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Ruthenium(II) complexes and their peptide nucleic acid bioconjugates for therapeutic and biosensing applications
thesisposted on 06.02.2017, 02:06 by Joshi, Tanmaya
The availability of a wide range of synthetically viable polypyridyl ligands and attractive physicochemical properties for the corresponding Ru(II)-diimine complexes renders them suitable for use as medicinal and imaging probes. The central theme of this thesis is the development of Ru(II)-polypyridyl bioconjugates for biosensing and therapeutic applications. Towards this end, a library of diverse Ru(II) complexes, with polypyridyl ligands judiciously selected to address the essential criteria for the targeted applications, and their model constructs based on peptide nucleic acid (PNA) backbone were prepared and screened for their photophysical, photochemical, electrochemical, and electrochemiluminescent properties. Furthermore, to assist in live cell applications, analytical studies on uptake and interaction of Ru(II)-PNA bioconjugates with lipid membranes were also performed. A family of carboxy functionalized ruthenium(II) dicarbonyl complexes of formula [Ru(L)(CO)2Cl2] (L = Me2bpy = 4,4'-dimethyl-2,2'-bipyridine; Me-bpyCHO = 4'-methyl-2,2'-bipyridine-4-carboxyaldehyde; Me-bpyCOOH = 4'-methyl-2,2'-bipyridine-4-carboxylic acid; CppH = 2-(pyridin-2-yl)pyrimidine-4-carboxylic acid; dppzcH = dipyrido[3,2-a:2',3'-c]phenazine-11-carboxylic acid) and [Ru(L)(CO)2Cl]+ (L = tpyCOOH = 6-(2,2':6',2''-terpyridine-4'-yloxy)hexanoic acid) were prepared which exhibited photoinduced CO release when irradiated around 310 nm, the wavelength for their maximum absorption. The 2,2'-bipyridine and 2,2':6',2''-terpyridine based complexes displayed better CO release properties (one equivalent per complex) than the corresponding dipyrido[3,2-a:2',3'-c]phenazine and pyridyl-pyrimidine counterparts. Investigations carried out on [Ru(Cpp-L- PNA)(CO)2Cl2] (Cpp-L-PNA = tert-butyl-N [2 (N-9-fluorenylmethoxycarbonyl)aminoethyl]-N-[6-(2-(pyridin-2yl)pyrimidine-4-carboxamido)hexanoyl]-glycinate) also demonstrated that CO release was unaffected by further conjugation of the parent ligand to carrier peptides and delivery vectors, such as a monomeric peptide nucleic acid (PNA) backbone in this case. Such Ru(II)-polypyridyl bis(carbonyl) complexes represent a promising class of photoactivatable CO releasing molecules (PhotoCORMs). A series of PNA-like-monomers containing Ru(II)-pyridylpyrimidine /dipyridoquinoxaline/dipyridophenazine complexes were prepared and characterised by IR and 1H NMR spectroscopy, mass spectrometry, electrochemistry and elemental analysis. These new compounds displayed electronic absorption and emission profiles typical of [Ru(tris(diimine))]2+ complexes, viz., a metal to ligand charge transfer (MLCT) band centered around 450 nm and an emission maximum in the 610-665 nm region following photoexcitation at 450 nm. The emission intensity and quantum yields for monomers incorporating dipyridophenazine or dipyridoquinoxaline units were found to be higher than for other Ru(II)-PNA-like monomers. The cyclic voltammetry revealed a reversible one electron RuII to RuIII oxidation process for these Ru(II)-PNA-like monomers. In comparison to the reversible redox potential for the [Ru(bpy)3]2+/[Ru(bpy)3]3+ system (888 mV vs Fc0/+), a positive shift in potential of up to 179 mV was observed for the Ru(II)-PNA monomers (935-1065 mV vs Fc0/+). The Ru(II)-PNA-like monomers displayed reasonably intense electrochemiluminescence (ECL) responses in the presence of a tripropylamine (TPA) co reactant, with the monomers [Ru(bpy)2(Cpp-L-PNA-OH)]2+ and [Ru(phen)2(Cpp-L-PNA-OH)] showing ECL-activity equivalent to [Ru(bpy)3]2+, regarded as the benchmark ECL emitter. Solid phase synthesis of bioconjugate constructs consisting of Ru(II)-tris(diimine) connected to PNA oligomers was achieved using a Ru(II)-PNA monomer, [Ru(bpy)2(Cpp-L-PNA-OH)]2+. Insertion of the Ru(II) fluorophore within a PNA sequence was demonstrated for the first time using this compound. The absorption spectrum for the Ru(II)-PNA conjugates displayed a broad MLCT transition band centered around 445 nm and an emission maximum at ca. 680 nm following 450 nm excitation. The absorption and emission response of the incorporated Ru(II)-polypyridyl unit were unaffected by duplex formation between the Ru(II)-PNA oligomer and the complementary DNA strand. However, the Ru-PNA•DNA duplexes exhibited greater thermal stability when compared to the corresponding non-metalated duplexes. The stronger electrostatic interactions between the Ru-PNA and polyanionic DNA oligomer, attributed to the additional positive charges introduced (Ru(II) unit and positively charged lysine/arginine), account for the enhanced duplex stability. The Ru(II)-PNA bioconjugates and their corresponding PNA•DNA duplexes were ECL active, producing intense ECL in the presence of a co-reactant (TPA) even at submicromolar concentrations. Quartz Crystal Microbalance with Dissipation (QCM-D) monitoring of interactions between PNA/peptide/Ru(II) conjugates and biomimetic membranes showed the unmodified PNA oligomer and its Ru(II) conjugate to traverse freely across the membrane in a trans-membrane manner without causing significant changes in membrane structure, for all lipid compositions. On the contrary, the Nuclear Localised Signal Peptide (NLS) conjugated PNA sequences showed membrane specific activities. In model mammalian membranes, rapid trans-membrane insertion was observed followed by a concentration dependent material removal (mainly from the membrane surface). The PNA sequences were found to cause greater disruption to the bacterial-mimetic membrane system. Strong interactions with the membranes also tend to cause irreversible structural changes, an effect prevailing in all model systems, suggesting similar activity mechanisms. The variations in the magnitude of the structural changes and disruptive tendency of PNAs are ascribed to their cationic charge and hydrophobicity along with the physical state of the model membrane used.