied efflux and permeation strains of Escherichia coli, revealing that installed structural motifs influenced bacterial permeation. Collectively, these probes provide a foundation for further studies examining how small structural changes affect accumulation in gram-negative bacteria.