Silicon-based chemistry: An original and efficient one-step approach to [18F]-Nucleosides and [18F]-Oligonucleotides for PET imaging

Article abstract of DOI:10.1002/chem.201003234

Take it eaSi! Nucleosides, dinucleotides, and one oligonucleotide, all modified by click chemistry, have for the first time been directly and very efficiently labeled with ¹⁸F by using a silicon-based, one-step approach that opens the way for t

Full text of DOI:10.1002/chem.201003234

DOI: 10.1002/chem.201003234
Silicon-Based Chemistry: An Original and Efficient One-Step Approach to
[18F]-Nucleosides and [18F]-Oligonucleotides for PET Imaging**
Jꢀrgen Schulz,[a] Delphine Vimont,[a] Thomas Bordenave,[b] Damien James,[b]
Jean-Marc Escudier,[c] Michꢁle Allard,[a] Magali Szlosek-Pinaud,*[b] and Eric Fouquet*[b]
Positron emission tomography (PET) is a powerful molec-
ular-imaging technique for physiological and biological in-
vestigations in various areas, such as oncology, cardiology,
and neurosciences, as well as for drug development. Due to
the increasing need of this technique for in vivo applications,
there is always a demand for the development of new trac-
ers and radiolabeling strategies.[1–3] PET uses short half-life
of our knowledge there is only one report of a 18F-labeling
attempt of an oligonucleotide aptamer.[9]
Traditional 18F-labeling methods require scrupulously dry,
very basic reaction conditions at high temperature; condi-
tions that are generally not suitable for biomolecules, such
as oligonucleotides.[3c] Therefore, the 18F labeling of those
molecules is always achieved by means of suitable prosthetic
groups labeled with 18F. This approach, however, requires a
multistep reaction sequence and may be time consuming,
leading to lower specific radioactivities (RAS).
radioisotopes, for example, 11C (t1/2 =20.4 min) and 18F (t1/2
=
109.6 min). Thus, rapid synthetic processes, including organic
transformations and purifications, are required to obtain
new radiopharmaceuticals. This chemistry becomes even
more challenging and ambitious when the goal is to label
biomolecules, such as nucleosides or oligonucleotides.
Indeed, nucleosides as PET tracers have recently been in-
vestigated by a number of researchers.[4] Among these trac-
ers, [18F]-FLT (fluorothymidine) was the second tracer to be
used, after [18F]-FDG (fluorodeoxyglucose), for routine clin-
ical PET. Furthermore, because of their excellent targeting
capacities and easy synthesis along with a high level of di-
versity, oligonucleotides are already extensively used in
vitro as ligands for nucleic acids (antisense oligonucleo-
tides), proteins, and small related molecules (aptamer oligo-
nucleotides).[5] Thus, the use of aptamers for in vivo imaging
appears especially promising, because of the wide range of
possibilities available to introduce variations in their struc-
ture through defined chemical modifications. However, only
few examples of oligonucleotide labeling for PET have been
reported so far, with 68Ga,[6] 11C,[7] and 18F,[8] and to the best
Furthermore, to simplify PET chemistry, there has been
recent interest in developing rapid, one-step labeling proce-
dures from shelf-stable, final-target precursors with aqueous
18FÀ, involving either fluoride–aluminium-chelate com-
plexes[10] or boron-[11] or silica-based[12] aqueous 18F capture.
In addition, there have been several improvements of the
nucleophilic SN2 fluorination applied to bioactive targets.[14]
However, all those methods have only been described for
direct 18F labeling of biomolecules, such as peptides or oligo-
saccharides, but not for oligonucleotides. In this context, we
chose to develop a similar approach to that of Ametameyꢀs
work,[13] which describes an efficient, one-step, no-carrier-
added nucleophilic 18F fluorination of peptides by using sili-
con–fluorine chemistry for nucleoside and oligonucleotides
18F labeling. To achieve that, we have envisaged to associate
two different bifunctional silicon building blocks with modi-
fied thymidines by click chemistry. Those monomers were
then either coupled to another nucleotide (T or G) to get si-
lylated dinucleotides or incorporated into a longer sequence
by using a DNA synthesizer. Each type of substrate (nucleo-
sides, dinucleotides, or oligonucleotides) was then engaged
into the 18F-fluorination reaction (Scheme 1).
