Radiosynthesis and 'click' conjugation of ethynyl-4-[18F]fluorobenzene - An improved [18F]synthon for indirect radiolabeling

Article abstract of DOI:10.1002/jlcr.3354

Reproducible methods for [¹⁸F]radiolabeling of biological vectors are essential for the development of new [¹⁸F]radiopharmaceuticals. Molecules such as carbohydrates, peptides and proteins are challenging substrates that often require multi-step indirect radiolabeling methods. With the goal of developing more robust, time saving, and less expensive procedures for indirect [¹⁸F]radiolabeling of such molecules, our group has synthesized ethynyl-4-[¹⁸F]fluorobenzene ([¹⁸F]2, [¹⁸F]EYFB) in a single step (14 ± 2% non-decay corrected radiochemical yield (ndc RCY)) from a readily synthesized, shelf stable, inexpensive precursor. The alkyne-functionalized synthon [¹⁸F]2 was then conjugated to two azido-functionalized vector molecules via CuAAC reactions. The first 'proof of principle' conjugation of [¹⁸F]2 to 1-azido-1-deoxy-β-d-glucopyranoside (3) gave the desired radiolabeled product [¹⁸F]4 in excellent radiochemical yield (76 ± 4% ndc RCY (11% overall)). As a second example, the conjugation of [¹⁸F]2 to matrix-metalloproteinase inhibitor (5), which has potential in tumor imaging, gave the radiolabeled product [¹⁸F]6 in very good radiochemical yield (56 ± 12% ndc RCY (8% overall)). Total preparation time for [¹⁸F]4 and [¹⁸F]6 including [¹⁸F]F^- drying, two-step reaction (nucleophilic substitution and CuAAC conjugation), two HPLC purifications, and two solid phase extractions did not exceed 70 min. The radiochemical purity of synthon [¹⁸F]2 and the conjugated products, [¹⁸F]4 and [¹⁸F]6, were all greater than 98%. The specific activities of [¹⁸F]2 and [¹⁸F]6 were low, 5.97 and 0.17 MBq nmol^-1, respectively. A new [¹⁸F]radiosynthon, [¹⁸F]ethynyl-4-fluorobenzene ([¹⁸F]EYFB), has been prepared for the indirect radiolabeling of sensitive molecules that are not amenable to standard radiolabeling conditions. [¹⁸F]EYFB was alkyne functionalized for use in 'click' chemistry, which was prepared in adequate yield and high purity from a single step using a readily synthesized inexpensive precursor. The utility of [¹⁸F]EYFB was investigated by 'click' conjugation to two azido-functionalized molecules (a glucose and matrix-metalloproteinase derivative). The radiochemical purity of [¹⁸F]EYFB and the conjugated products were all ≥99%.

Full text of DOI:10.1002/jlcr.3354

Research article
Received 16 July 2015,
Revised 23 September 2015,
Accepted 29 September 2015
Published online 3 November 2015 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3354
Radiosynthesis and ‘click’ conjugation of
18
ethynyl-4-[ F]fluorobenzene — an improved
‡
18
[ F]synthon for indirect radiolabeling
a
a
b†
a
*
Maxine P. Roberts, Tien Q. Pham, John Doan, Cathy D. Jiang,
Trevor W. Hambley,b Ivan Greguric,a and Benjamin H. Frasera
Reproducible methods for [18F]radiolabeling of biological vectors are essential for the development of new [18F]
radiopharmaceuticals. Molecules such as carbohydrates, peptides and proteins are challenging substrates that often require
multi-step indirect radiolabeling methods. With the goal of developing more robust, time saving, and less expensive
procedures for indirect [18F]radiolabeling of such molecules, our group has synthesized ethynyl-4-[18F]fluorobenzene
([18F]2, [18F]EYFB) in a single step (14 2% non-decay corrected radiochemical yield (ndc RCY)) from a readily synthesized,
shelf stable, inexpensive precursor. The alkyne-functionalized synthon [18F]2 was then conjugated to two azido-functionalized
vector molecules via CuAAC reactions. The first ‘proof of principle’ conjugation of [18F]2 to 1-azido-1-deoxy-β-D-glucopyranoside
(3) gave the desired radiolabeled product [18F]4 in excellent radiochemical yield (76 4% ndc RCY (11% overall)). As a second
example, the conjugation of [18F]2 to matrix-metalloproteinase inhibitor (5), which has potential in tumor imaging, gave the
radiolabeled product [18F]6 in very good radiochemical yield (56 12% ndc RCY (8% overall)). Total preparation time for
[
18F]4 and [18F]6 including [18F]FÀ drying, two-step reaction (nucleophilic substitution and CuAAC conjugation), two HPLC
purifications, and two solid phase extractions did not exceed 70min. The radiochemical purity of synthon [18F]2 and the
conjugated products, [18F]4 and [18F]6, were all greater than 98%. The specific activities of [18F]2 and [18F]6 were low,
5.97 and 0.17MBq nmolÀ1, respectively.
