Photoactivated 1,3-dipolar cycloaddition for the rapid preparation of 18F labelled radiotracers

Article abstract of DOI:10.1016/j.tet.2011.05.122

An ¹⁸F-labelled 2,5-diaryl tetrazole reagent has been prepared and reacted with substituted alkene dipolarophiles through a photoactivated 1,3-dipolar cycloaddition reaction. The radiobioconjugation reaction furnished the desired product in 5 m

Full text of DOI:10.1016/j.tet.2011.05.122

Tetrahedron 67 (2011) 5572e5576
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18
Photoactivated 1,3-dipolar cycloaddition for the rapid preparation of F labelled
radiotracers
ˇ
*
ꢀ
ꢀ
David Thonon , Eve Goukens, Geoffroy Kaisin, Jerome Paris, Jessica Flagothier, Andre Luxen
Cyclotron Research Center, Liege University, Sart-Tilman B.30, 4000 Liege, Belgium
a r t i c l e i n f o
a b s t r a c t
An 18F-labelled 2,5-diaryl tetrazole reagent has been prepared and reacted with substituted alkene
dipolarophiles through a photoactivated 1,3-dipolar cycloaddition reaction. The radiobioconjugation
reaction furnished the desired product in 5 min with radiochemical conversions of 85e95% at room
temperature. Remarkably, for the activated dipolarophiles, these results were obtained in highly dilute
Article history:
Received 6 April 2011
Received in revised form 26 May 2011
Accepted 30 May 2011
Available online 2 June 2011
solutions (10e100 mM).
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Photoclick chemistry
Fluorine-18
2,5-Diaryl tetrazole
Radiolabelling
Radiopharmaceutical
1. Introduction
[
18F]arylazides. The photogenerated [18F]arylnitrene reacts with nu-
cleophilic groups present on the biocompound. The poor labelling
efficiency, which was attributed to the short lifetime of the in-
termediate [18F]arylnitrene, has precluded the development of this
photochemical approach.10 Recently, Lin and co-workers reported
a bioorthogonal photoinducible 1,3-dipolar cycloaddition reaction
(‘photoclick’) for rapid and highly selective modification of proteins
without the need for catalysts (Fig. 1).11e13 Specifically, an aryl tet-
razole moiety was extremely rapidly photolysed to generate a nitrile
imine intermediate, which efficiently reacts with an alkene dipolar-
ophile. Moreover, the pyrazoline cycloadduct formed shows strong
fluorescence in the region of 487e538 nm.
Positron emission tomography (PET) is a well-established and
still growing non-invasive imaging modality, ideally suited to
in vivo imaging of molecular processes thanks to its high sensitivity
(nano to picomolar range) and quantitative nature. Fluorine-18 is
by far the most used positron emitting radionuclide in PET due to
its availability and its physical and nuclear properties.1 However,
the poor nucleophilicity of the fluoride anion prevents its direct
incorporation into water soluble biomolecules, e.g., peptides, pro-
teins, oligonucleotides, lipids. Indeed, stability issues and side
reactions preclude the use of harsh conditions usually employed to
label small and stable compounds. Thus, the further development
of molecular imaging is hindered by these difficulties to label more
complex and promising molecules.2
Despite recent progress,3,4 the search for a rapid, selective and
generic method for radiolabelling biomolecules continues. The cop-
per (I)-catalysed 1,2,3-triazole formation involving azides and ter-
minalalkyneshasprovento beavaluable tool forefficient fluorine-18
labelling of various biomolecules.5e7 Nevertheless, the employment
of cytotoxic copper salts hampers its general use. The labelling of
biomolecules with fluorine-18 by photochemical conjugation has
been only tentatively explored in the past.8e10 All efforts have
focused on the preparation of [18F]arylnitrene through irradiation of
* Corresponding author. Tel.: þ32 43662334; fax: þ32 43662946; e-mail address:
Fig. 1. Photoactivated 1,3-dipolar cycloaddition between a 2,5-diaryl tetrazole and
a substituted alkene dipolarophile.
