UWS Academic Portal Development of a neutral diketopyrrolopyrrole phosphine oxide for the selective bioimaging of mitochondria at the nanomolar level Abelha,

Development of novel bioimaging materials that exhibit organelle specific accumulation continues to be at the forefront of research interests and efforts. Among the various subcellular organelles, mitochondria, which are found in the cytoplasm of eukariotic cells, are of particular interest in relation to their vital function. To date, most molecular architectures that target mitochondria utilise delocalised lipophilic cations such as triphenylphosphonium and pyridinium. However, the use of such charged motifs is known to be detrimental to the working function of the mitochondrial transmembrane potential and there remains a strong case for development of neutral mitochondrial fluorescent probes. Herein, we demonstrate for the first time the exploitation of diketopyrrolopyrrole-based chemistries for the realisation of a neutral fluorescent probe that exhibits organelle specific accumulation within the mitochondria at the nanomolar level. The synthesised probe, which bears a neutral triphenylphosphine oxide moiety, exhibits a large Stokes shift and high fluorescence quantum yield in water, both highly sought-after properties in the development of bioimaging agents. In-vitro studies reveal no interference with cell metabolism when tested for the human MCF7 breast cancer cell and nanomolar subcellular organelle colocalisation with commercially available mitochondrial staining agent Mitotracker Red. In light of its novelty, neutral structure and the preferential accumulation at nanomolar concentrations we anticipate this work to be of significant interest for the increasingly larger community devoted to the realisation of neutral mitochondrial selective systems and more widely to those engaged in the rational development of superior organic architectures in the biological field. Introduction Over the last two decades there has been increasing interest in the rational development of novel bioimaging materials which exhibit organelle specific accumulation.[1–8] Among those subcellular organelles of interest, mitochondria, which are found in the cytoplasm of eukaryotic cells and are responsible for aerobic metabolism, are of particular note and significant efforts have been devoted towards the realisation of novel staining agents.[5–8] Mitochondria denote the principal energy-producing compartment in most eukaryotes. Their function is critical in a number of vital process such as adenosine triphosphate (ATP) production, central metabolism and apoptosis,[9,10] and irregularities in these functions are intimately linked to degenerative diseases such as Alzheimer’s and Parkinson’s as well as cardiac complications and cancer.[6–10] Currently, a range of diagnostic imaging techniques, such as radioisotope labelling, magnetic resonance imaging and positron emission tomography, are employed for monitoring subcellular organelles.[6,11] Recently, approaches exploiting fluorescence emission have been developed for the investigation of specific organelles such as mitochondria, representing a powerful class of imaging strategies, on account of their ability to translate molecular recognition into highly discriminative and easily detected optical signals.[6,12,13] The mitochondrion, which is structurally characterised by a double membrane separated by the intermembrane space, exhibits a highly negative transmembrane potential as a result of the process of mitochondrial respiration whereby the proton pumps in the inner mitochondrial membrane transport protons onto the intermembrane space.[11,14–17] This large potential distinguishes it from other subcellular organelles and has therefore inspired the successful development of optically active probes that exploit it to selectively target this subcellular organelle. Thus, architectures containing delocalised lipophilic cations (DLCs) such as triphenylphosphonium or pyridinium cation, which are present in the majority of mitochondria targeting probes to date and are believed to improve membrane transport and increase mitochondria probe accumulation, have been widely used to ensure selectivity towards this subcellular compartment attracted by the negative potential of the membrane, whilst maintaining an otherwise non-polar framework in most cases.[6– 8,18] In this regard, a number of organic dyes such as fluorescein,[19,20] coumarin[21,22] and rhodamine[23,24] have been utilised, as the fluorescent tag to which DLCs are anchored, in the development of novel fluorescent probes. However, their biological applications are often limited by their photophysical and/or biological properties. Importantly, and despite the large efforts devoted to the development of charged fluorescent probes as selective staining agents for mitochondria, the known negative [a] Dr T. F. Abelha, Prof. C. Alexander School of Pharmacy The University of Nottingham University Park NG72RD Nottingham, UK. [b] Dr G. Morris, Dr A. J. McLean, Dr C. J. McHugh School of Computing, Engineering and Physical Sciences University of the West of Scotland PA12BE Paisley, UK. E-mail: callum.mchugh@uws.ac.uk [c] Dr S. M. Lima, Dr L. H. C. Andrade Grupo de Espectroscopia Óptica e Fototérmica Universidade Estadual de Mato Grosso do Sul CP 351, 79804-970 Dourados, MS, Brazil. [d] Dr J. Calvo-Castro School of Life and Medical Sciences University of Hertfordshire AL109AB Hatfield, UK. E-mail: j.calvo-castro@herts.ac.uk Supporting information for this article is given via a link at the end of the document.