Keywords: [18F]Fluorine; click chemistry; radiochemistry; [18F]synthons; matrix metalloproteinase inhibitors (MMPIs); positron
emission tomography (PET)
Introduction
aLifeSciences Division, Australian Nuclear Science and Technology Organisation
(ANSTO), Locked Bag 2001, Kirrawee DC, NSW 2232, Australia
Positron emission tomography (PET) provides detailed three-
dimensional information on functional processes in vivo and
has important diagnostic applications in oncology, neurology,
and cardiology. Although numerous radioisotopes (copper-64,
gallium-68, zirconium-89, and iodine-124) are available for PET,
bSchool of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia
*Correspondence to: Tien Q. Pham, LifeSciences Division, Australian Nuclear
Science and Technology Organisation (ANSTO), Locked Bag 2001, Kirrawee DC,
NSW 2232, Australia.
fluorine-18 remains very popular because of
a unique
E-mail: tien.pham@ansto.gov.au
combination of physical and chemical properties. These include
low positron emission energy (0.202 MeV), excellent decay
profile (97% β + emission), advantageous half-life (109 min),
availability in nucleophilic (FÀ) or electrophilic (F+) form, and
similar steric and electronic properties to the hydroxyl group.
The use of nucleophilic [18F]fluoride is considerably more
widespread than electrophilic [18F]fluorine because of higher
specific activity products, superior radiolabeling selectivity, and
ready availability. However, direct radiolabeling with [18F]fluoride
often employs harsh conditions including higher temperatures
(>100 °C) that are often incompatible with large biomolecules.1
Biomolecules are typically radiolabeled via indirect methods
where [18F]fluoride is first incorporated into a [18F]radiolabeled
‘synthon’ containing a functional group that reacts with the
biomolecule under relatively mild conditions.2 A number of [18F]
synthons are available for indirect radiolabeling such as, but not
limited to, N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) and
p-nitrophenyl-2-[18F]fluoropropionate ([18F]NFP) (Figure 1). The
†John Doan current address: Royal Prince Alfred Hospital, PET & Nuclear Medicine.
‡Additional supporting information may be found in the online version of this
article at the publisher’s web-site.
Abbreviations: Acetonitrile, (CH3CN); 4-azidophenacyl-[18F]fluoride,
([18F]APF); decay corrected, (dc); dichloromethane, (CH2Cl2); N,
N-diisopropylethylamine, (DIPEA); dimethylformamide, (DMF);
dimethylsulfoxide, (DMSO); equivalent, (eq.); ethyl acetate, (EtOAc);
[18F]fluoroethylazide, ([18F]FEA); high performance liquid
chromatography, (HPLC); high-resolution mass spectrometry, (HRMS);
hour, (h); low-resolution mass spectrometry, (LRMS); metalloproteinase
inhibitors, (MMPIs); p-nitrophenyl-2-[18F]fluoropropionate, ([18F]NFP);
non-decay corrected, (ndc); nuclear magnetic resonance, (NMR);
positron emission tomography, (PET); radiochemical yield, (RCY);
retention time, (tR); room temperature, (rt); solid phase extraction,
(SPE); N-succinimidyl-4-[18F]fluorobenzoate, ([18F]SFB); trifluoroacetic
acid, (TFA).
J. Label Compd. Radiopharm 2015, 58 473–478
Copyright © 2015 John Wiley & Sons, Ltd.

M. P. Roberts et al.
following features to enable practical, straightforward use of this
new [18F]synthon. These include:
(1) An aromatic ring for improved UV detection and increased
stability in vivo,
(2) An ammonium cation leaving group for straightforward
purification, preferably by SPE for time reduction,
(3) An alkyne functionality that acts both as weakly activating
group (electron withdrawing group) and functional group
for subsequent ‘click’ conjugation reactions,
(4) Low volatility to avoid loss during heating, hence maximize
radiochemical yield, and
(5) An inexpensive radiolabeling precursor that is prepared in a
single step and isolated by filtration from commercially
available 4-ethynyl-N,N-dimethylaniline.
Figure 1. Examples of
[
18F]synthons for radiolabeling, conjugated via:
(a) acylation, (b) aliphatic ‘click’ and (c) aromatic/heteroaromatic ‘click’ reaction.