dthonon@ulg.ac.be (D. Thonon).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.05.122

D. Thonon et al. / Tetrahedron 67 (2011) 5572e5576
5573
It has been shown that this reaction can be carried out in various
solvents including water and other protic solvents.14 Thanks to the
high quantum yield of the photolysis (0.5e0.9), a simple hand held
bench top UV lamp was sufficient to initiate the reaction.14 In the
conditions evaluated by Lin, the reaction was highly regioselective
and no competitive reactions with other functional groups like
nitrile, aldehyde or proteinaceous groups were observed. Typically,
the dipolarophile was an electron-deficient alkene such acrylate,
acrylamide or fumarate. Simple alkenes, such as 1-decene afforded
pyrazoline with moderate yields.14 The functionalization of bio-
molecules by such moieties is practicable. For example, site-specific
incorporation of alkene-containing amino acids into proteins has
been reported by several groups15e18 and synthesis of maleimide
peptide has also been described.19
the main unlabelled impurity was alcohol 5, which is the main
degradation product of the 5e7 mg of precursor 6 used.
Scheme 2. Synthesis of tetrazole tosylate 6 and radiolabelling. Reagents and conditions
(a) NaOH, ClCH2CH2OH, n-BuOH/H2O, 54%. (b) 1. PhSO2NHNH2, EtOH. 2. PhN2Cl, pyr,
37%. (c) TosCl, Et3N, 36%. (d) Cs19F, DMSO, 79%. (e) KF[18F]-K-222/K2CO3, DMSO, 69%.
Thus, this photoactivated cycloaddition seems particularly well
adapted for the fast conjugation of radionuclides to biomolecules in
dilute media. In this article, we report the first method to label dipo-
larophile compounds with fluorine-18 through the photoclick reaction.
As this impurity presents a tetrazole moiety, it is imperative to
separate this product from [18F]7 to avoid unwanted dipolarophile
consumption. This separation can be carried out efficiently by semi-
preparative HPLC. The collected HPLC peak was diluted with water,
trapped on a Sep-Pak cartridge and [18F]7 was eluted with 1 ml of
acetonitrile (the decay-corrected radiochemical yield was 75% for
the purification step). The concentration of non-radioactive 7
2. Results and discussion
Initially, we focused on the introduction of fluorine-18 on
a nitrophenyl tetrazole derivative (Scheme 1). This compound was
not ideally suited for this application as the position of the nitro
leaving group is poorly activated. Moreover, Lin and co-workers
showed that the cycloaddition rate constant was about 100-fold
lower for monoaryl tetrazole compared to diaryl tetrazole.12,13
Nevertheless, compound 1 can be prepared easily in a single step,
which is attractive in a proof of concept perspective.20
present in the solution was determined by HPLC (20e90 m
M).y
As fumarates are known to be good dipolarophiles, dieth-
ylfumarate was used to demonstrate the potential of this approach.
First, a study was conducted on the non-radioactive compound 7 to
produce the standard 9 and to check the experimental conditions
(Scheme 3). The reaction was carried out in a PBS buffer (pH 7.4,
50 mM)/CH3CN mixture (1:1) at room temperature in an open
quartz vial. The reaction medium was gently stirred and photo-
activated through two flexible light guides (See Experimental
section). The concentration of tetrazole 7 was fixed at 0.1 mM to
ensure proper HPLC detection.
N
N
N
N
N
N
N
N
Under these conditions, 1 min of photoactivation proved suffi-
cient to completely consume tetrazole 7.
18F
NO2
In the absence of a dipolarophile, tetrazole 7 was totally con-
verted under these conditions to a more polar compound, which
did not react thereafter with a dipolarophile even if added in huge
excess. Moreover, as this compound did not show absorption
around 350e400 nm, we can assume that this product is not the
nitrile imine intermediate but the nitrile imine/H2O adduct de-
scribed by Song.11 In the presence of diethylfumarate, this product
was not detected and only the desired compound 9 was present,
exhibiting a maximum absorption wavelength around 350 nm.
Complete conversion was obtained for diethylfumarate concen-
trations between 0.15 and 10 mM.
Compound 9 was used as a reference compound for the radio-
synthesis of [18F]9. [18F]7 was reacted with diethylfumarate 8 under
the conditions described above for the preparation of 9. With
concentrations of diethylfumarate between 1 and 10 mM and after
5 min of photoactivation and stirring, radio-HPLC analysis showed
a major peak (>95%) at a retention time corresponding to the
standard 9 (Table 1, entry 1e2). Thus, the lifetime of the in-
termediate nitrile imine photogenerated does not seem problem-
atic, contrary to what was observed with [18F]arylnitrenes.10 No
degradation product was detected after 4 h. A minor radioactive
peak was also detected at a retention time corresponding to the
nitrile imine/H2O adduct (Fig. 3).
