Solids and liquids NMR of biological metal complexes

Metal-ions like sodium, magnesium, zinc and cobalt are important cofactors for biomolecular structure and function. Folding processes of nucleic acids or proteins, the tertiary structure stabilization, and catalysis are accredited to metal-ions. This proposal targets the solid-state and solution NMR study of metal-ion interactions to porphyrinoid systems, RNA or DNA duplexes and quadruplexes. Using solution and solid-state NMR techniques, the metal-ions can be localized due to exploiting dipolar interactions in conjuction with quadrupolar or paramagnetic interactions.Classical NOE-spectroscopy and through the-bond heteronuclear correlation can be used between spin 1/2 nuclei to measure distances in solution state. These methods are however only of very limited use for quadrupole nuclei (spin>1/2, 23Na, 25Mg, 59Co, 51V, 43Ca) or paramagnetic systems. For paramagnetic systems pseudo-contact shifts, relaxation enhancement, and cross-correlated Curie-Spin relaxation can deliver additional structure information in solution. In the solid phase, site specific assignment can be done using novel multiple quantum magic angle spinning recoupling (e.g. MQMAS or MQMAS-REDOR) techniques, which were described by the author of this proposal previously. Based on these studies on model systems bigger complexes, like the above mentioned biologically relevand porphyrinoids and DNA or RNA oligoplexes will be investigated in the proposed research. In addition computational approaches will be done to compare the spectroscopic parameters (chemical shift anisotropy, quadrupolar coupling constant, asymmetry parameter) obtained from experimental methods with theoretical predictions and to refine the structure models. The porphyrinoid samples will be prepared at the Institute of Organic Chemistry at the University of Linz as well as the RNA/DNA -quadruplexes and - duplexes in a close collaboration with the Department of Molecular Biology at University of South Bohemia Budweis in the Czech Republic.The solid-state NMR experiments can be done in a collaboration with the New York University (Chemistry Department) and the New York Structural Biology Center. Liquid state NMR experiments will be run in Linz.

Two main groups of porphyrin compounds can be distinguished according to their functional qualities. The first group is characterized by selectively uptake by tumor cells and interaction with nuclear cell DNA. This interaction leads to inhibition of cell proliferation by induction of apoptosis, necrosis or autophagy without light activation. The porphyrinoid backbone is the structural element necessary for selective accumulation of these drugs in tumor cells. Besides a modification of DNA interaction, the introduction of new meso-substituents can influence the penetrance and localization of porphyrin compounds into the cell. The second group are used as photosensitzers in PDT producing reactice oxygen species (ROS) after activation with light of the appropriate wavelength. Photodynamic therapy (PDT) is used in clinical oncology (PDT) for more than 25 years. Using the advantage of a photosensitizing drug (PD) stimulated by light of the appropriate wavelength followed by damage of a target tissue, PDT is used in clinical treatment of both pre-cancer and cancer lesions. The drug application is usually systemic nevertheless the activation is achieved through precise application of light and therefore is considered to be more local rather than systemic. Although a group of drugs is used in clinical routine, there are only a few novel porphyrin compounds that achieve the advantages of being fully water soluble and having a stimulation wavelength rather in near the infrared spectrum to increase the penetration depth. ROS are known to interact with cells in many ways leading to cell damage and consequently cell death. The most important are DNA strand cleavage and destruction of mitochondrial membranes, which leads to apoptosis or necrosis. Nevertheless little is known about the exact intracellular mechanism leading to cell death. In conclusion there is a substantial need to design selectively tailored fully water soluble porphyrins and to elucidate their intracellular mechanism in porphyrin induced cell death. In the past stand-alone FWF-project (FWF-P18384; "Solid state and liquid NMR of biomolecular metal complexes") many porphyrinoid derivatives, with altered substitution pattern, were synthesized and characterized systematically. The interaction modes of porphyrins with DNA were studied with solution NMR Spectroscopy and Circular Dichroism Spectroscopy. Their tumoristatic effects were evaluated on the small intestinal neuroendocrine tumor (SI-NET) cell line KRJ-I and the medullary thyroid carcinoma (MTC) cell line MTC-SK, both known for their poor response to standard chemo- and radiotherapy. The antiproliferative effects of the synthesized porphyrin compounds were evaluated, nevertheless the underlying cellular pathways involved in the antiproliferative response in both tumor cell lines are still unknown. As a consequence, the investigation of intracellular signaling pathways is one of the main objectives of the further research work of my research group.