The results herein describe the successful radiosynthesis
and conjugation of [18F]2 to an azido-functionalized carbohydrate
3 (1-azido-1-deoxy-β-D-glucopyranoside) and a matrix metallo-
proteinase inhibitor 5 (3-(hydroxylcarbamoyl)-3(S)-hydroxy-2(R)-
(cyclopentylmethyl)propionyl-L-tert-leucine-N-2-azidoethylamide) that
has potential for development as a tumor PET imaging agent. A
comparison of the efficiency of radiolabeling the matrix
metalloproteinase inhibitor 5 to that achieved by Doan et al.14
is also described.
radiosynthesis of the aforementioned [18F]synthons, and many
others, often require multiple synthesis and purification steps3
followed by conjugation to the molecule of interest through
acylation/alkylation reactions.4 However, in more recent times,
the further development of [18F]synthons has produced more
efficient radiosyntheses. For example, the radiosynthesis of [18F]
NFP has recently been reported in 26% yield from one-step in
45 min, followed by conjugation to RGD peptides5 as well as the
radiosynthesis of [18F]F-Py-TFP (6-[18F]fluoronicotinic acid 2,3,5,6-
tetrafluorophenyl ester), that was prepared in 60–70% yield from
a single 10 min reaction, before being easily purified by Sep-Pak
and also conjugated to a RGD peptide.6
Experimental procedures
General
All reagents were purchased from Sigma-Aldrich and used without
further purification. Aqueous [18F]HF was produced on an IBA Cyclone®
A popular alternative strategy for indirect [18F]radiolabeling is
the CuAAC or ‘click’ reaction, and the growing applications of ‘click
chemistry’ in PET studies has been recently reviewed.4,7 The CuAAC
reaction can be performed under aqueous conditions, is highly
chemoselective, and provides conjugates in high radiochemical
yield and purity. The triazole nitrogen atoms formed during the
CuAAC reaction are also weak hydrogen bond acceptors, which
increases hydrophilicity of the products.8 To date, the alkyl-based
synthons, such as, [18F]fluoroethylazide ([18F]FEA), [18F]4-
fluorobutyne and [18F]5-fluoropentyne (Figure 1)9 have been
reported; however, their high volatility, lack of UV absorption,
challenging purification, and susceptibility to defluorination limit
their use as [18F]synthons. More recently, some research groups
are shifting focus from alkyl-based synthons toward aromatic
[18F]fluorine-based synthons that are less susceptible to in vivo
radiodefluorination.10 Thonon et al.11 reported the radiosynthesis
of the azido-aromatic synthon 1-(azidomethyl)-4-[18F]-
fluorobenzene, via a four-step 75-min synthesis (34% decay
corrected (dc) radiochemical yield (RCY)), which was then
successfully conjugated to an alkyne-functionalized amino acid
and an alkyne-functionalized neuropeptide. Wester et al.12
reported the azido-aromatic synthon 4-azidophenacyl-[18F]
fluoride ([18F]APF) in up to 70% yield from a single 15-min reaction
for subsequent protein conjugation. Furthermore, Daumar et al.13
reported the radiosynthesis of the novel aromatic synthon, 6-[18F]
fluoro-2-ethynylpyridine, obtaining a 28% dc RCY in a single-step
10-min synthesis, followed by click conjugation to an azido-
functionalized peptide (Figure 1).
18 twin cyclotron (ANSTO, Camperdown, Australia) using the 18O(p, n)18
F
nuclear reaction. C18 Sep-Pak® Plus & Lite SPE cartridges were
purchased from Waters (Rydalmere, NSW, Australia), activated with
ethanol (2 mL), then washed with H2O (20 mL) prior to use; C18 Strata®
X SPE cartridges were purchased from Phenomenex (Lane Cove, NSW,
Australia), activated with ethanol (2 mL), and washed with H2O (20 mL);
Affinimp® SPE cartridges were purchased from Polyintell Intelligent
Polymers(Chaussée du Vexin, Val-de-Reuil, France), activated with CH3CN
(4 mL). Nuclear magnetic resonance (NMR) spectra were recorded using a
Bruker Avance DPX-400 spectrometer (LabX, Midland, ON, Canada)
operating at 400.13 MHz for 1H NMR and at 100.61 MHz for 13C NMR.
Low-resolution mass spectrometry was performed on a Micromass ZQ
quadrupole mass spectrometer (LabX), and high-resolution mass
spectrometry was performed at the University of Wollongong, Australia
using a Bruker Daltonics BioApex-II 7 T FTICR spectrometer (LabX) equipped
with an off-axis analytical electron spray ionization source. HPLC QC (quality
control) percentage purity was calculated by integrating the area under the
peak using EMPOWER software (Waters); details are available in Supporting
Information. The radiosynthesis of [18F]2, [18F]4, and [18F]6 were confirmed
by co-injection with non-radioactive standards (2, 4, and 6, respectively)
using HPLC analysis. Radioactivity was measured with a Capintec R15C dose
calibrator (Capintec, Ramsey, NJ, USA).
