Kinetic Spin Chemistry Study of Key Radicals of Biomolecules

The CIDNP (Chemically Induced Dynamic Nuclear Polarization) method has been widely used to study reactions between a photo-excited dye and bio molecules in order to learn something about the exposure of polarizable groups in the environment of bio molecules. After photo excitation radical ion pairs are formed. The aim of the project is to get detailed information about the structure, the reactivity and the spin behavior of such short living intermediates consisting of triplet states and radicals. As photo synthesizer various substances (dyes), like 2,2,-bipyridyl, flavin mononucleotides, carboxybenzophenones, bipyrazine and anthraquinone sulfonic acids will be used in combination with amino acids tyrosine, trypthophane, histidine, methionine etc. The excited states of the photo sensitizer molecules react reversibly with the amino acid side chains on the surface of the proteins to generate dye-protein radical ion pairs, in which spin-selective singlet-triplet transitions lead to nuclear polarization and magnetic field effects (MFE). These effects can be detected by CIDNP- and MARY-techniques. To clarify the whole reaction mechanism including electron- and pH-dependent proton transfer reactions, additional laser flash photolysis (LFP) investigations are necessary. The corresponding rate constants can be obtained from concentration dependent quenching reactions. From the combination of all these spectroscopic methods we expect detailed insights into the reactions mechanisms involved in these biochemical reactions. The experimental results will be compared with theoretical calculations based on modern diffusion theories.

Free radicals, resulting from electron transfer, are often observed in living biological systems to give rise to many important, functional processes. Nucleic acids and their building blocks are involved in the pathological damage of DNA due to electron removal from DNA bases. This makes the characterization of the radicals and the study of their reactions with nucleotides extremely important. Unfortunately, optical techniques often suffer from insufficient spectral resolution and do not allow unambiguous identification of the radical intermediates of purine nucleotide, while their direct detection by Electron Spin Resonance Spectroscopy (ESR) under physiological conditions is usually problematical because of very short lifetimes of such radicals. We found only limited examples of the detection of radicals derived from purines in solution by means of ESR-spectroscopy. In order to study the chemical reactivity of two purine nucleotides, adenosine-5?-monophosphate (AMP) and guanosine-5?-monophosphate (GMP), towards 3,3?,4,4?-benzophenone tetra-carboxylic acid (TCBP) in the triplet-excited state in aqueous solutions of different pH at room temperature and to provide further information on the acid-base properties of radical intermediates observed in the photo-oxidation reactions of purines in aqueous solution, we have combined two techniques, namely the Time-Resolved laser flash photolysis (LFP) and the Time-Resolved Chemically Induced Dynamic Nuclear Polarization (CIDNP). We have been able to study the kinetics of the photo-induced oxidation of GMP and AMP and we got information to provide a basis for understanding the rates of electron transfer within these molecules. The pH-dependence of the kinetics is now well understood.