1
2
Scheme 1. Attempts to label 2-(4-nitrophenyl)-2H-tetrazole 1. Reagents and condi-
tions (18F/K-222/K2CO3 or F-TEAF, 90e180 ꢀC).
18
Unfortunately, all attempts to label this compound (18F/K-222/
K2CO3 or 18F-TEAF) at temperatures between 90 and 180 ꢀC were
unsuccessful, as only 18F fluoride and the decomposition product of
the 2-(4-nitrophenyl)-2H-tetrazole precursor were recovered.
These results encouraged us to switch to the more convenient
tetrazole precursor 6. This compound features an aliphatic tosylate
leaving group which is known to be easily substituted.21 Moreover,
the diaryl tetrazole moiety should favor a fast cycloaddition reaction.
The tosylate 6 was synthesized in three steps starting from 4-
hydroxybenzaldehyde (Scheme 2). The key step was the Kakehi
diaryltetrazole synthesis from aryl aldehyde 4 and an arene di-
azonium salt.22,23 The standard compound 7 was obtained by
reacting tosylate 6 with anhydrous caesium fluoride in DMSO. We
found that 6 was efficiently labelled in DMSO at 95 ꢀC in 5 min with
standard KF[18F]-K-222/potassium carbonate conditions. Identifi-
cation of [18F]7 was based on radio-HPLC analysis showing the
same retention time as the corresponding non-radioactive refer-
ence compound 7 (Fig. 2). Radiochemical conversions obtained
were between 75 and 90%. Increasing the reaction time or tem-
perature did not improve yields. Modifications of the nature of the
solvent or of the reagent’s quantities (K-222, K2CO3 or precursor 6)
were not considered. The mean decay-corrected radiochemical
yield was 69ꢁ5% (n¼4). This value is mostly limited by trapping of
y
Formation of non-radioactive 7 in non carrier-added synthesis is linked to the
presence of stable isotope originating from the radionuclide production, solvents,
chemicals and lines used to transfer radioactivity from cyclotron. Usually, the ratio
19F/18F is around 100e1000/1.
[
18F]Fꢂ on the walls of the glass vial used for the radiolabelling
reaction. HPLC analysis of the crude reaction media showed that

5574
D. Thonon et al. / Tetrahedron 67 (2011) 5572e5576
Fig. 2. Radio-HPLC diagram (top) of [18F]7 (
g-trace) and HPLC diagram of reference compound 7 (254 nm).
This was the only peak detected when no fumarate was added to
the reaction medium. When the diethylfumarate concentration was
decreased below 1 mM, radiochemical conversions became de-
pendent on the concentration of non-radioactive compound 7
present in solution (Table 1).
The quantity of non-radioactive 7 can be decreased by using an
aliquot of the purified [18F]7 solution. With this approach, we have
obtained radiochemical conversions superior to 85% in 5 min with
diethylfumarate concentrations of 10 mM (2 nmol in 200 ml, entry
8). This outcome is of great interest, particularly in the biomarker
discovery and development field where new biomolecules are
available in very low quantities. Conventional labelling methodol-
ogies require larger amount of biocompounds, limiting their bio-
evaluation in vivo. As this ‘aliquot’ approach leads to discarding
a significant amount of the radioactive [18F]7 produced, it is espe-
cially appropriate in the case of animal studies where low levels of
activity are required (typically 5e10 MBq for mouse imaging). En-
couraged by these results, we have prepared a model peptide fea-
turing an electron-deficient alkene. The terminal NH2 group of
peptide 10 was coupled with acrylic acid to furnish an ‘acrylamide-
like dipolarophile’ peptide (Scheme 4).
Scheme 3. Dipolar cycloaddition between fluorotetrazole and diethylfumarate.
Reagents and conditions (rt, PBS/CH3CN (1:1), hn).