Chemistry
4-Ethynyl-N,N,N-trimethylbenzenaminium trifluoromethanesulfonate (1)
Methyl trifluoromethanesulfonate (0.95 mL, 8.61 mmol, 1.25 eq.) was
added to 4-ethynyl-N,N-dimethylaniline (1.00 g, 6.89 mmol, 1.00 eq.) in
CH2Cl2 (3 mL), and the solution stirred at rt for 2 h. The precipitate formed
was collected by filtration and washed with CH2Cl2 (3 × 1 mL) to obtain
the product as a white powder (1.61 g, 77%). 1H NMR (400.13 MHz,
(CD3)2CO): δ (ppm) 8.13 (d, J = 9.20 Hz, 2H), 7.74 (d, J = 9.20 Hz, 2H), 3.92
(s, 1H), 3.87 (s, 9H); 13C NMR (100.61 MHz, (CD3)2CO): δ (ppm) 148.10,
134.43, 125.35, 121.82, 82.05, 57.65; LRMS (ES+) m/z 160.08 [M]+; LRMS
Given the aforementioned challenges with alkyl-[18F]synthons
and promising aromatic [18F]synthons, our group has developed
radiosynthon [18F]2 and performed two proof of concept CuAAC
experiments. [18F]2 and its precursor were designed with the
www.jlcr.org
Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 473–478

M. P. Roberts et al.
(ESÀ) m/z 148.88 [CF3O3S]À; HRMS (TOF MS AP+) m/z [M]+ C11H14
calculated 160.1126; found 160.1118; HPLC QC purity 96.25%.
N
the reaction vessel in a slurry of iced H2O (~60 s) and venting the mixture,
mobile phase (1 mL, 50/50/0.1, CH3CN/H2O/TFA, v/v) was added and the
vial shaken to aid washing down the walls of the reaction vial. The mixture
was purified by HPLC (Luna C18 column 250 × 10 mm, 10 μm; isocratic
CH3CN/H2O/TFA 50/50/0.1, v/v; 6.0 mL/min, tR 13 min, λ 254 nm). HPLC
collected [18F]2 was diluted with H2O (12 mL), transferred to a C18 Strata
X SPE cartridge (Phenomenex), washed with additional H2O (10 mL) and
remained cartridge trapped for the subsequent elution into the click
conjugation reaction. [18F]2 was obtained in 14 2% collected ndc RCY,
n = 3 (RCY was calculated based on [18F]radioactivity eluted from the
cartridge into the subsequent reaction vessel).
(2R,3R,4R,5R)-2-(4-(4-Fluorophenyl)-1H-1,2,3-triazol-1-yl)-6-(hydroxymethyl)
tetrahydro-2H-pyran-3,4,5-triol (4)
1-Ethynyl-4-fluorobenzene (71 mg, 0.59 mmol, 1.2 eq.) was dissolved in
n-butanol (6 mL) and degassed using nitrogen gas. In a separate reaction
vessel
3 (100 mg, 0.49 mmol, 1 eq.), sodium ascorbate (190 mg,
0.98 mmol, 2 eq.) and copper (II) sulfate (120 mg, 0.49 mmol, 1 eq.) were
dissolved in H2O (3 mL). The vessel was degassed using nitrogen gas
and then the two reaction vessels were combined and stirred at room
temperature for 16 h under a nitrogen atmosphere. H2O (15 mL) was
added to the reaction mixture and the precipitate formed was removed
by filtration. The filtrate was purified directly using automated flash
chromatography (Grace Reveleris 12 g C18 column; flow rate 24 mL/min;
solvent system 1/99% CH3CN/H2O, increased to 80/20%). The colorless
fractions containing the product (eluted at 45/55% CH3CN/H2O) were
evaporated under reduced pressure then lyophilized to afford a white
solid (90 mg, 56%). 1H NMR (400.13 MHz, (CD3OD): δ (ppm) 8.54 (s, 1H),
7.86 (m, 2H), 7.18 (m, 2H), 5.66 (d, 1H, J = 9.20 Hz), 3.93 (m, 2H), 3.74 (m,
1H), 3.59 (m, 3H); 13C NMR (100.61 MHz, (CD3OD): δ (ppm) 162.81 (d, JF-
C = 246.37 Hz), 146.58, 127.31 (d, JF-C = 8.23 Hz), 126.64 (d, JF-C = 3.38 Hz),
119.91, 115.42 (d, JF-C = 22.01 Hz), 88.35, 79.77, 77.07, 72.69, 69.51, 60.99;
LRMS (ES+, 40 V) m/z [M + Na]+ C14H16FN3NaO5: calculated 348.10; found
347.98; HRMS (TOF MS AP+) m/z [M + Na]+ C14H17FN3NaO5: calculated
348.0972, found 348.0989; HPLC QC purity 99.25%.
