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 ~100ms; 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: 550nm, DACP-1: 555nm, DACP-2: 590nm) enabled eventual cell studies (confocal microscopy) and animal studies. The limits of detection (LOD, 1: 270nM DACP-1: 10.1nM DACP-2: 17.0nM), 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.
Keywords: Biothiol; Confocal; Coumarin; Fluorescence; Green channel; Mouse model; Red channel; Reduced glutathione; Selenide; Selenium.
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