Fluorescent probes for the labelling of cardiomyocyte and mitochondrial proteins

Organophosphates are a known danger to human health with an array of biochemical targets and complex toxic effects. Phenyl saligenin phosphate (PSP) is an organophosphate and related phenoxy substituted organophosphates irreversibly bind to a variety of important enzymes causing the condition known as organophosphate induced delayed neuropathy (OPIDN). These compounds also potentially cause cardiac muscle damage but little is known about how this class of compounds effect cardiomyocytes. The identification of proteins targeted by these toxins is of interest. This study investigates the synthesis and application of novel fluorescently labelled organophosphates to investigate organophosphate interaction with H9c2 cardiomyoblasts. The synthesis of probes with BODIPY, pyrene and rhodamine fluorescent component is explored with success in preparation of pyrene and rhodamine probes. The fluorescent behaviour of three rhodamine labelled phenyl saligenin phosphates probes with alkyl and PEG linkers is examined and the cytotoxicity of the probes assessed by monitoring MTT reduction, and LDH release from exposed H9c2 cardiomyoblasts. All three analogues showed cytotoxicity (4 h exposure; 100 μM). These probes were found to bind to proteins within mitochondria and spots from 2D-gel electrophoresis experiments, visualised at 532 nm, which are undergoing MS analysis. This project also describes progress towards a photoreactive-fluorescent probe to identify the site of action of the potassium channel opener, diazoxide, which activates cardioprotective pathways. The drug is believed to target mitochondrial KATP channels, but the protein structure of these channels has yet to be elucidated. The fluorescence labelling of whole cardiac cells with a dansyl fluorescent -benzophnone-photoreactoive diazoxide probe has been demonstrated and the progress toward a rhodamine fluorescent analogue is presented.