(2R,3R,4R,5R)-2-(4-(4-[18F]Fluorophenyl)-1H-1,2,3-triazol-1-yl)-6-
(hydroxymethyl) tetrahydro-2H-pyran-3,4,5-triol ([18F]4)
Compound 3 (1.1 mg, 5.36 μmol), sodium ascorbate (23.0 mg, 116 μmol),
copper iodide (4 mg, 19.3 μmol), 2,6-lutidine (14 μL, 120.9 μmol) and H2O
(0.4 mL) were added to a reaction vial warmed at 80 °C for 1 min, or until
the solution turned pale yellow. [18F]2 was eluted from the C18 Strata X
SPE cartridge with CH3CN (0.6 mL) into the reaction vial and stirred at
80 °C for 10 min. H2O (1.5 mL) was added to the mixture, and it was
filtered through a syringe filter before HPLC purification (Waters Atlantis
Prep T3 C18 column, 250 × 10 mm, 5 μm, isocratic CH3CN/H2O/TFA,
50/50/0.1, v/v, 2.0 mL/min, tR 15.5 min, λ 254 nm), obtaining 76 4%
collected ndc RCY, n = 3 (11% overall two step).
(2S,3R)-2-(Cyclopentylmethyl)-N1-((R)-1-(2-(4-(4-[18F]fluorophenyl)-
1H-1,2,3-triazol-1-yl)ethylamino)-3,3-dimethyl-1-oxobutan-2-yl)-
N4,3-dihydroxysuccinamide ([18F]6)
(2S,3R)-2-(Cyclopentylmethyl)-N1-((R)-1-(2-(4-(4-fluorophenyl)-1H-
1,2,3-triazol-1-yl)ethylamino)-3,3-dimethyl-1-oxobutan-2-yl)-N4,3-
dihydroxysuccinamide (6)
Compound 5 (2.2 mg, 5.31 μmol), sodium ascorbate (23 mg, 116.10 μmol),
copper iodide (3.8 mg, 19.25 μmol), 2,6-lutidine (14 μL, 120.88 μmol) and
H2O (0.4 mL) were added to a reaction vial and warmed to 45 °C for
1 min, or until the solution turned pale yellow. [18F]2 was eluted from the
C18 Strata X SPE cartridge with CH3CN (0.6 mL) into the reaction vial and
stirred at 45 °C for 10 min. H2O (1.5 mL) was added to the mixture, that
was then syringe filtered before HPLC purification (Waters XTerra Prep
MS C18 column, 350 × 7.8 mm, 10 μm, isocratic CH3CN/H2O/TFA 35/65/
0.1, v/v, 3.0 mL/min, tR 14.5 min, λ 233 nm), obtaining 56 12% collected
ndc RCY, n = 3 (8% overall two step).
1-Ethynyl-4-fluorobenzene (35 mg, 0.28 mmol, 1.2 eq.) and 5 (100 mg,
0.23 mmol, 1 eq.) was dissolved in n-butanol (6 mL) and degassed using
nitrogen gas. In
a separate reaction, sodium ascorbate (94 mg,
0.46 mmol, 2 eq.) and copper (II) sulfate (60 mg, 0.23 mmol, 1 eq.) were
dissolved in H2O (3 mL). This vessel was degassed and then the two
reaction vessels were combined. The reaction was stirred at room
temperature for 16 h under an atmosphere of nitrogen. H2O (15 mL)
was added and the resultant yellow precipitate was removed by filtration
and the filtrate evaporated under reduced pressure. The yellow film was
purified by preparative HPLC (isocratic 35/55/10, CH3CN/H2O/100 mM
NH4HCO3, pH 8, 5 mL/min, T3 Atlantis 250 × 10 mm, tR 19.12 min, λ
254 nm) then lyophilized to afford the product as a white solid (48 mg,
38%). 1H NMR (400.13 MHz, (CD3)2SO): δ (ppm) 10.56 (s, 1H), 8.86, (s,
1H), 8.48 (s, 1H), 8.24 (bs, 1H), 7.86 (m, 2H), 7.49 (d, 1H, J = 7.20 Hz), 7.27
(m, 2H), 5.33 (d, 1H, J = 6.40 Hz), 4.44 (m, 2H), 4.14 (d, 1H, J = 7.20 Hz),
3.73 (t, 1H, J = 6.40 Hz), 3.53 (m, 2H), 2.65 (m, 1H), 1.71 (m, 1H), 1.59-
0.85 (m, 10H), 0.80 (s, 9H); 13C NMR (100.61 MHz, (CD3)2SO): δ (ppm)
173.12, 171.23, 169.32, 162.18 (d, JF-C = 195.38 Hz), 145.91, 127.93,
127.57 (d, JF-C = 6.54 Hz), 122.00, 116.27 (d, JF-C = 17.30 Hz), 71.68, 60.54,
49.52, 49.12, 37.88, 35.04, 34.23, 33.06, 32.28, 27.02, 25.15; LRMS (ES+,
40 V) m/z [M + H]+ C26H38FN6O5: calculated 533.29; found 533.09; m/z
[M + Na]+ C26H37FN6NaO5: calculated 555.27; found 555.08; HRMS (TOF
MS AP+) m/z [M + H]+ C26H38FN6O5: calculated 533.2888; found
533.2918; HPLC QC purity 98.51%.
