A 'click chemistry' approach to the efficient synthesis of modified nucleosides and oligonucleotides for PET imaging

Article abstract of DOI:10.1016/j.tetlet.2009.12.120

Different thymidine derivatives bearing either an iodoaryl moiety at the 5′ position or a dialkylsilyl group at the 3′ position have been efficiently synthesized as precursors for carbon-11 or fluorine-18 labeling, respectively. Furthermore, iodoarylated thymidine derivatives have been incorporated into oligonucleotides giving an original way to label them with carbon-11.

Full text of DOI:10.1016/j.tetlet.2009.12.120

Tetrahedron Letters 51 (2010) 1230–1232
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A ‘click chemistry’ approach to the efficient synthesis of modified
nucleosides and oligonucleotides for PET imaging
Damien James a, Jean-Marc Escudier b, Eric Amigues a, Jürgen Schulz c, Christiane Vitry a,
a,
Thomas Bordenave a, Magali Szlosek-Pinaud a, , Eric Fouquet
*
*
a Université de Bordeaux,UMR CNRS 5255, Institut des Sciences Moléculaires, 351 Cours de la Libération, Talence F-33405, France
b Université Paul Sabatier, UMR CNRS 5068, LSPCMIB, 118 rte de Narbonne, Toulouse F-31062, France
c Université de Bordeaux, UMR CNRS 5231, LIMF, 146 rue Léo Saignat, Bordeaux F-33076, France
a r t i c l e i n f o
a b s t r a c t
Different thymidine derivatives bearing either an iodoaryl moiety at the 50 position or a dialkylsilyl group
at the 30 position have been efficiently synthesized as precursors for carbon-11 or fluorine-18 labeling,
respectively. Furthermore, iodoarylated thymidine derivatives have been incorporated into oligonucleo-
tides giving an original way to label them with carbon-11.
Article history:
Received 16 November 2009
Revised 16 December 2009
Accepted 21 December 2009
Available online 28 December 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Positron Emission Tomography (PET) is a powerful imaging
technique for clinical, medical, and biological investigations in var-
ious areas such as oncology, cardiology, and neurosciences. Due to
the increasing need of this technique in in vivo biochemistry and
medicine, the development of new tracers and radiolabeling strat-
egies is always requested.1–3 Nucleosides as PET tracers have
recently been investigated by a number of institutions.4 Among
them, the [18F]-fluorothymidine (FLT) has even become the second
tracer, after [18F]-fluorodeoxyglucose (FDG), for routine clinical
PET. Furthermore, aptamers (‘adaptable oligomers’) can be gener-
ated against any small molecules or protein targets using a com-
pletely synthetic method (SELEX).5 Thus, the use of aptamers for
in vivo imaging is specially promising because of the wide range
of possibilities available to introduce variations in their structure
through defined chemical modifications. Only a very few examples
of oligonucleotides labeling for PET were reported with gallium-
68,6 carbon-11,7 and fluorine-18,8 and to the best of our knowledge
there is only one report of a fluorine-18 labeling attempt to an oli-
gonucleotide aptamer.9
synthesis of modified thymidines bearing either an iodoaryl moi-
ety or a dialkylsilyl group, and then the incorporation of iodoary-
lated thymidines into oligonucleotides (Fig. 1).
The 50-iodoarylated thymidines 4, 7, and 9 were all synthesized
from thymidine aldehyde 1, prepared according to the Pfitzner–
Moffatt procedure,16,17 and by using a 3+2 cycloaddition of an azide
with an alkyne so called ‘click’ chemistry as conjugation mode
between the arm bearing an iodoaryl moiety and the nucleoside
(Scheme 1). Two ways were explored by using either the azido-
thymidine 2 or the propargyl-thymidine 5.18 Thus, 2 was obtained
in two steps: the first one being a Sakuraï reaction between 1 and
the
x-bromoallyltrimethylsilane, easily prepared itself from x-
hydroxyallyltrimethyl-silane,19 leading after treatment with TiCl4,
to the 50-C(S)-bromopentenyl thymidine with
a 55% overall
yield.20,21 Then, the azido group needed for ‘click chemistry’ was
introduced by action of sodium azide in anhydrous dimethylform-
amide at 80 °C providing 2 in 89% yield. The ‘click reaction’ was then
realized using the alkyne 3 as the partner, prepared from 4-iodophe-
However, the chemistry of carbon-11 is nowadays representing
an essential tool for the clinical research purpose.10 In that way we
recently developed a modified version of the Stille coupling allow-
ing an efficient transfer of alkyl groups,11 and the preparation of a
T
O
Carbon-11 labelling
OR
I
[
11C]-methylstannate reagent12 as well as its application to the
T
HO
R = H, or ODN
O
labeling of functionalized quinolines.13 In addition to the organo-
tin-mediated carbon-11 transfer reaction, the use of organosilicon
chemistry for fluorine-18 labelling of peptides has been recently
described by Ametamey’s group.14,15 We then envisaged first the
OH
T = Thymine
T
HO
O
Fluorine-18 labelling
O
t-Bu
Si H
t-Bu
* Corresponding authors. Tel.: +33 054 000 2829; fax: +33 054 000 6283.
E-mail addresses: m.szlosek@ism.u-bordeauxl.fr (M. Szlosek-Pinaud), e.fouquet@
ism.u-bordeaux1.fr (E. Fouquet).
Figure 1. Modified nucleosides or oligonucleotides for PET imaging.
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.12.120

