A fast, simple, and reproducible automated synthesis of [ 18F]FPyKYNE-c(RGDyK) for αvβ3 receptor positron emission tomography imaging

Article abstract of DOI:10.1002/jlcr.1948

[¹⁸ F]FPyKYNE-c(RGDyK) was successfully synthesized by the Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition of alkynes to azides using [ ¹⁸ F]FPyKYNE as a prosthetic group in an overall radiochemical yield of 12%-18% (decay-correcte

Full text of DOI:10.1002/jlcr.1948

Research Article
Received 15 July 2011,
Revised 29 September 2011,
Accepted 7 October 2011
Published online 16 November 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1948
A fast, simple, and reproducible automated
synthesis of [18F]FPyKYNE-c(RGDyK) for avb3
receptor positron emission tomography
imaging
Ana C. Valdivia,a Miriam Estrada,a Tayebeh Hadizad,a Duncan J. Stewart,a,b
Rob S. Beanlands,a,b and Jean N. DaSilvaa,b
*
[
18 F]FPyKYNE-c(RGDyK) was successfully synthesized by the Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition of alkynes to
azides using [18 F]FPyKYNE as a prosthetic group in an overall radiochemical yield of 12%–18% (decay-corrected) and
>99.5% chemical and radiochemical purities in 125 min including quality control. This simple, fully automated two-step,
two-reactor approach consists of a quick and convenient purification of the prosthetic group using silica gel cartridges
and its subsequent use for the labeling of the azido-c(RGDyK) peptide via click chemistry.
Keywords: avb3 integrin receptors; 18F-labeling; [18F]-FPyKYNE; c(iRGDyK); PET
click chemistry approach. This approach will facilitate the routine
production of RGD derivatives for PET imaging research.
Introduction
Angiogenesis or the formation of new blood vessels plays a crucial
role in tumor vasculature and in various human heart diseases
including cardiomyopathy, atherosclerosis, peripheral artery
Experimental
disease, ischemia, inflammation, and atherosclerotic lesions.1–3 In
General methods
a cardiovascular setting, angiogenesis is triggered by hypoxia
and ischemia, and its major action in tissues is the restoration of
2-Nitro-hydroxypyridine, 5-chloropent-1-yne, pentane, diethyl
perfusion and oxygenation. Among other important biomarkers
ether, acetonitrile (MeCN), dimethylformamide, dimethyl sulfoxide
of angiogenesis, cell surface integrin receptors are of particular
(DMSO), (+)L-sodium ascorbate, copper sulphate (CuSO4), and
interest. Between all of the integrins discovered to date, integrin
KryptofixW222 (K222: 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]
avb3 is the most extensively studied. Integrin avb3 receptors bind
hexacosane) were purchased from Sigma–Aldrich (Canada Ltd.).
to peptides containing the amino acid sequence arginine-glycine-
Azido-cyclic RGDyK (Azido-cyclic Arg-Gly-Asp-D-Tyr-Lys) was
aspartic acid (RGD) present in the extracellular matrix. The goal of
purchased from FutureChem Co. Ltd. (Seoul, South Korea). Solid
phase extraction cartridges ChromafixW were purchased from
Macherey–Nagel (Duren, Germany) and Sep-PakW Plus silica
cartridges 55–105 mm were acquired from Waters (USA). All
pro-angiogenic therapy in the treatment of ischemic diseases is
to stimulate the growth of new blood vessels in order to improve
tissue vascularization and function.1–4 However, the lack of a
reliable, easily applicable, and noninvasive technique to assess
other chemicals were purchased from standard commercial
the presence of angiogenesis is a limiting factor in evaluating the
sources (VWR or Fisher Scientific) and used without further
purification. Semipreparative HPLC for the labeled peptide was
therapeutic efficacy of angiogenesis targeted therapies.1–3
Several groups have investigated the imaging of integrin avb3
performed using a C18-reverse-phase column (Luna, 10 mm,
receptors with RGD containing peptide-based positron emission
tomography (PET) tracers.5–21 Most of these radiolabeled peptides
show high affinity and selectivity for the avb3 integrin receptor
aNational Cardiac PET Centre, University of Ottawa Heart Institute, 40 Ruskin
Street, Ottawa, ON, Canada
in vitro and receptor-specific accumulation in tumors in vivo. The
most evaluated of these radiotracers is 18F-Galacto-RGD22, which
bDivision of Cardiology (Department of Medicine) and Department of Cellular
and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada
has also been investigated in humans.19,20 The long, complex,
multistep synthetic approach renders their production difficult to
bring to a clinical setup. We present here a simple and convenient
strategy consisting of a two-step, two-reactor synthesis of an
18F-labeled RGD peptide in a fully automated process using the
*Correspondence to: Jean N. DaSilva, National Cardiac PET Centre, University of
Ottawa Heart Institute, 40 Ruskin Street, Ottawa, ON, Canada K1Y4W7.
E-mail: jdasilva@ottawaheart.ca
J. Label Compd. Radiopharm 2012, 55 57–60
Copyright © 2011 John Wiley & Sons, Ltd.