Purity, stability, and specific activity measurement
Radiochemical purity, stability, and specific activity measurements were
determined by analytical HPLC QC (Supporting Information). The specific
activity values were obtained by measurement of the radioactivity
injected and the no-carrier added [19F]UV absorbance associated with
the corresponding [18F]radioactive peak. The concentration was then
obtained by comparison of the UV area under the curve to the non-
radioactive standard concentration curve.
Results and discussion
Chemistry
Synthesis of the non-radioactive standard 4 was achieved in a
single step from commercially available starting materials. 1-
Azido-1-deoxy-β-D-glucopyranoside 3 was dissolved in water
and added to 1-ethynyl-4-f luorobenzene 2 in n-butanol. To
this solution was added sodium ascorbate, 2,6-lutidine and
copper (II) sulphate. The solution was stirred at room
temperature for 16 h and then the crude reaction mixture
purified directly by reverse phase flash chromatography to
give the desired standard 4 in 56% yield (Scheme 1). The
non-radioactive standard 6 was synthesized in a similar
Radiochemistry
Ethynyl-4-[18F]fluorobenzene ([18F]2)
An aqueous [18F]HF solution (0.1-2 GBq) was added to a reaction vial
containing a solution of potassium carbonate (4.5 mg in 45 μL H2O,
32.3 μmol) and K222 (Kryptofix 2.2.2) (12.1 mg, in 121 μL CH3CN,
32.3 μmol). The solvent was evaporated under a stream of nitrogen at
100 °C under vacuum, and the residue azeotropically dried three times
by repeated addition and evaporation of anhydrous CH3CN (3 × 1 mL).
To the activated K18F.K222 complex was added 4-ethynyl-N,N,N-
trimethylbenzenaminium trifluoromethanesulfonate 1 (5 mg, 16.15 μmol)
manner described previously, except precursor
5 was
in DMF (1 mL), and the mixture heated at 150 °C for 5 min. After cooling prepared via a nine-step synthesis (Supporting Information),14
J. Label Compd. Radiopharm 2015, 58 473–478
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org

M. P. Roberts et al.
Scheme 1. Synthesis of non-radioactive standards 4 and 6, reagents: (a) 3, or (b) 5, CuSO4, sodium ascorbate, 2,6-lutidine, n-butanol/H2O 2/1, room temperature, 16 h.
and then purified by preparative HPLC to give the desired Radiochemistry
standard 6 in a 38% yield (Scheme 1). The synthesis of the
[18F]2 was synthesized under classical radiolabeling conditions
4-ethynyl-N,N,N-trimethylbenzenaminium trifluoromethanesulfonate
by nucleophilic aromatic [18F]fluorination, and subsequently
radiolabeling precursor 1 was achieved as outlined in Scheme 2.
4-Ethynyl-N,N-dimethylaniline was dissolved in dichloromethane
(CH2Cl2); methyl triflate was added, and the solution was stirred
was ‘click’ conjugated using a CuAAC reaction to 3 and 5 to
produce [18F]4 and [18F]6, respectively (Scheme 2).
at room temperature for 2 h. The resulting precipitate was
Radiosynthesis of ethynyl-4-[18F]fluorobenzene ([18F]2)
collected by filtration and washed with CH2Cl2 to give the
desired radiolabeling precursor 1 in 77% yield.15
Radiosynthesis of [18F]2 was performed with no-carrier-added
[18F]fluoride through the activated K18F.K222 complex.
