Partner und Internationale Organisationen
(Englisch)
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A, B, CZ, DK, FIN, F, D, GR, H, I, NL, N, PL, P, SI, E, S, CH, GB
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Abstract
(Englisch)
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The COST project C99.0055 'Generalized labeling of radiopharmaceutically relevant biomolecules with [99mTc(OH2)3(CO)3]+ through integrated, monodentate ligand groups: a [2+1] approach. Basic investigations and applications' investigates basic mechanistic behavior of the [M(OH2)3(CO)3]+ (M=Tc, Re) complex as relevant for ligand substitution reaction with bidentate and monodentate ligands. These basic investigations are complemented by synthetic studies for the generalized labeling of biomolecules with ligands that have been evaluated partially in former and more fundamental studies. The labeling of a biomolecule with [99mTc(OH2)3(CO)3]+ requires the substitution of at least one water ligand. The absolute upper rate limit is therefore the water self exchange rate in [99Tc(OH2)3(CO)3]+ or [Re(OH2)3(CO)3]+. This crucial factor allows to predict, if the labeling of a biomolecule will occur with a reasonable rate and if it is convenient for a practical labeling process in routine application. We have measured during the last period this self exchange rate for [Re(OH2)3(CO)3]+ in dependence of the pH-value with NMR techniques at the EPF Lausanne (Prof. A. Merbach). It turned out that the water self exchange rate occurs along two different pathways, one is pH-dependent the other is not. The rate constant of pathway 1 (pH independent) was found to be 6.3 ± 0.1 10-3 s-1 whereas pathway 2 is 27±5 s-1 respectively. This strong pH dependence clearly implies that water exchange at physiological pH must be fast which rationalizes the application of [99Tc(OH2)3(CO)3]+ for labeling of biomolecules at least on the base of water self exchange. The mechanism is supposed to be Id based on DS#. It can be expected that the corresponding rate constants for Tc are by a factor of at least 10 larger than those for rhenium. Corresponding studies are planned for the last part of the project. The rate of ligand substitution with chelators interesting to be applied in biomolecules have been studied as well. Ligands were in particular bipy, phen, dimethylsulfide and thiourea the coordinating groups of which are integral part of many useful ligands. The rates of ligand substitution were found to be in a similar range between 5.8 10-3 M·s-1 (for phen) and 41.5 10-3 M·s-1 (for thiourea). According to these rates, ligand substitution at low concentration occurs at a reasonable rate within the temperature scale of water. Obviously, this experience was made before based on qualitative observations but the results now allow a rationalized design of appropriate ligands and sequence of reactivity for groups of ligands. Additionally, the CO exchange was studied in order to compare the rate of exchanged with those already known for 99Tc. The exchange of CO with 13CO was followed over a several months period. Beside the exchange, several new compounds were detected which correspond essentially to higher carbonylated species [Re(OH2)3-n(CO)3+n]+. A broad additional signal that appeared in 13C NMR spectrum is interesting but could not be assigned so far. The [2+1] principle of labeling biomolecules comprises a bidentate anionic or neutral ligand and a monodentate ligand. The biomolecule can be attached to either of the two but preferentially to the monodentate ligand for convenience. We have performed qualitative studies for the complex formation with many anionic bidentate ligands in the former period (see last abstract). The versatility of the approach could principally be proven and was applied i.e. to derivatized thymidine. The kinetic studies with bipy and phen mentioned above have been extended to acetyl-acetonate and 8-hydroxychinolin (8-Hox) as well, both potentially very versatile bidentate anionic systems. Studies for the mechanism of substitution are currently undertaken, the x-ray structure of [Re(OH2)(acac)(CO)3] confirmed the presence of water after substitution rather than the presence of a halide. The same structural features as for acetyl-acetonate were also found for hydroxy-chinolin and the x-ray structure of [Re(OH2)(Ox)(CO)3] could be elucidated. Beside the ligands investigated in a first part of the project, these ligands might be very useful extension since they are easily derivatized linked to biomolecules. On the side more directed towards practical application, our results were transformed to biologically oriented working groups within the COST B12 action. Several have adopted the [2+1] principle introduced through this project to their own biomolecules. In collaboration with the 'Forschungszentrum Rossendorf, Germany' (Dr. H.-J. Pietzsch) central nervous system (CNS) receptor ligands have been derivatized with a bidentate ligand and the remaining coordination site was substituted with various highly lipophilic monodentate ligands like isocyanides under conditions relevant for practical application. Labeling with this mixed ligand method was successful and biological studies are currently underway. Our contribution in this collaboration is basically technology transfer, initial investigations and the characterization of the chemical compositions of some the products formed. In a further collaboration, we attached histidin through its N-terminus to peptides. These peptides were then labeled with [M(OH2)3(CO)3]+ (M= 99mTc, Re) and various monodentate ligands coordinated to the remaining site. Monodentate chelators were in particular imidazole, pyrazine and isocyanides. Biological studies with these peptides labeled through the generalized [2+1] approach are also on the way. This part of the project is in collaboration with the Klinikum Rechts der Isaar, TU München, (Dr. H.-J. Wester).
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