Fluorescence-Based Detection of Fatty Acid β-Oxidation in Cells and Tissues Using Quinone Methide-Releasing Probes

Abstract

Detection of metabolic activity enables us to reveal the inherent metabolic state of cells and elucidate mechanisms underlying cellular homeostasis and growth. However, a fluorescence approach for the study of metabolic pathways is still largely unexplored. Herein, we have developed a new chemical probe for the fluorescence-based detection of fatty acid β-oxidation (FAO), a key process in lipid catabolism, in cells and tissues. This probe serves as a substrate of FAO and forms a reactive quinone methide (QM) as a result of metabolic reactions. The liberated QM is covalently captured by intracellular proteins, and subsequent bio-orthogonal ligation with a fluorophore enables fluorescence analysis. This reaction-based sensing allowed us to detect FAO activity in cells at a desired emission wavelength using diverse analytical techniques including fluorescence imaging, in-gel fluorescence activity-based protein profiling (ABPP), and fluorescence-activated cell sorting (FACS). The probe was able to detect changes in FAO activity induced by chemical modulators in cultured cells. The probe was further employed for fluorescence imaging of FAO in mouse liver tissues and revealed the metabolic heterogeneity of FAO activity in hepatocytes by the combination of FACS and gene expression analysis, highlighting the utility of our probe as a chemical tool for fatty acid metabolism research.

Benzylic Trifluoromethyl Accelerates 1,6-Elimination Toward Rapid Probe Activation

“…33 However, a recent study on activity-based protein labeling employed quinone methide intermediates generated upon 1,6-elimination of benzylic fluoride. 34 This brings forth α-F-benzylation in probe engineering as an unproductive strategy in terms of probe activation, necessitating the discovery of alternate elimination pathways. Our pursuit of finding this alternative elimination mechanism leveraged thermodynamic analyses of potential leaving groups at the benzylic (α) position, some considered atypical in organic synthesis.…”

Deconvoluting nitric oxide–protein interactions with spatially resolved multiplex imaging

“…The strategy of analyte-triggered protein labeling has been proposed to interrogate analyte-regulated signaling pathways. 37–40 However, integrating this strategy with activity-based sensing to provide dual-functional probes capable of fluorogenically imaging NO and covalently labeling proteins remains challenging, which requires a unique group that is intrinsically inert but can be specifically activated by NO to label proteins, and that a fluorogenic signal should be yielded accompanying this labeling event. Herein, we reported probe NOP-1 fulfilling these requirements.…”

The first specific probe for pyrrolidine with multifunction by the interaction mechanism of atomic economic reaction

“… 11 The optical properties of the probe are changed by the chemical reaction between the analyte and the probe, 12 so it has great advantages in specificity. 13 Among the reactive fluorescent probes, usually, the probes with the interaction mechanism of the decomposition-type reaction are common, 14 and have been used to specifically detect H 2 O 2 , 15 H 2 S, 16 and enzymes 17 in living cells. In contrast, the reactive probes that are directly combined with the analyte to produce a substance are relatively rarely reported, 18 and most of them are used to detect inorganic small molecule species, such as SO 2 , 19 NO, 20 H + , 21 Hg 2+ , 22 etc.…”