D. James et al. / Tetrahedron Letters 51 (2010) 1230–1232
1231
OH
N
N
T
OH
O
N
O
a, b
T
c, d
N3
O
OH
I
4
O
OSiPh2t-Bu
2
3
I
T
O
O
I
OSiPh2t-Bu
N
N
OH
f, g
T
N
1
O
N3
OH
I
6
7
OH
T
e
O
OSiPh2t-Bu
5
N
N
OH
T
O
N
O
f, g
O
O
N3
OH
I
O
8
I
9
Scheme 1. Synthesis of 50-iodoarylated thymidines 4, 7, and 9. Reagents, conditions, and yields: (a) (i)
x-bromoallyltrimethylsilane, BF3.Et2O, CH2Cl2, ꢀ78 °C to rt, 4 h; (ii)
TiCl4, CH2Cl2, 0 °C, 5 min, 55%; (b) NaN3, DMF, 80 °C, 3 h, 95%; (c) 1-iodo-4-(prop-2-ynyloxy)benzene 3, CuSO4 (20 mol %), sodium ascorbate (60 mol %), t-BuOH/H2O (3:1), rt,
12 h, 89%; (d) NH4F, MeOH, 60 °C, 24 h, 86%; (e) propargyl bromide, Zn (activated), THF, 10 °C, 5 min, 91% (S/R: 70:30); (f) CuSO4 (20 mol %), sodium ascorbate (60 mol %), t-
BuOH/H2O (3:1), rt, 12 h, 84% starting from 6 and 80% from 8; and (g) NH4F, MeOH, 60 °C, 24 h, 85% starting from 6 and 79% from 8.
nol and propargyl bromide via a Williamson reaction, providing the
correspondingtriazolylderivativein89%yield. Finally, thedeprotec-
tion of the silylated group, with NH4F in methanol at 60 °C, allowed
us to get the desired iodoarylated thymidine 4 in 86% yield. On the
other hand, the alkyne-thymidine 5 was obtained in 91% yield, in a
70:30S/R ratio, bytreating1 withpropargylbromide, inthepresence
of activated zinc. At this stage, the two diastereoisomers are separa-
ble by column chromatography, but we decided to use the mixture
for the next steps. 5 was then engaged into two ‘click reactions’
involving two azides according to a procedure described by Thom-
son et al.:22 6 prepared from 4-iodobenzylic alcohol and 8 prepared
by azidation of the alcohol resulting from the Williamson reaction
between monotosylated diethylene glycol23 and 4-iodophenol.
Thus, the corresponding triazoles were obtained in 84% and 80%
yields, respectively, and the deprotection of the 30 position, using
NH4F in MeOH led, finally, to 7 and 9 in 85% and 79% yields,
respectively.
introduction of an alkyne group at the 30 position of a thymidine
was done in three steps, involving the protection of the primary
alcohol in 50 with TBDPS, the propargylation, using the propargyl
bromide, and finally the deprotection of the 50 position leading to
the desired compound 10, in a 61% overall yield.24 On the other
hand, the suitable azide partner was obtained in two steps from
4-bromobenzyl alcohol 11, prepared following a variation of Schre-
iber’s Letter.25 The first one was the introduction of the silyl group,
by treating the lithiated derivative with di-tert-butylchlorosilane,
leading to compound 12 in 50% yield. Then, the ‘click reaction’ be-
tween 10 and 12 was done under classical conditions, leading to
the desired 30-silylated thymidine 13 in 77% yield. Of course, the
two strategies are versatile authorizing labeling with carbon-11
or fluorine-18 in both 30 and 50 positions. Indeed, azide 12 can react
with propargyl-thymidine 5 giving access to the 50-silylated thymi-
dines and in the same way azide 6 can react with propargyl-thymi-
dine 10 giving the 30-iodoarylated thymidines.
Besides the compounds envisaged for the carbon-11 labeling,
we have also developed a synthesis, involving the ‘click chemistry’,
to modify a thymidine at the 30 position with a di-tert-butylsilyl
group in order to label it with fluorine-18 (Scheme 2). Thus, the
After obtaining modified monomers, the next step was to intro-
duce the corresponding phosphoramidites into oligonucleotides
(Scheme 3). Thus we have envisaged to synthesize 10-mer oligonu-
cleotidic sequences, composed of the four bases and including
T
T
T
HO
HO
t-Bu
HO
O
O
a,b,c
f
O
H
Si
N
N
t-Bu
OH
O
O
t-Bu
H Si
t-Bu
N
N3
12
10
13
d,e
OH
Br
11
Scheme 2. Synthesis of 30-silylated thymidine 13. Reagents, conditions, and yields: (a) TBSCl, pyridine, rt, 24 h, 95%; (b) NaH, propargylbromide, THF, rt, 12 h, 76%; (c) PTSA,
MeOH, rt, 24 h, 97%; (d) (i) NaH, THF, 50 °C, 3 h, (ii) t-BuLi, THF, ꢀ78 °C, (iii) ClSiH(t-Bu)2, 50%; (e) DBU, DPPA, DMF, 60 °C, overnight, 80%; and (f) 1-(azidomethyl)-4-(di-tert-
butylsilyl)-benzene 12, CuSO4 (20 mol %), sodium ascorbate (60 mol %), t-BuOH/H2O (3:1), rt, overnight, 77%.

