Structure–Function Study of a Family of Membrane‐Targeted Azobenzene Phototransducers With Bacillus subtilis

Membrane‐targeted azobenzenes enable light‐based control of membrane‐associated cellular processes with high spatiotemporal precision. Their reversible trans–cis isomerization provides tunable control over molecular geometry and polarity, which can modulate cell membrane potential‐dependent biological functions. Despite extensive work in eukaryotes, structure–property relationships for membrane‐targeted azobenzenes remain largely unexplored in prokaryotes, where membrane electrostatics and the electrical potential component of the proton motive force are tightly coupled to bioenergetics, metabolism, and adaptive responses, including antibiotic susceptibility. Building on our previous finding that the amphiphilic azobenzene Az‐2Pyr (also known as ZIAPIN2) inserts into bacterial membranes and elicits a light‐driven response distinct from that observed in eukaryotic cells, here we show a comparative study to connect molecular design to photoswitching and bacterial outcomes. We study three amphiphilic azobenzenes with an identical core and a systematic variation in cationic pyridinium arms, from one (Az‐1Pyr) to four (2Pyr‐2Pyr), to probe the roles of charge density and molecular symmetry. We first characterize their optical properties by steady‐state and ultrafast optical spectroscopy, resolving spectral signatures and kinetics of trans–cis photoisomerization. We then quantify bacterial affinity via cellular uptake, lethality, and ζ‐potential. Finally, we show that photoswitching combined with bacterial association modulates antibiotic susceptibility.