[a] Dr. J. Schulz, Dr. D. Vimont, Prof. M. Allard
Universitꢁ de Bordeaux, LIMF, UMR5231, 146, rue Lꢁo Saignat
Bordeaux, 33076 (France)
First, we synthesized two different bifunctional silicon
building blocks, 2 and 3, with an azide or an alkyne moiety,
respectively, starting from 4-di-tert-butylsilyl-benzylalcohol
(1) obtained from 4-bromobenzylalcohol according to our
previously described procedure (Scheme 2).[15] The di-tert-
butylsilyl group has been reported to be the best substituent
[b] T. Bordenave, Dr. D. James, Dr. M. Szlosek-Pinaud, Prof. E. Fouquet
Universitꢁ de Bordeaux, ISM, UMR5255
Synthesis—Bioactive Molecules group
351, Cours de la liberation, Talence, 33405 (France)
Fax : (+33)540006286
E-mail: m.szlosek@ism.u-bordeaux1.fr
e.fouquet@ism.u-bordeaux1.fr
À
on the silicon atom to stabilize the Si F bond under physio-
logical conditions.[16] Then, 2 and 3 were engaged in micro-
wave-radiation-mediated click reactions with 3’-propargyl-
[c] Dr. J.-M. Escudier
Universitꢁ Paul Sabatier, SPCMIB, UMR5068
Modified Nucleic Acids group
118, rte de Narbonne, Toulouse, 31062 (France)
AHCTUNGTRENNUNG
thymidine (4)[15] and 5’-azidothymidine (5)[17], respectively,
leading to 6 in 99% yield and 7 in 82% yield (Scheme 2).
To evaluate the compatibility of our labeling conditions
with the phosphodiester linkage and the presence of a
[**] PET=positron emission tomography
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201003234.
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Chem. Eur. J. 2011, 17, 3096 – 3100

COMMUNICATION
Scheme 1. Silicon-based 18F radiolabeling of nucleosides and oligodeoxynucleotides (ODNs).
purine base, such as guanine, known to be sensitive to acidic
conditions at high temperatures, we first synthesized two
model dinucleotides (T–T and T–G) by the phosphoramidite
method.[18] The dinucleotides were prepared by starting
from 6 and coupling to the commercially available phos-
phoramidites of thymidine or guanosine (3.4 equiv) with ex-
cellent yields. Then the dimethoxytrityl (DMTr) group was
removed under acidic conditions (trifluoroacetic acid
(TFA)) and the cyanoethyl group and the protecting group
of the guanine were removed by using either triethylamine
(T) or ammonia (G), leading to 8 and 9 in 88 and 81%
yields, respectively, over the two steps (Scheme 2).
Similarly to what has been reported by Ametamey et al.,
À
that is, hydrogen as leaving group (Si H), we observed that,
most of the time, the addition of acetic acid did not exert
any dramatic effect, except in two cases (Table 1, compare
Table 1. 18F radiolabeling of 6, 7, 8, and 9.[a]
Entry Precursor (amount [mg]) T [8C] Acetic acid [ml] RCY [%][b]
1
2
3
4
5
6
7
8
9
10
11
12
13
14
6 (6)
6 (6)
6 (6)
7 (6)
7 (6)
7 (6)
8 (7)
8 (6)
8 (6)
8 (8)
9 (7)
9 (7)
9 (6)
9 (8)
60
60
165
60
60
165
60
3
0
3
3
0
3
3
0
3
3
3
0
3
3
43
11
30
34
33
32
22
19
24
33
23
16
23
36
Finally, we chose to prepare a decameric ODN composed
of the four bases and including a silylated thymidine (T*).
60
To use
a DNA synthesizer and the phosphoramidite
100
165
60
60
100
165
method, the easiest solution was to prepare phosphorami-
dite 10 from 7 and introduce it towards the end of the syn-
thesis, at the 5’ position, leading to ODN 11: 5’-d(T*GACT-
GACGC)-3’.
When silylated-modified thymidines, dinucleotides and
one ODN, had been obtained, the next step was to investi-
gate the 18F fluorination by using potassium fluoride in the
presence of kryptofix-2.2.2 in DMSO at different tempera-
tures (60, 100, or 1658C) and with or without acetic acid
(3 mL) as an additive.[13a] The highest temperature of our
study was chosen on the basis of preliminary promising re-
sults with more robust substrates at 1658C, even if we could
anticipate that such conditions could be less compatible with
oligonucleotides. Furthermore, all experiments started with
high activities of 18F (100–130 GBq), corresponding directly
to the production conditions of real radiopharmaceuticals,
whenever these conditions are generally known to decrease
the yields of the radiosynthesis due to the radiolysis phe-
nomenon.