Table 1
Effect of reagent concentrations on the radiosynthesis of [18F]9 (5 min, rt)
Entry
Diethylfumarate 8
7
Radiochemical conversion
1
2
3
4
5
6
7
8
9
10 mM
1 mM
30
30
30
44.5
3.8
30
3.8 mM
1.13
30
m
m
m
M
M
M
96
96
85
45
92
89
43
86
5
100
100
100
100
m
m
m
m
M
M
M
M
m
M
m
m
M
M
10
10
10
m
m
m
M
M
M
m
M
m
M
Scheme 4. Dipolar cycloaddition between fluorotetrazole and alkene/peptide
(peptide¼Gly-Gly-Arg-Arg-Pro-Tyr-Ile-Leu-COOH (rt, PBS/CH3CN (1:1), h
n).
This peptide (5.5 mM, 1.1
(36.5
M) under the conditions described to prepare [18F]9 and 9.
m
mol) was conjugated with [18F]7
m
After 5 min of photoactivation, 85% of the activity was converted
into [18F]11. At lower peptide concentrations (0.5e1 mM), radio-
chemical conversions decreased severely (w30e40%), although
this was not the case with diethylfumarate. This means that for
Fig. 3. Radio-HPLC diagram (top) of [18F]9 (
compound 9 (350 nm).
g-trace) and HPLC diagram of reference
relatively available biomolecules (>1
mmol per radiosynthesis)

D. Thonon et al. / Tetrahedron 67 (2011) 5572e5576
5575
functionalization by acrylamide-like dipolarophiles is sufficient,
while in other cases more activated dipolarophiles are required.
curing system incorporates two flexible light guides, which are fixed
at a 5 mm distance from the quartz tube. The lamp power was
400 W. An integral filter removes unwanted infrared portion of the
spectrum to avoid overheating the work area. 2-(4-Nitrophenyl)-
2H-tetrazole was synthesized according to Gaponik.20 Peptide syn-
theses were carried out on an automated solid phase peptide syn-
thesizer PS3 (Protein Technologies) by an Fmoc strategy employing
HBTU as the coupling reagent. Acrylic acid was used to functionalize
3. Conclusions
In conclusion, we have developed a very fast and efficient bio-
conjugation method for fluorine-18 labelling through photo-
activated 1,3-dipolar cycloaddition. A novel [18F]fluorotetrazole
prosthetic group was prepared with a d.c yield of 52%. The effi-
ciency of the method was demonstrated with diethylfumarate and
a model alkene/peptide. The conjugation proceeds with high rates
and yields of 85e95%. As the reaction is easy to set-up (room
temperature, no work-up, compatible with water and oxygen) its
automation seems unproblematic provided that a suitable UV
source has been mounted in the automation system. In addition, as
the reaction is clean and no inorganic catalysis is used, there is no
need for intensive purification (no HPLC purification of the conju-
gated radiocompound) and the quality control remains limited.
This is of prime importance regarding the relatively short time
available for radiotracer production due to the half-life of fluorine-
18 (w110 min). Thus, we have shown that photoactivated 1,3-
dipolar cycloaddition can be successfully used for fast preparation
of 18F labelled compounds even at very low concentrations. Current
work in our laboratory is investigating the use of alternative elec-
trophilic groups to functionalize biocompounds (e.g., maleimide,
vinyl ketone.) and reach the performance obtained with dieth-
ylfumarate. The purification of intermediate [18F]7 on Sep-Pak
cartridges instead of time consuming HPLC separation is also un-
der investigation. In the perspective of future in vivo applications,
the influence of the polysubstituted pyrazoline prosthetic group on
the radiopharmaceutical pharmacokinetics should be taken into
account. Hydrophilic linkers or substituents of appropriate polarity
grafted on to the prosthetic group could be used to tune the overall
hydrophilicity. We strongly believe that such an uncatalyzed re-
action between 1,3-dipoles and dipolarophiles will become a gen-
eral class of reaction for radiopharmaceutical preparation.
the N-terminal moiety. Enantiomerically pure
obtained from IRIS Biotech.
L-amino acids were
4.2. Syntheses
4.2.1. 4-(2-Hydroxyethoxy)benzaldehyde(4). 4-Hydroxybenzal-
dehyde 3 (12.2 g, 0.1 mmol) and 4.4 g of NaOH (0.11 mmol) were
dissolved in a mixture of 200 ml of n-BuOH and 30 ml of water. This
solution was brought to reflux and a solution of 24.1 ml of 2-
chloroethanol (0.3 mmol) in 50 ml of n-BuOH was added over
a period of 90 min. The reaction mixture was refluxed overnight.
