Didactic approach recounting advances and limitations in novel glutathione and cysteine detection (reduced GSH probe) with mixed coumarin, aldehyde, and phenyl–selenium chemistry

Abstract We describe the pertinent research steps and analysis, many of which are chemical, to achieve a novel molecular probe for glutathione (GSH) which has been published and patented based on two recent articles: “Exceptional time response, stability and selectivity in doubly-activated phenyl selenium-based glutathione-selective platform” and “Enhanced Doubly Activated Dual Emission Fluorescent Probes for Selective Imaging of Glutathione or Cysteine in Living Systems” ( Kim et al., 2015 ; Mulay et al., 2018 ). The papers involve coumarin probes. Reaction/detection unfolds with aminothiol attack at an electrophilic ring carbon position. An adjacent –CHO group is heavily involved in resonance aspects of the C–Se position, as well as the binding of the pendant N–group; the coumarin lactone carbonyl also allows for resonance to be achieved (vide infra). The leaving group, –SePh, while precedented in some systems, depends on electronic tuning ( Fig. 1 ). For 1, the response times with GSH was ~ 100 ms; a 100-fold fluorescence increase is observed (Compound 1). The probe also reacts with cysteine (Cys) and homocysteine (Hcy), albeit differently. For glutathione probing, the greater wavelength maxima (1: 550 nm, DACP–1: 555 nm, DACP–2: 590 nm) enabled eventual cell studies (confocal microscopy) and animal studies. The limits of detection (LOD, 1: 270 nM DACP–1: 10.1 nM DACP–2: 17.0 nM), as measured using the 3σ/k method. We provide a didactic presentation from probe conception to probe in vivo testing, etc., with additional considerations presented; a variety of factors/issues (2.1–2.28) help maintain a realistic sequence, a flow from wider to narrower, of the factors that go into developing medical, biological and neurodegenerative disease-related probes, meant to help other researchers follow our intention, gain perspective, and overcome current limitations.