[a] 18F labeling experiments were carried out in DMSO (300 mL) for
15 min. [b] The RCYs are not decay corrected.
entry 1 and 2, and Table 2, compare entry 2 and 3). Never-
theless, the radiochemical yields (RCYs) are consistently
higher when acetic acid is used. Finally, optimization of the
reaction conditions, especially in terms of temperature, led
to RCYs of the 18F incorporation of up to 43% (not decay
corrected) with any type of substrates (nucleoside/dinucleo-
tides/oligonucleotides). Thus, with nucleosides 6 (Table 1,
entry 1) and 7 (Table 1, entry 4) 608C was sufficient to ach-
ieve high 18F incorporation, whereas 1658C lead to similar
RCYs (Table 1, entry 6), or even lower (Table 1, entry 3,
Scheme 3). With dinucleotides 8 and 9, a higher reaction
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E. Fouquet, M. Szlosek-Pinaud et al.
Scheme 3. 18F fluorination of nucleosides 6 and 7 and dinucleotides 8 and
9.
both dinucleotides, whereas at 1658C RCYs were improved
to 33 and 36%, respectively (Table 1, entries 10 and 14).
These results have thus demonstrated the perfect compati-
bility of our conditions of fluorination with the phospho-
diester linkage as well as the presence of a more fragile
purine base, such as guanine.
Finally, to test the practical utility of this 18F-fluorination
method, 18F labeling of the model ODN 11 was performed
under similar conditions (Figure 1). Firstly, as previously de-
scribed for the dinucleotides, 608C led to the lowest RCY
(Table 2, entry 1). But, as anticipated, 1658C was not the
ideal temperature for such a fragile substrate (Table 2,
entry 4). Finally, the best compromise was found to be
1008C (Table 2, entry 2), giving [18F]-16 with excellent RCY
(36%) and purity (Figure 1). This represents a unique exam-
ple of direct 18F labeling of oligonucleotides at the last step
of the synthesis.
To conclude, this is a unique and efficient protocol for the
18F fluorination of nucleosides, leading, in particular, to 12
in 43% not-decay-corrected yield with specific activity over
370 GBqmmolÀ1 and more than 95% radiochemical purity
accompanied with a high product purity. Undoubtedly, this
work represents a very promising alternative to the actual
[18F]-FLT radiotracer. Biological evaluations of 12 are cur-
rently underway. Furthermore, this method offers a unique
approach to label oligonucleotides. This hypothesis was first
validated by the results obtained with dinucleotides 8 and 9,
showing that our methodology was perfectly compatible
both with the phosphodiester linkage and the presence of
purine bases in the sequence. Then, those conditions of 18F
fluorination were applied successfully and very efficiently to
an ODN, constituting the unique example of a direct 18F la-
beling of an oligonucleotide at the last step of the synthesis.
Thus, 16 was obtained in 36% not-decay-corrected yield,
along with a high specific activity (over 259 GBqmmolÀ1)
and more than 95% radiochemical purity by using the pro-
duction conditions of radiopharmaceuticals used clinically as
Scheme 2. Silylated thymidines 6 and 7, dinucleotides 8 and 9, and ODN
11. a) 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), diphenylphosphoryl
ACHTUNGTRENNUNG
azide (DPPA), DMF, RT, overnight, 99%; b) NaH, propargyl bromide,
THF, overnight, RT, 91%; c) CuSO4 (20 mol%), sodium ascorbate
(60 mol%), tBuOH/H2O (3/1), 1008C (microwave radiation 50 W),
10 min, 99% starting from 4 and 82% from 5; d) 1H-tetrazole, 30 min,
RT, then collidine, I2, 1 h, RT, 98% with B=T, 99% with B=G; e) TFA
(3%), CH2Cl2, 15 min, then Et3N, CH3CN, 1 h, 608C, 88% with B=T or
NH4OHsol (25% in H2O), 558C, overnight, 81% with B=G; f) 2-cyano-
ACHTUNGTRENNUNGethyl(N,N-diisopropylamino) chlorophosphoramidite, (iPr)2EtN, THF,
45 min, RT, 60%; g) DNA synthesizer.