The solvent was evaporated in vacuo, and the residue was purified
by silica gel dry flash chromatography (Petroleum ether/EtOAc
manual gradient from 0% to 100% EtOAc) to afford compound 4
(9.0g, 54%) as a colourless oil, which solidified upon standing
overnight. 1H NMR (400 MHz, CDCl3)
d 9.83 (s, 1H), 7.78 (d, 2H,
J¼8 Hz), 6.96 (d, 2H, J¼8 Hz), 4.11 (t, 2H, J¼4 Hz), 3.94 (t, 2H,
J¼4 Hz), 1.87 (br s, 1H). 13C NMR (100 MHz, CDCl3)
d 190.8, 163.6,
132.0, 130.3, 114.8, 69.5, 61.2.
4.2.2. 2-(4-(2-Phenyl-2H-tetrazol-5-yl)phenoxy)ethanol (5). 4-(2-
Hydroxyethoxy)benzaldehyde 4 (715 mg, 4.31 mmol) and 813 mg
of benzenesulfonohydrazide (4.73 mmol) were dissolved in 30 ml
of ethanol. The reaction mixture was stirred for 1 h at room tem-
perature. The solvent was removed in vacuo to furnish a colourless
solid. The solid was redissolved in 50 ml of pyridine (solution A). In
parallel, a solution of NaNO2 (886 mg, 12.84 mmol) in 4 ml of water
was added dropwise to a cooled mixture (dry-ice ethylene glycol
cooling bath, between ꢂ10 ꢀC and ꢂ20 ꢀC) of aniline (906 mg,
9.74 mmol) dissolved in 8 ml water/ethanol (1:1) and 4.36 ml
concentrated HCl to give a solution of phenyldiazonium chloride.
This solution was added dropwise over 20 min to solution A, which
was maintained between ꢂ10 and ꢂ20 ꢀC (dry-ice ethylene glycol
cooling bath). After this, the red solution was stirred for 3 h at room
temperature. The solution was extracted with ethyl acetate/water
and the organic layers were collected and concentrated under re-
duced pressure. The residue was purified by silica gel column
chromatography (PE/EtOAc 1:5) to furnish 450 mg of a dark pink
4. Experimental section
4.1. General
All solvents and chemicals were of analytical grade and used
without further purification. The [18O]-enriched water was pur-
chased from Cambridge Isotope Laboratories. Mass spectra were
recorded with a Finnigan TSQ7000 mass spectrometer (Thermo-
ElectronCorp.) operating in full-scan MS mode with an ESIþ source
and with a Bruker Daltonics micrOTOF spectrometer (TOF-ES-MS).
1H and 13C NMR spectra were recorded on a Bruker DRX 400 (1H at
400 MHz and 13C at 100 MHz). 1H and 13C spectra were referenced to
TMS using the 13C or residual proton signals of the deuterated sol-
vents as internal standards. HPLC analyses were run on a Waters
Alliance System (996 PDA detector and NaI(Tl) scintillation detector
from Eberline) controlled by the Empower program. Analyses were
performed on an XBridge C18 column from Waters (250ꢃ4.6 mm,
solid (37%). 1H NMR (400 MHz, CDCl3)
d
8.22 (d, 2H, J¼8 Hz), 8.19 (t,
2H, J¼8.5 Hz), 7.59 (t, 2H, J¼7.6 Hz), 7.51 (t, 1H, J¼7.9 Hz), 7.07 (d,
2H, J¼8.9 Hz), 4.19 (t, 2H, J¼4 Hz), 4.04 (t, 2H, J¼4 Hz).1.83 (br s,1H).
13C NMR (100 MHz, CDCl3)
d 165.7, 161.2, 137.7, 130.4, 130.3, 129.4,
120.9, 120.5, 115.7, 70.1, 62.1. HRMS (ESI) m/z [MþH] calcd for
C15H15N4O2, 283.1189; found 283.1190.
