A general method for labeling siRNA by click chemistry with fluorine-18 for in vivo PET imaging Flagothier J 1, Mercier F 1, Kaisin G 1, Teller N 2, Warnock G 1, Plenevaux A 1, Luxen A 1 1 ULg – Liège University – Cyclotron Research Center – Liège – Bât. B30, Allée du 6 août, 8 - Belgium 2 Eurogentec SA – Building 2 – Liège Science Park – rue Bois St-Jean 5 – Belgium Introduction Positron emission tomography (PET) is a high- resolution, sensitive, functional imaging technique that permits repeated, non invasive assessment and quantification of specific biological and pharmacological processes in humans. [1] Fluorine-18 appears often as the radionuclide of choice for the preparation of short-lived positron-emitter radiotracers due to its physical and nuclear characteristics. [2] Fluorine-18 labeling of biomolecules such as peptides, [3] oligosaccharides, and oligonucleotides [4] (ONs) requires very mild reaction conditions. Today, the method of choice for a highly efficient fluorine-18-labelling of ONs is the conjugation of a prosthetic group, carrying the radioisotope, with a reactive function of the ONs. For the conjugation reaction of the prosthetic group with the ON, we selected click reaction and more specifically the Cu(I) catalyzed formation of 1,2,3- triazole using Huisgen 1,3-dipolar cycloaddition of terminal alkynes with azides (Figure 1). This reaction is highly regioselective leading to 1,4-disubstituted 1,2,3-triazoles and can be performed in different solvents with very high yield. [5-7] Conjugations with ONs are usually performed at 3’-ends using a well chosen linker in order to limit degradation by exonucleases. [8] Here we report three alkyne-bearing linkers, the synthesis of siRNA modified by two these linkers and click chemistry to finally obtain [ 18 F]siRNA. The siRNA used in this case is an inhibitor of MPP-12 protein. MMP-12 is considered to Figure 1 : Coupling between ONs and prosthetic group using click chemistry. Results Three linkers 1, 2 and 3 have been synthesized in our laboratory. The linkers must fulfill a number of requirements: an alkyne for click reaction with the [ 18 F]synthon, a secondary alcohol with a succinic arm to connect to the CPG solid support and a primary alcohol protected by DMTr group to start the ON sequence (Figure 2). The [ 18 F]siRNA was purified by semi-preparative HPLC and formulated before IV injection in mice (Figure 3 and 4). Synthesis has also been realized with linker 2. The conditions of reaction are identical to that for the double-stranded siRNA 8 synthesis and for the click chemistry. Conclusion We have prepared three new universal linkers which allow the introduction of an alkyne function at the 3’-end of ONs. Under click conditions, this alkyne modified ON can then react with an azide function of a prosthetic group carrying the fluorine radioisotope, 1-(azidomethyl)-4-[ 18 F]-fluorobenzene which is obtained using a remote controlled synthesizer FastLab TM. The [ 18 F]siRNA has been injected intravenously in normal mice and an asthmatic mouse model and the biodistribution has been determined by µPET/CT. The tracer was rapidly removed by the renal system with high activity in the kidneys and bladder. High uptake was also seen in the liver and intestines. The next step will be administration by inhalation to target directly the lungs. Two articles about synthesis of [ 18/19 F]siRNA are in progress in our laboratory. References [1]: Schubiger P.A. et al.; Chem. Rev. 108: (2008) [2]: Pike V.W. et al.; Eur. J. Org. Chem. 17: (2008) [3]: Olberg et al.; Bioconjug. Chem. 19: (2008) [4]: Viel T. et al.; J. Label. Compd. Radiopharm. 50: (2007) [5]: Kolb, H. C. et al.; Angew. Chem. Int. Ed. Engl. 40: (2001) [6]: Gil, M.V. et al.; Synthesis (2007) [7]: Victoria D. Bock et al.; Eur. J. Org. Chem (2006) [8]: Stetsenko D.A. et al.; Bioconjug. Chem. 12: (2001) [9]: Garbaki N. et al.; Pulm. Pharmacol.Ther. 22: ( 2009) [10]: Thonon D. et al.; Bioconjug. Chem. 20: (2009) Acknowledgements We gratefully acknowledge Dr N. Teller (Eurogentec, Liège, Belgium) for support on ON chemistry and the solid support functionalization, and the Région Wallone for financial support (OligoPETprojects). Cyclotron Research Center Figure 2: Linkers used for the functionalization of CPG Scheme 1: Functionalization of CPG solid support and siRNA synthesis The following oligonucleotide sequences (synthesized by Eurogentec (Liège, Belgium)) were used: 5-UCACUUACAGGAUCUAUAA-3 (sense) and 5-UUAUAGAUCCUGUAAGUGA-3 (antisense). [9] This siRNA should target the lungs, the major site of MPP-12 expression. To obtain the siRNA [ 18/19 F]11, a click chemistry reaction was realized (Scheme 2). The prosthetic group used was 1-(azidomethyl)-4-[ 18 F]- fluorobenzene. [10] The radiosynthesis of the synthon [ 18/19 F]10 has been fully automated on a FASTLab TM synthesizer from GE HealthCare. Figure 3: µPET/CT image of biodistribution of [ 18 F]siRNA in normal mouse. Scheme 2: Click reaction Figure 4: µPET/CT image of biodistribution of [ 18 F]siRNA in asthmatic mouse model. No differences were apparent between normal and asthmatic mouse. After IV injection, selective binding of [ 18 F]siRNA in the lungs was limited. The siRNA synthesis has been realized on the solid support modified by the linkers 1 and 2. The sense- strand and the antisense-strand have been synthesized with modified alkyne solid support and a normal solid support, respectively. After cleavage and annealing, we obtained the ON modified by the linker (Scheme 1). be highly associated with inflammatory lung disease. [9]