Table 2. 18F radiolabeling of ODN 11.[a]
Entry
Amount [mg]
T [8C]
Acetic acid [ml]
RCY [%][b]
1
2
3
4
1
1
1
3.7
60
100
100
165
3
3
0
3
1
36Æ2[c]
9
7.6
[a] 18F labeling experiments were carried out in DMSO (300 mL) for
15 min. [b] The RCYs are not decay corrected. [c] Number of experi-
ments=3.
temperature was clearly required (Scheme 3). Thus, at 608C
(Table 1, entries 7 and 11) and 1008C (Table 1, entries 9 and
13), almost identical RCYs, around 23%, were observed for
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18F-Labeled Nucleosides and Oligonucleotides
COMMUNICATION
tions. The semi-preparative HPLC system was used with a Sykam S1021
pump system equipped with UV-variable wavelength Knauer and Ge ra-
diodetector. UV chromatograms were recorded at 254 nm.
All fluorination experiments were run on a commercial TRACERLab
ꢄ
FxFÀN (Ge Healthcare).
ACTHNUTRGNEUNG
No-carrier-added 18F was produced through the 18O(p,n)18F nuclear reac-
tion by irradiation of enriched [18O]-H2O. 18F production was achieved by
bombardment: 80–100 min; 40 mA; target volume 1,8–2,5 mL, 100–
130 GBq are obtained.
18F was trapped on an anion-exchange resin cartridge (Sep-Pak QMA
light, Waters). The cartridge was eluted with a solution of Kryptofix
(K2.2.2., 22 mg) and potassium carbonate (7 mg) in H2O (0.3 mL) and
CH3CN (0.3 mL). Solvents were removed by heating at 1008C for 8 min
and applying a gentle stream of nitrogen. During this time, CH3CN (4ꢃ
0.25 mL) was added and evaporated to give the dry K
plex.
[18F]F/K2.2.2. com-
ACHTUNGTRENNUNG
The peak of the 18F-labeled products was confirmed by comparison with
HPLC retention times of the nonradioactive reference molecule 16 as
well as precursor 11.
Typical procedure for synthesis of [18F]-16: A solution of 11 (1–3 mg)
and glacial acetic acid (3 mL) in anhydrous DMSO (300 mL) was added to
the dry KACHTUNGTRNEUNG
[18F]F/K2.2.2. complex. After heating at the determined temper-
ature (60, 100, or 1658C) for 15 min, the reaction mixture was diluted
with CH3CN (2 mL) and H2O (3 mL). This solution was injected into a
semi-preparative HPLC (isocratic, CH3CN/triethylammonium acetate
(TEAA) 50 mm 35:65, 2.5 mLminÀ1) and the product peak (tR =19.0 min)
was easily separated from the starting material (tR =24.0 min). Then 16
was formulated in an aqueous physiological solution (0.9% NaCl solu-
tion) and the purity of 16 was checked by analytical HPLC (isocratic,
CH3CN/TEAA 50 mm 33:67, 1.0 mLminÀ1, tR =19.65 min), and RCY
(7.6–36%, not decay corrected) were calculated as the ratio of the radio-
activity of pure 16 to the initial radioactivity of 18F (EOB).
Figure 1. 18F fluorination of ODN 11. A) HPLC profile (radiodetection)
of the radiosynthesis of [18F]-16 (1008C, 15 min DMSO, CH3COOH).
[a] Excess of 18FÀ. B) Quality control of [18F]-16 (UV absorption and radi-
odetection).
Acknowledgements
PET tracers (starting at around 100 GBq). These promising
results also open the way for the labeling of related mole-
cules, such as aptamers that are envisaged as an access to
new specific PET tracers. Alternatively, with awareness of
the drawbacks that may arise due to the modification of the
global lipophilicity of the radiotracer,[19] we and others[20]
have engaged studies on the modification of the spacer to
balance, if necessary, the introduction of the hydrophobic si-
lylated aromatic moiety. Thus, the diversification of silylat-
ed/azido building blocks, introduced through a direct func-
tionalization of oligonucleotides[21] with an alkyne arm by
using click chemistry, are currently ongoing to study the
impact of those modifications both on the 18F fluorination
step and on the physico–chemical properties of the corre-
sponding radiotracers.
This work was supported by the CNRS (grant given to D.J.). The authors
are grateful to the “plateforme de synthꢅse d’oligonucleotides modifiꢁs
de l’interface Chimie Biologie de l’ITAV” of Toulouse for providing fa-
cilities for oligonucleotide synthesis.
Keywords: 18F labeling · click chemistry · nucleosides ·
oligonucleotides · silicon
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Received: November 10, 2010
Revised: January 10, 2011
Published online: February 10, 2011
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Chem. Eur. J. 2011, 17, 3096 – 3100