1232
OH
D. James et al. / Tetrahedron Letters 51 (2010) 1230–1232
ODMTr
Acknowledgments
T
T
O
O
a, b, c
P4 : (a) 78%, (b) 80%, (c) 50%
R
R
OSiPh2t-Bu
O
P
P5 : (a) 76%, (b) 81%, (c) 33%
This work was generously supported by the CNRS (grant given
NC
O
P7 : (a) 82%, (b) 77%, (c) 80%
P9 : (a) 74%, (b) 53%, (c) 52%
to D.J.). The authors are grateful to the ‘Plateforme de Synthèse
d’oligonucléotides modifiés de l’Interface Chimie Biologie de l’IT-
TAV’ de Toulouse for providing facilities for oligonucleotide
synthesis.
N(i-Pr)2
4, 5, 7, 9
P4, P5, P7, P9
DNA Synthesizer
Supplementary data
5'-d(T4GACTGACGC)-3' ODN 4
5'-d(T5GACTGACGC)-3' ODN 5
5'-d(T7GACTGACGC)-3' ODN 7
5'-d(TGACT7GACGC)-3' ODN 7'
5'-d(T9GACTGACGC)-3' ODN 9
Supplementary data (experimental details of chemistry and full
characterizations of compounds 1–13 as well as the oligonucleo-
tides ODN4, ODN5, ODN7, ODN7 and ODN9) associated with this
article can be found, in the online version, at doi:10.1016/
j.tetlet.2009.12.120.
0
Scheme 3. Synthesis of phosphoramidites P4, P5, P7, and P9. Reagents and
conditions: (a) DMTrCl, AgNO3, collidine, THF, rt, 12 h; (b) TBAF, THF, rt, 12 h;
and (c) (2-cyanoethyl)(N,N-diisopropylamino)chlorophosphite, (i-Pr)2EtN, THF, rt,
45 min.
References and notes
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either phosphoramidites P4, P5, P7, or P9 (the modified thymidine
being noted T* in the sequence) at the 50 position (50-d(T*GAC-
TGACGC)-30) or even in the middle of the sequence with P7 (50-
d(TGACT*GACGC)-30). To do that, each monomer was converted
first into the corresponding phosphoramidites, in a three-step pro-
cedure (Scheme 3).
The first one was the protection of the 50 position with a DMTr
(p-dimethoxytrityl) group.21 The second step was the deprotection
of the hydroxyl group at the 30 position, and finally, the third one
was the introduction of the phosphoramidite function at the 30 po-
sition leading to P4, P5, P7, and P9. Oligonucleotides were then syn-
10. Allard, M.; Fouquet, E.; James, D.; Szlosek-Pinaud, M. Curr. Med. Chem. 2008, 15,
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thesized on
1 lmol scale by a standard phophophoramidite
methodology. On the basis of trityl assays and materials recovered,
P4, P5, P7, and P9 were incorporated with the same efficiency as the
commercial phosphoramidites. The five oligonucleotides were
then deprotected and removed from the solid support with con-
centrated ammonia. After HPLC purification, the isolated yields
were in the range of those classically obtained for modified oligo-
nucleotides. ODN4, ODN5, ODN7, and ODN9 were characterized by
mass spectroscopy (MALDI-TOF).
In conclusion, 50- and 30-modified original thymidines, envis-
aged as precursors for either carbon-11 or fluorine 18 labeling,
can be prepared in a very efficient way by using ‘click chemistry’
as the conjugation step. By the way, we have already obtained good
results in terms of methyl transfer with 4, 7, and 9 by applying our
modified Stille’s coupling reaction11,12 and preliminary results,
concerning the fluorination of 13, were obtained by applying the
conditions described by Ametamey’s group12 are particularly
encouraging. These results will be reported in due course. Iodoary-
lated monomers have been successfully introduced into oligonu-
cleotides, leading to various substrates usable to investigate our
[
11C]-methyl group transfer methodology. Finally ODN5, the oligo-
nucleotide in which the modified thymidine bears a pendant ter-
minal alkyne, will be used as a versatile substrate to test direct
‘click reactions’18,26 either with 6, 12 or with other suitable azido
partners offering a very original and powerful way to get various
precursors, from the same oligonucleotide, either for the carbon-
11 or for fluorine-18 labeling.
26. Pourceau, G.; Meyer, A.; Vasseur, J.-J.; Morvan, F. J. Org. Chem. 2009, 74, 1218–
1222.