Leveraging In Situ Chlorination for Resolving Subcellular Chlorinative Stress

Abstract Hypochlorous acid (HOCl) is a key effector in host defense, serving as a potent mediator of the innate immune response. However, dysregulation of HOCl production contributes to tissue damage and chronic inflammation. Existing HOCl indicators are constrained by limited conjugation feasibility for subcellular targeting, narrow emission tunability, poor aqueous compatibility that requires high organic cosolvent content, or a lack of chlorination-specific sensing mechanisms. Here, we identify a previously unrecognized HOCl-mediated chlorination of 2,6-unsubstituted BODIPY scaffolds that produces pronounced increases in fluorescence emission wavelength, intensity, and fluorescence lifetime. We leveraged this reaction to develop a series of HOCl indicators, termed HOClsense dyes, for resolving chlorinative stress at the subcellular level. These HOClsense dyes provide switchable fluorescence turn-on and turn-off detection modes across tunable emission wavelengths, accompanied by redshifted responses. Strategic alkylation of the pyridyl group further enabled click-based functionalization, allowing the cellular distribution of HOClsense dyes to be tailored for subcellular imaging. Using HOClsense dyes, we resolved PAMP-induced extracellular and lysosomal HOCl and identified elevated basal HOCl levels and MPO-dependent hyperresponsive HOCl generation in Niemann–Pick disease cell models.