F-18 radiolabelling of biologically interesting molecules via click-chemistry

The doctorate project is focused on the development and standardization of a general method for F-18 radiolabelling of biologically interesting molecules (e.g. peptides and oligomers), to carry out in vivo imaging assays by PET (Positron Emission Tomography). The selected procedure deals with the introduction of a terminal alkynyl group into the biological substrate, and the synthesis of a functionalizable F-18 molecule containing an azide moiety. Once introduced the radioisotope, the following Huisgen dipolar cycloaddition between the alkyne and the azide affords the desired radiolabelled biomolecule. After problematic and non-producing attempts in employing commercial available or described azides, a fully new fluorinated azide was designed. It is composed by a bifunctionalized aryl bringing, not directly linked to the ring, an azide group at one side, and a PEG chain terminated by a fluoride atom at the other. The aim is to obtain a precursor with suitable characteristics of versatility, easy handling, stability, and reactivity, necessary to make efficient conjugation to biomolecules, and to minimize non-specific binding during PET exam. The synthetic work was carried-out in BIOMETRA Department labs of Milan University, in collaboration with the professor Patrizia Ferraboschi research group, while the radiochemistry activities were performed in Tecnomed Foundation (Milano-Bicocca University) labs. Various synthetic pathways were tested in order to obtain an azidic precursor useful for F-18 introduction, and for the subsequent cycloaddition reaction. In particular, a strategy of consecutive orthogonal protection-deprotection steps brought to the synthesis of three different precursor types (tosylate, mesylate, and iodinated), and the fluorinated reference standard. Starting from commercially available methyl 4-(bromomethyl)benzoate, the ester group was reduced to alcohol. The alcohol residue was protected as tetrahydropyranyl ether, while bromine was employed as leaving group to introduce a triethylene glycol chain. After protection of the terminal hydroxyl as acetate, benzylic hydroxyl was transformed to mesylate, and finally to azide. Removal of the acetate made possible the introduction of the mentioned leaving groups useful for the fluorination reaction. F-18 radiolabelling tests, carried-out on a dedicated automated synthesis module, demonstrated that the best precursor, in terms of radiolabelling yield and effectiveness of purification by separation to the substrate, was the iodinated one. Purification of radioactive azide through cartridges procedure resulted in 51% radiochemical yield non-corrected for the decay (radiochemical purity 93%). It was then carried-out the fluoroazide conjugation with a low-cost biological model, propargylglycine. The cold click-chemistry reaction was performed in a water/acetonitrile/DMSO solution, in presence of copper (II) sulfate, and sodium ascorbate, bringing successfully to the corresponding 1,2,3-triazole. The radioactive analogue reaction showed the effectiveness of the conversion to the radioactive triazole (52% conversion, non-corrected for the decay) within 30 minutes. In future developments, a resources scale-down for the radioactive procedure will be implemented. Moreover, the triazole final purification method will be optimized, and this methodology will be applied to biologically relevant molecules (e.g. the propargylglycine S-enantiomer or interesting tool-peptides such as RGD or NGR peptides). In vivo imaging will be performed on a dedicated small animal PET scanner, in collaboration with the pre-clinical research group directed by professor Rosamaria Moresco (Milano-Bicocca University).