4.2.3. 2-(4-(2-Phenyl-2H-tetrazol-5-yl)phenoxy)ethyl 4-methylb-
enzenesulfonate (6). Tetrazole alcohol 5 (50 mg, 0.177 mmol) was
dissolved in 5 ml of CH2Cl2. p-Toluenesulfonyl chloride (67 mg,
0.354 mmol) and 50 mg (0.496 mmol) of triethylamine were added
to this solution and the reaction mixture was stirred under inert
atmosphere (N2) overnight at room temperature. The solution was
extracted with ethyl acetate/water and the organic layers were
collected and concentrated in vacuo. The residue was purified by
silica gel column chromatography (PE/EtOAc 1:9 to eliminate im-
purities, then PE/EtOAc 1:3 to elute the targeted compound).
Compound 6 was obtained as a pink solid (28 mg, 36%). 1H NMR
3.5
given in parentheses) at 1 mL/min. Preparative HPLC was performed
). Solid
m) with MeOH and H2O containing 0.1% TFA mixture (proportions
on an Xbridge Prep C18 column from Waters (250ꢃ10 mm, 5
m
phase extraction cartridges were purchased from Waters. TLC
analyses were performed on silica gel 60 F254 plates, with EtOAc as
mobile phase. A Bioscan TLC scanner model AR2000 was used for
analysis of the 18F labelled compounds. Radiochemical yields are
given decay-corrected and are based on the activity eluted from the
QMA cartridge. All photoinduced reactions were carried out in
quartz tubes (solvent volume 200 mle2000 ml) with magnetic stir-
ring under ambient atmosphere. The source of photons was a spot-
lite curing system from UVP (Upland, CA). This portable ultra-violet
(400 MHz, CDCl3)
(d, 2H, J¼8.3 Hz), 7.55 (t, 3H, J¼8.4 Hz), 7.37 (d, 2H, J¼8.4 Hz), 6.93
d
8.20 (d, 2H, J¼8.4 Hz), 8.16 (d, 2H, J¼8 Hz), 7.85

5576
D. Thonon et al. / Tetrahedron 67 (2011) 5572e5576
(d, 2H, J¼8 Hz), 4.43 (t, 2H, J¼4 Hz), 4.24 (t, 2H, J¼4 Hz), 2.47 (s, 3H).
13C NMR (100 MHz, CDCl3)
164.9, 159.8, 145.0, 136.9, 132.9, 129.9,
129.7, 129.6, 128.6, 128.0, 120.4, 119.8, 114.9, 68.0, 65.5, 21.7. HRMS
(ESI) m/z [MþH] calcd for C22H21N4O4S, 437.1278; found 437.1277.
medium was then diluted with acetonitrile (2 ml) and water
(1.3 mL). Labelling efficiency was checked by radio-TLC (silica gel,
EtOAc; Rf values: [18F] fluoride¼0; [18F]7¼0.7). HPLC Analysis:
tR¼19.2 min (MeOH/H2O 70/30 0.1% TFA). Radiochemical yield
(decay-corrected)¼69ꢁ5% (n¼4). [18F]7 was purified on an XBridge
Prep column (5 ml/min, 80% MeOH, 20% H2O, 0.1% TFA,
tR¼10.7 min). The collected peak was diluted with 20 ml of water
and trapped on a Sep-Pak Vac cartridge (200 mg, 3cc, tC18, Waters).
The cartridge was rinsed with 5 ml of water and [18F]7 was eluted
with 1 ml of acetonitrile. The purified sample was analyzed by
analytical HPLC. Radiochemical purity of [18F]7 was more than 98%.
Decay-corrected radiochemical yield for purification (HPLCþcar-
d
4.2.4. 5-(4-(2-Fluoroethoxy)phenyl)-2-phenyl-2H-tetrazole
(7). Tosylate 6 (93 mg, 0.213 mmol) was dissolved in 2 ml of an-
hydrous DMSO. Anhydrous caesium fluoride (97.2 mg, 0.639 mmol)
was added and the mixture stirred and heated at 80 ꢀC for 18 h in
a sealed vial. The solution was extracted with ethyl acetate/water
and the organic layers were collected and concentrated in vacuo.
The residue was purified by silica gel column chromatography (PE/
EtOAc 1:9 to eliminate impurities, than PE/EtOAc 1:5 to elute the
searched compound). Compound 7 was obtained as a colourless
tridge) was 75%. Specific activity: 7e25 GBq/mmol at EOB.