Optimization of the radiosynthesis of [18F]2 (Figure 2) was
performed by reacting 1 with K18F.K222 complex under various
reaction conditions including solvent (CH3CN, DMF, and DMSO),
temperature (100 and 150 °C), time (5, 10, and 15 min) and
precursor quantity (10, 5, 2 mg).
All optimization experiments started with a precursor mass of
10 mg. At the specified time points, small aliquots (~100 μL) of
the reaction mixture were analyzed by HPLC and the RCY
compared (all radiochemical yields were calculated by collecting
the HPLC product peak and measuring the amount of
radioactivity collected). After the last time point, HPLC mobile
phase was added to the remaining reaction mixture and shaken
to aid washing the sides of the reaction vial, before the entire
contents was also analyzed by HPLC and the RCY compared.
Reaction conditions using DMF at 150 °C for 5 min was found
to be optimal and was further developed to decrease the
precursor 1 mass to 5 mg (14 2% collected ndc RCY, n = 3).
Scheme 2. Synthesis of 1, radiosynthesis of [18F]2 and click conjugated products
18F]4 and [18F]6, reagents: (a) methyl triflate, CH2Cl2, room temperature, 2 h; (b)
18F.K222 complex, DMF, 150 °C, 5 min; (c) (i) 3, 80 °C, or (ii) 5, 45 °C, sodium
ascorbate, copper iodide, 2,6-lutidine, CH3CN/H2O 2/1, 10 min.
[
K
Figure 2. Summary of reaction yield optimization (ndc RCY), of synthon [18F]2, based on aliquot samples (5–15 min) and total remaining reaction mixture by HPLC; the
effects of varying solvent, temperature, time, and precursor mass, n = 3.
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J. Label Compd. Radiopharm 2015, 58 473–478

M. P. Roberts et al.
The decreased precursor amount resulted in lower yields available azido-functionalized carbohydrate 3, producing [18F]4.
based on the 5–15 min aliquot HPLC injections, however This could then be directly compared to a similar literature
comparable yields based on the overall remaining reaction radiolabeling19 where 3 was click conjugated to synthon [18F]4-
mixture, which was a more accurate representative of the fluorobutyne to give [18F]7, which was prepared in a three-step
reaction. Across the range of experimental conditions tested, reaction in a 30% dc RCY (Scheme 3). [18F]4-Fluorobutyne was
the HPLC yields of the whole remaining reaction mixture were reported not isolated because of its volatility and was used for
unsurprisingly much lower than the small aliquot injections, as conjugation immediately without purification. Hence, the RCY
losses from adherence of radiochemical products to the wall of could not be compared directly to that of [18F]2; however, [18F]
the reaction vessel were accounted for by the washing step.
2 required a shorter synthesis time, fewer synthetic steps, and
The discrepancy between the aliquot and whole remaining was able to be isolated because of its higher boiling point. The
reaction yields may also be due to the loss of volatile [18F] subsequent click conjugation of [18F]2 and 3 was performed
methylfluoride that was not apparent in the small aliquot HPLC under similar conditions to those described in the literature
injections. The possibility of [18F]methylfluoride formation was (copper(I) iodide, sodium ascorbate, and 2,6-lutidine in CH3CN/
anticipated, but the predicted high stability of [18F]2 due to the H2O)19 to produce [18F]4 in 76 4% ndc RCY (11% overall two
ethynyl functional group being only weakly activating was step). The main advantage of [18F]2 over [18F]4-fluorobutyne
considered more favorable than a higher RCY that would likely was the elimination of a time-consuming and difficult distillation.
be obtained from a more activated ring system (e.g., 2- or 4- Instead, [18F]2 was purified by HPLC, then easily trapped and
substituted nitrobenzene or pyridine).16
eluted from a Strata X SPE cartridge for the subsequent
The superiority of the trimethylammonium group is conjugation step, resulting in a comparable overall RCY to that
demonstrated with the use of less activating substituents, such of [18F]-4-fluorobutyne.