A. C. Valdivia et al.
250 Â 10 mm, Phenomenex, USA) with a radiation detector FPYKYNE-c(RGDyK)
using acetonitrile/ammonium formate 0.1 M, 25/75, flow rate
To a mixture of FPyKYNE (0.001 g, 5.6 mmol) and azido-c(RGDyK)
peptide (0.001 g, 1.2 mmol) in 600 mL of DMSO/water (1/1), CuSO4
(1.2mmol in water) and sodium ascorbate (3.8 mmol in water)
were added and this mixture was heated at 40ꢀC. The conversion
of the azido-c(RGDyK) to the FPYKYNE-c(RGDyK) was followed by
analytical HPLC, with all the azide-peptide (tR = 3 min) converted
to FPyKYNE-c(RGDyK) (tR = 6.9 min) after 20 min. Purification of
FPyKYNE-c(RGDyK) (tR = 8 min) was achieved by semipreparative
HPLC. The identity of FPyKYNE-c(RGDyK) was characterized by
electrospray ionization mass spectrometry. Calculated for
C35H47N12O9 [M + H]: 958.443; found: 958.446.
5 mL/min, UV at 254 nm. Analytical HPLC was carried out on a
C18-reverse-phase column (Luna, 10 mm, 4.6 Â 10 mm, Phenom-
enex, USA) using acetonitrile/ammonium formate 0.1 M, 25/75,
flow rate 2 mL/min, UV at 254 nm. Furthermore, two additional
reverse-phase analytical columns Luna C18 ODS and SCX
(10 mm, 4.6 Â 25 mm, Phenomenex, USA) were used for product
identification. Nuclear magnetic resonance (NMR) was per-
formed using a 500 MHz Bruker Avance (Bruker BioSpin Ltd.,
ON, Canada) instrument and mass spectrometry analyzed with
a quadrupole time-of-flight (QTOF) spectrophotometer.
2-Nitro-3-pent-4-yn-1-yloxypyridine
[
18F]FPyKYNE-c(RGDyK)
The nitro precursor for 18F-labeling was synthesized as previously
described by Kuhnast et al.23 The 1H and 13C NMR data matched The fully automated radiosynthesis and semipreparative HPLC of
those reported earlier.23 EI-MS (m/z): calculated for C10H10KN2O3:
[
18 F]FPyKYNE-c(RGDyK) was carried out in a dual reactor
245.033; found 245.030 [M+ K].
TRACERlabW FX N Pro, (GE Healthcare) (Figure 1). [18 F]Fluoride
was trapped on an anion exchange resin and eluted from the
cartridge into reactor-1 with a solution of K2CO3 (0.2 mmol) and
K222 (0.4 mmol) in 95% MeCN in water. The resulting solution
was then concentrated to dryness with a stream of N2 at 110ꢀC
2-Fluoro-3-pent-4-yn-1-yloxypyridine, (FPyKYNE)
A mixture of potassium fluoride (KF) (0.1 g, 1.74 mmol) and K222 under vacuum for 3 min. This procedure was repeated twice to
(0.6g, 1.74 mmol) in anhydrous DMSO (5mL) was stirred at 150ꢀC afford the dried K[18 F]F-K222 complex in reactor-1. The precursor
for 10 min under argon. Subsequently, the 2-nitro-3-pent-4-yn-1- (2-nitro-3-pent-4-yn-1-yloxypyridine) (6.0 mg, 0.03 mmol) in
yloxypyridine (0.3g, 1.45 mmol) was added to the solution. The DMSO (600 mL) was added to reactor-1, and the reaction mixture
reaction mixture was left stirring for 1 h. After cooling, water was heated at 155ꢀC for 10 min. The reaction vessel was cooled
(100 mL) was added and the mixture was extracted with ethyl ace- to 35ꢀC using a stream of air and the mixture transferred onto
tate (100 mL Â 3). The organic phases were combined, dried over a column of silica gel cartridges (n = 3) connected in series. Our
Na2SO4, and the solvent was concentrated under vacuum followed strategy for the purification of [18 F]FPyKyNE is based on the dif-
by purification using flash column chromatography with hexanes/ ference in the retention factor (Rf) between the 2-nitro-3-pent-4-
ethyl acetate (8.5/1.5) to afford FPyKYNE (0.27 g, 1.49 mmol, 86%) yn-1-yloxypyridine used as the precursor (Rf = 0.22) and FPyKYNE
as crystalline oil. The 1H and 13C NMR data matched those reported (Rf = 0.57) in pentane/ether, 1/1. [18 F]FPyKYNE was eluted using
earlier.23 19F-NMR: dF: À84.7899 (referenced with respect to 5.5 mL of pentane/ether (1/1) into reactor-2, with the unreacted
trifluoroacetic acid at À76.55 ppm). EI-MS (m/z): calculated for precursor trapped on silica, and the solvent evaporated under
C10H10FNO 179.075; found 179.076 [M+]
a stream of N2 at 33ꢀC. A second elution of [18 F]FPyKYNE from
Figure 1. Graphical display of the TRACERlab FX N Pro.
www.jlcr.org
Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 57–60