PBS buffer (50 mM, pH 7.4) was added to a [18F]7 acetonitrile
solution to obtain a 1:1 solvent mixture. Dipolarophile (dieth-
ylfumarate or peptide 10) was added to obtain concentrations de-
solid (48 mg, 79%). 1H NMR (400 MHz, CDCl3)
d 8.20 (d, 2H,
J¼7.6 Hz), 8.18 (t, 2H, J¼7.8 Hz), 7.59 (t, 2H, J¼8 Hz), 7.51 (t, 1H,
J¼7.9 Hz), 7.06 (d, 2H, J¼8 Hz), 4.81 (dt, 2H, 3JHH¼4 Hz, 2JHF¼53 Hz),
scribed in the results and discussion section for 10 mM: 2
100 mol/100 l of solution of
l of solution of [18F]7, for 1 mM: 0.2
18F]7, for 100 l of solution of [18F]7, for 10
M: 20 nmol/100 M:
2 nmol/100
l of solution of [18F]7. The mixture was irradiated with
mmol/
3
3
4.30 (dt, 2H, JHH¼4 Hz, JHF¼27 Hz). 13C NMR (100 MHz, CDCl3)
m
m
m
d
165.0, 160.3, 137.0, 129.7, 129.5, 128.7, 120.4, 119.8, 115.0, 81.8 (d,
[
m
m
m
JCeF¼167 Hz), 67.1 (JCeF 20 Hz) HRMS (ESI) m/z [MþH] calcd for
m
C15H14N4O2, 285.1146; found 285.1148.
a portable ultra-violet curing system for 5 min and readily analyzed
by radio-HPLC. The reaction medium was diluted with 20 ml of
water and fixed on a Sep-Pak Vac cartridge to remove the PBS buffer
and acetonitrile (200 mg, 3cc, tC18, Waters). The excess dipolar-
ophile was not removed during this operation. The cartridge was
washed with 5 ml of water and dried with a nitrogen flow. [18F]9
and [18F]11 were eluted with 1 ml of ethanol. [18F]9 and [18F]11
were isolated with radiochemical purity superior to 95%.
4.2.5. Diethyl
azole-4,5-dicarboxylate,
0.051 mmol) was dissolved in 800
3-(4-(2-fluoroethoxy)phenyl)-4,5-dihydro-1H-pyr-
(9). Fluorotetrazole (14.6 mg,
l of EtOAc. 14.8 mg of dieth-
7
m
ylfumarate (0.086 mmol) were added to this solution and the
mixture was irradiated with a portable ultra-violet curing system
for 4 h. The solvent was removed under reduced pressure to give
a crude product. This solid was purified by semi-preparative HPLC
(MeOH/H2O 0.1% TFA, 70/30, 4 ml/min) to furnish 7.2 mg of a col-
Acknowledgements
ourless solid (0.017 mmol, 33%). 1H NMR (400 MHz, CDCl3)
d 7.78 (d,
2H, J¼8.9 Hz), 7.29 (t, 2H, J¼7.2 Hz), 7.15 (d, 2H, J¼7.8 Hz), 6.93 (t,
ꢀ
This work was supported by the Region Wallone (Neofor and
3
3H, J¼7.6 Hz), 5.14 (d, 1H, J¼5 Hz), 4.79 (dt, 2H, JHH¼4 Hz,
Keymarker projects), the IISN of Belgium (convention 4.4511.08)
and the FRS-FNRS Belgium. G.K. is research fellow at the FRS-FNRS
Belgium. We thank Geoffrey Warnock for useful discussions.
2JHF¼48 Hz), 4.54 (d,1H, J¼5 Hz), 4.29 (m,1H), 4.20 (m, 5H),1.20 (m,
6H). 13C NMR (100 MHz, CDCl3)
d 169.9, 168.9, 159.1, 144.1, 143.5,
130.3, 129.6, 124.6, 120.0, 114.5, 113.3, 81.8 (d, JCeF¼170 Hz), 67.1 (d,
JCeF¼21 Hz), 66.1, 62.2, 62.1, 56.1, 13.8, 13.7 HRMS (ESI) m/z [MþH]
calcd for C23H26N2O5, 429.1820; found 429.1820.
References and notes
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€
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900
ml of H2O/CH3CN/DMSO 1:1:1. 5.5 mg of peptide 10
(0.006 mmol) were added to this solution and the mixture was
irradiated with a portable ultra-violet curing system for 2 h. The
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viously conditioned under carbonate form. Then, 700 ml of a 50:50
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6
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