LD and ER Targeting Cysteine Fluorescent Sensing Driven A/B-ring-Naphthalene/Indole Simultaneously Substituted Water Hypersensitive Flavonol: Simultaneous Dual-Colour Visualization of LD and ER, and Precisely Controlled Linear CO Delivery

Abstract

The first cysteine (Cys) fluorescence sensing driven flavonol, HIBC (3-hydroxy-2-(1-methyl-1H-indol-3-yl)-4H-benzo[g]chromen-4-one), was developed as a single fluorescent probe (SFprobe) and a photoCORM. This compound is characterized by the simultaneous substitutions at both the A- and B-rings with naphthalene and indole, respectively. Notably, HIBC exhibits remarkable hypersensitivity to minor variations in water fractions (f_w, vol % in acetonitrile) ranging from 0% to 15%, enabling in situ real-time visualization of lipid droplets (LDs) and the endoplasmic reticulum (ER) with high spatial resolution through distinct dual-colour fluorescence without any crosstalk. Under O₂, HIBC facilitates the delivery of precisely controlled amounts of CO gas within a therapeutic and safe dosage range to living systems via visible light irradiation. This control is achieved by modulating either the intensity or duration of the irradiation light, or by adjusting the dosage of photoCORM. Its fluorescence allows for in situ real-time imaging and tracking of intracellular distribution while monitoring CO delivery progress. HIBC is generated from the sensing reaction between precursor IBCA (2-(1-methyl-1H-indol-3-yl)-4-oxo-4H-benzo[g]chromen-3-yl acrylate) and Cys. In PBS buffer containing only 30% DMSO, IBCA can rapidly detect and image both endogenous and exogenous Cys within just 250 seconds. It demonstrates high selectivity—particularly against homocysteine (Hcy) and glutathione (GSH)—and sensitivity, achieving detection limits as low as 87 nM in living HeLa cells and zebrafish across a wide linear concentration range of 0–10 μM (0–2 equiv.). Importantly, IBCA also targets LDs and ER while monitoring fluctuations in Cys levels during periods of ER stress. Both IBCA and HIBC, along with all photoreaction products, exhibit negligible toxicity while demonstrating good permeability in live HeLa cells and zebrafish. The HIBC we developed represents a pioneering instance of a flavanol driven by Cys fluorescence sensing that features simultaneous substitutions at both the A-and B-rings with naphthalene and indole moieties, respectively. Compared to unsubstituted flavonol, these structural improvements not only lead to a substantial red shift of 88 nm in the absorption peak but also significantly enhance overall performance. It serves not only as an exceptionally water hypersensitive SFprobe for simultaneous dual-colour fluorescence visualization of LDs and ER but also functions effectively as a photoCORM. This work provides not only a method for precise control over CO release but also facilitates the development of user-friendly molecular tools for further investigation into the roles played by LDs, ER, Cys and CO in biological processes; their interactions; relationships among them; along with potential applications in clinical diagnosis.