Organophosphorus compounds as fluorescent probes for cell imaging

Abstract

Small molecules containing fluorescent moieties can be used as a means of studying cell structure and function, as a result of the high sensitivity of fluorescence microscopy. This technique allows one to obtain specific information about the cell and has recently attracted considerable interest by many research groups. This work presents three projects in which the main aim was to develop multi-modal imaging agents. They will possess a fluorescent group and also another moiety which provides predictable biological properties. Our interest is centred on two types of fluorophore: Polycyclic Aromatic Hydrocarbons (PAHs) and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY). The first project describes the synthesis of the phosphorus analogues of the biologically-active indazole core which remain rare in the literature. The synthesis presented here shows the versatility of our approach and allows for substitution on the phenyl ring of the newly formed phosphindole core simply by changing the nitrile used. Position 3 of the phosphindoles was also varied to bear different aromatic groups; the chosen aromatic systems were phenyl, naphthyl and anthracyl. These were chosen in order to prepare a fluorescent and biologically-active core. In practice, the phosphindoles showed near zero quantum yields. Photoinduced Electron Transfer and Dexter Energy Transfer seem to be the plausible responsible phenomena behind this lack of fluorescence. Temperature was found to be a key variable in the synthesis of phosphindoles since a temperature below 110 °C in the last step led to the formation of two chlorothiophosphonates. One of these unexpected chlorothiophosphonates showed strong activity against Bacillus subtilis and Streptococcus pyogenes. The second project describes the synthesis of the pyrene-based ligand 109, which is significant as it was based on an air-stable alkyl primary phosphine. This remarkable stability is provided by the electronic properties that both the pyrene and the butyl linker ii

confer on the corresponding primary phosphine. The tridentate ligand 109 was obtained following a double hydrophosphination reaction of the primary phosphine, and 109 was subsequently used to create complexes with the transition metals from groups 9 and 10. These demonstrated demonstrated weak fluorescence despite the presence of a metallic core. The presence of the DNA intercalating pyrene unit and the presence of the square-planar Pt centre in complex 116 required an assessment of the cytotoxicity of the complex. In assays, 116 was shown to exert similar cytotoxicity towards bone osteosarcoma (U2OS) and transformed mammary cancer (HMLER) cell lines as the anticancer drug Cisplatin. The advantage of complex 116 is that it contains an intercalating function, a potential cytotoxic platinum centre and moderate/mild loss of fluorescence for cell imaging by optical microscopy. The final project discussed in this thesis is the synthesis of a phosphonium salt containing BODIPY as fluorophore, bound to a macrocycle which is able to undergo complexation reactions with d-block metals. This is another example of a molecule capable of multi-modal functionality, since phosphonium salts have been shown to target mitochondria. Positively charged compounds freely diffuse across the negatively charged mitochondrial membrane and the BODIPY moiety allows for imaging of the compound’s fate by optical microscopy. Finally, the tetraamine macrocycle of the molecule allowed ligand 154 to be reacted with [Cu(OAc)2] which gave the fluorescent Cu(II) complex 155. This complex is interesting because it proves that coorduination to Cu is possible. The next step in this research would be to prepare the 64Cu analogue, which would be a candidate for Positron Emission Tomography (PET) imaging. In this manner, 64Cu-154 would be a fluorescent organelle-specific PET imaging agent.