A general method for labeling siRNA by click chemistry with fluorine-18 for
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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

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A general method for labeling siRNA by click chemistry with fluorine-18 for in vivo PET imaging

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A general method for labeling sirna by click chemistry with fluorine 18 for in vivo pet imaging

A general method for labeling siRNA by click chemistry with fluorine-18 for in vivo PET imaging

Flagothier J1, Mercier F1, Kaisin G1, Teller N2, Warnock G1, Plenevaux A1, Luxen A1

1ULg – Liège University – Cyclotron Research Center – Liège – Bât. B30, Allée du 6 août, 8 - Belgium

2Eurogentec SA – Building 2 – Liège Science Park – rue Bois St-Jean 5 – Belgium

Cyclotron Research Center


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

be highly associated with inflammatory lung disease. [9]

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 highyield.[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[18F]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.


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[18/19F]11, a click chemistry reaction was realized (Scheme 2). The prosthetic group used was 1-(azidomethyl)-4-[18F]-fluorobenzene.[10] The radiosynthesis of the synthon[18/19F]10has been fully automated on a FASTLabTM synthesizer from GE HealthCare.

Threelinkers1, 2 and 3 have been synthesized in ourlaboratory. The linkers must fulfill a number of requirements: an alkyne for click reactionwith the [18F]synthon, a secondaryalcoholwith a succinic arm to connect to the CPG solid support and a primaryalcoholprotected by DMTr group to start the ON sequence(Figure 2).

Figure 3: µPET/CT image of biodistribution of [18F]siRNA in normal mouse.

Figure 2: Linkers used for the functionalization of CPG

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).

Scheme 2: Click reaction

The [18F]siRNAwaspurified by semi-preparative HPLC and formulated before IV injection in mice (Figure 3 and 4).

Synthesis has also been realizedwith linker 2. The conditions of reaction are identical to that for the double-strandedsiRNA8 synthesisand for the click chemistry.

Figure 4: µPET/CT image of biodistribution of [18F]siRNA in asthmatic mouse model.

No differences were apparent between normal and asthmatic mouse. After IV injection, selective binding of [18F]siRNA in the lungs was limited.

Scheme 1: Functionalization of CPG solid support and siRNA synthesis


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-[18F]-fluorobenzene which is obtained using a remote controlled synthesizer FastLabTM. The [18F]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 [18/19F]siRNA are in progress in our laboratory.


We gratefully acknowledge Dr N. Teller (Eurogentec, Liège, Belgium) for support on ON chemistry and the solid support functionalization, and the RégionWallone for financial support (OligoPETprojects).


[1]: Schubiger P.A. et al.; Chem. Rev. 108: 1501-1516 (2008) [2]: Pike V.W. et al.; Eur. J. Org. Chem. 17: 2853-2873 (2008) [3]: Olberg et al.; Bioconjug. Chem. 19: 1301-1308 (2008) [4]: Viel T. et al.; J. Label. Compd. Radiopharm. 50: 159-1168 (2007) [5]: Kolb, H. C. et al.; Angew. Chem. Int. Ed. Engl. 40: 2004-2021 (2001) [6]: Gil, M.V. et al.; Synthesis 1589-1620 (2007) [7]: Victoria D. Bock et al.; Eur. J. Org. Chem. 51-68 (2006) [8]: Stetsenko D.A. et al.; Bioconjug. Chem. 12: 576-586 (2001) [9]: Garbaki N. et al.; Pulm. Pharmacol.Ther. 22: 267-278 ( 2009) [10]: Thonon D. et al.; Bioconjug. Chem. 20: 817-823 (2009)

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