A. C. Valdivia et al.
A)
40.00
35.00
30.00
25.00
20.00
15.00
10.00
5.00
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
Minutes
0.060
0.050
0.040
0.030
0.020
0.010
0.000
B)
UV (254 nm)
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00 11.00
Minutes
Figure 2. (A) Radioactivity and (B) UV (254 nm) chromatograms of [18 F]FPyKYNEc(RGDyK).
the cartridges was performed with 3 mL of the pentane/ether
[
18 F]FPyKYNE was carried out in a mixture of DMSO/water in the
solution into reactor-2. After complete evaporation of the presence of CuSO4 and sodium ascorbate (Figure 2). [18 F]FPyKYNE-
solvent in reactor-2, azido-c(RGDyK) (0.5 mg, 2.8 mmol), CuSO4 c(RGDyK) was purified by reverse-phase semipreparative HPLC and
(4 mmol), sodium ascorbate (12 mmol) were added to a final obtained in 12%–18% radiochemical yield (decay-corrected from
volume of 1 mL in a mixture of DMSO/water (1/1). The reaction
mixture was left reacting at 65ꢀC for 20 min and subsequently
purified by semipreparative HPLC. The fraction containing
[
H2N
18 F]FPyKYNE-c(RGDyK) peptide was collected. The solvent
NH
was evaporated under vacuum at 80ꢀC and reformulated in
saline using an in-house rotary evaporator system. Analytical
HPLC was performed to determine the chemical and radioche-
mical purities of [18 F]FPyKYNE-c(RGDyK), as well as for the
determination of the specific activity using the reference
compound FPyKYNE.
HN
18F
O
N
O
N3
H
N
N
NH
H
O
NH
C
O
O
H
O
N
O
N
H
HO
O
Results and discussion
CuSO4
Na Ascorbate
OH
DMSO:Water (1:1)
60 °C
FPyKYNE-c(RGDyK) peptide (standard) was synthesized via ‘click
chemistry’ reaction between the azido-c(RGDyK) peptide and
FPyKYNE. [18 F]FPyKyNE was prepared via aromatic nucleophilic
substitution in reactor-1 with a modification performed to the
purification method. Instead of utilizing HPLC, a rapid purification
was performed using three silica gel cartridges connected in
series, acting as a flash column chromatography for ease of
separation. Elution of pure [18 F]FPyKyNE was performed using a
50% mixture of pentane and ether into reactor-2. These low
boiling points solvents can be easily evaporated. On average,
100–200 mCi was obtained after evaporation of the eluent in
about 20–25 min (20%–35% decay-corrected radiochemical
H2N
HN
NH
18F
O
N
N
O
H
N
N
N
N
NH
H
O
NH
O C
O
H
O
N
O
N
HO
H
O
OH
yield). Subsequent labeling of the azido-c(RGDyK) peptide with Scheme 1. 18 F-Labeling of the azido-c(RGDyK) with [18 F]FPyKYNE via click Chemistry.
J. Label Compd. Radiopharm 2012, 55 57–60
Copyright © 2011 John Wiley & Sons, Ltd.
www.jlcr.org

A. C. Valdivia et al.
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obtained with >99% radiochemical purity (Scheme 1). The identity
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Conclusion
A fast, reproducible and simple two-step automated method was
successfully developed for the preparation of [18 F]FPyKYNE-c
(RGDyK) peptide. Our strategy includes rapid purification of the
prosthetic group [18 F]FPyKYNE and its subsequent attachment to
an azido-c(RGDyK) derivative via ‘click chemistry’ in high yield,
radiochemical and chemical purities. The straightforward synthesis
of the novel [18 F]FPyKYNE-c(RGDyK) can be brought to a routine
clinical setup using the approach presented here.
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Acknowledgements
The authors thank the radiochemistry staff. This work was
funded by an Heart and Stroke Foundation of Ontario Program
Grant in Molecular Function and Imaging (HSFO GIA #PGR6242).
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Conflict of Interest
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Wester, W. A. Weber, M. Schwaiger, J. Nucl. Med. 2005, 446, 1333–1341.
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Wester, R. Haubner, M. Schwaiger, Clin. Cancer Res. 2007, 13, 6610–6616.
[21] M. Lei, M. Tian, H. Zhang, Curr. Med. Imaging Rev. 2010, 6, 33.
The authors have declared that there is no conflict of interest.
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Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 57–60