as bromine or iodine (Hammett constant ~0.23), as opposed to
The optimized conditions for the radiolabeled carbohydrate
more activating substituents such as cyanide or nitrogen dioxide [18F]4, the procedure described by Kim et al.19 for the
(Hammett constant ~0.65 and ~0.8, respectively).1 However, it radiosynthesis of [18F]7 and for [18F]8 described by Doan
has been reported that in the presence of less activating et al.14 (Scheme 3) provided the starting point for the click
substituents with low Hammett constants (σ) para to the conjugation of [18F]2 to the matrix metalloproteinase inhibitor
trimethylammonium leaving group, aliphatic substitution 5. Similar reaction conditions (copper iodide, sodium ascorbate,
undesirably occurs on the N(CH3)3 group, and [18F]methylfluoride 2,6-lutidine in CH3CN/H2O for 10 min), except a lower reaction
formation competes with the desired aromatic [18F]radiolabeling.1 temperature (45 °C) and an alternate base diisopropylethylamine
The Hammett constant σ of the ethynyl group 0.23, similar to that (DIPEA), were utilized for radiolabeling the matrix metalloproteinase
of other less activating substituents such as bromine or iodine17 inhibitor 5 to produce [18F]6. The initial HPLC purification
gives rise to low RCYs of only 12–15% due to the aforementioned conditions employed were those described by Doan et al. for
competing reaction, hence the RCY obtained herein is within the the purification of the alkyl-analog [18F]8 14; however, in our
expected range.1
hands, the basic mobile phase conditions resulted in [18F]6
The suitability of the trimethylammonium triflate leaving largely streaking and broadening over an extended period of
group, as opposed to halogen groups, in the presence of the less time. This made collecting [18F]6 in a concentrated volume
activating ethynyl substituent was also assessed using the non- difficult and the losses on the HPLC column led to low RCYs
radioactive standard 2 to synthesize [18F]2 via 19F/18F halogen (~5–10% ndc). It is difficult to explain this occurrence because
exchange. Using the same reaction conditions as before (DMF, the radiosynthons are similar in structure with the only difference
150 °C, 5 mg, 5 min), the radiosynthesis of [18F]2 was successful, being a small aliphatic chain ([18F]8) versus an aromatic ring ([18F]
however in a lower yield (RCY 3% ndc) compared to the 6). Nevertheless, optimization of the HPLC purification conditions
trimethylammonium precursor 1 (RCY 14 2% ndc). These showed that the use of an acidic mobile phase corrected the
results demonstrated that other less activating halogens product peak broadening issue, enabling [18F]6 to be collected
(bromine, chlorine, or iodine) would most likely have not been as one sharp peak over a short timeframe. This significantly
suitable and concurs with the literature that the trimethylammonium improved the RCY (56 12% ndc) (8% overall two step) such that
leaving group is superior to halides, particularly in the presence of it was comparable to that obtained by Doan et al. for [18F]8 (51%
less activating substituents such as the ethynyl group in this case.1,18 dc RCY).
In an attempt to circumvent time-consuming purification by
HPLC, SPE (phenomenex C18 Strata™ X, Waters C18 Sep-Pak®
(Plus & Lite), and Polyintell Affinimip®) was also investigated for
ease and to decrease total synthesis time. However, the chemical
and radiochemical purity obtained from the various SPE
attempts were not satisfactory for general use and for the
subsequent click conjugation, hence HPLC purification was
utilized. Following HPLC purification, [18F]2 was transferred to a
C18 Strata™ X SPE cartridge for concentration and solvent
exchange required for subsequent elution into the click
conjugation reaction.
Radiosynthesis of [18F]4 and [18F]6
Scheme 3. Radiosynthesis of literature click conjugated products [18F]7 and [18F]
8, reagents: (a) [18F]-4-fluorobutyne, 3, sodium ascorbate, copper iodide, 2,6-
lutidine, CH3CN/H2O, 1/1, 90 °C, 10 min; (b) 5, [18F]-5-fluoropentyne 45 °C, sodium
ascorbate, copper iodide, DIPEA, CH3CN/H2O 1/1, 10 min.
To investigate the utility of synthon [18F]2, it was first decided, as
a proof of concept, to click conjugate it to a commercially
J. Label Compd. Radiopharm 2015, 58 473–478
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org

M. P. Roberts et al.
Purity, stability, and specific activity
indirect [18F]radiolabeling of several biological vectors and the
results from these studies will be reported in due course.
The purity and stability of synthon [18F]2 was analyzed by HPLC
QC at time points t = 0 and 180 min. No significant decrease in
radiochemical purity was observed (99.4 to 99.3%) over
180 min. The radiochemical purity of the radiolabeled products
[18F]4 and [18F]6 at was found to be 99.0 and >99.9%,
respectively. Chemical purity determined by UV analysis of
[18F]4 and [18F]6 revealed absence of the starting azide or
reaction side products.
Acknowledgements
We thank Iveta Kurlapski for chemical and radiochemical
technical assistance. The support of the Australian Research
Council through grant no. DP1094232 is gratefully
acknowledged.
The specific activities of [18F]2 and [18F]6 in our initial
References
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Supporting information
and large molecules for PET radiotracer/radiopharmaceutical Additional supporting information may be found in the online
development. We are currently utilizing and evaluating [18F]2 for version of this article at the publisher’s web-site.
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Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 473–478