Diapositive 1

1
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
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
oligonucleotides4 (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 18FsiRNA.
The siRNA used in this case is an inhibitor of
MPP-12 protein. MMP-12 is considered to
be highly associated with inflammatory lung
disease. 9
Figure 1 Coupling between ONs and prosthetic
group using click chemistry.
Results
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
18/19F11, a click chemistry reaction was
realized (Scheme 2). The prosthetic group used
was 1-(azidomethyl)-4-18F-fluorobenzene.10
The radiosynthesis of the synthon 18/19F10 has
been fully automated on a FASTLabTM synthesizer
from GE HealthCare.
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 18Fsynthon, 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).
Figure 3 µPET/CT image of biodistribution of
18FsiRNA 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 18FsiRNA 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.
Figure 4 µPET/CT image of biodistribution of
18FsiRNA in asthmatic mouse model.
No differences were apparent between normal and
asthmatic mouse. After IV injection, selective
binding of 18FsiRNA in the lungs was limited.
Scheme 1 Functionalization of CPG solid support
and siRNA synthesis
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)-4-18F-fluorobenzene which is
obtained using a remote controlled synthesizer
FastLabTM. The 18FsiRNA 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 18/19FsiRNA are in progress in our
laboratory.
References 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)
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).