Radiosynthesis and bioconjugation of [18F]FPy5yne, a prosthetic group for the18F labeling of bioactive peptides

Article abstract of DOI:10.1002/jlcr.1561

A new ¹⁸F-based prosthetic group has been prepared for the labeling of azide-modified peptides for use in PET imaging. 2-[ ¹⁸F]fluoro-3-(hex-5-ynyloxy)pyridine ([¹⁸F]FPy5yne, [ ¹⁸F]-1) was prepared via efficient nucleophilic heteroaromatic substitution of either the corresponding 2-nitro (2) or 2-trimethylammonium trifluoromethanesulfonate pyridine (3). Best radiochemical yield of [ ¹⁸F]FPy5yne from 2 was 91% by radioTLC (15 min, 110°C, DMSO). From 3, best radiochemical yield by radioTLC was 93% (15 min, 110°C, MeCN). HPLC-purified [¹⁸F]FPy5yne was ligated to model peptide N ₃-(CH₂)₄-CO-YK-RI-OH by way of Cu ^I-mediated Huisgen [3+2] cycloaddition in the presence of copper-stabilizing ligand tris(benzyl-triazolylmethyl) amine (TBTA) and N,N-diisopropylethylamine (DIEA). Bioconjugate radiochemical yields were obtained in average yields of 89%78.6% (n = 4), as judged by radioHPLC. Best non-decay-corrected, collected radiochemical yield of modified peptide from end-of-bombardment was 5.8% (18.7% decay-corrected), with a total preparation time of 160 min from start of synthesis. Copyright

Full text of DOI:10.1002/jlcr.1561

Research Article
Received 3 July 2008,
Revised 23 September 2008,
Accepted 1 October 2008
Published online 6 November 2008 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1561
Radiosynthesis and bioconjugation
of [18F]FPy5yne, a prosthetic group for
the F labeling of bioactive peptides
18
a
Ã
b
a
´
James A. H. Inkster, Brigitte Guerin, Thomas J. Ruth,
and Michael J. Adama
A new 18F-based prosthetic group has been prepared for the labeling of azide-modified peptides for use in PET imaging.
2-[18F]fluoro-3-(hex-5-ynyloxy)pyridine ([18F]FPy5yne, [18F]-1) was prepared via efficient nucleophilic heteroaromatic
substitution of either the corresponding 2-nitro (2) or 2-trimethylammonium trifluoromethanesulfonate pyridine (3). Best
radiochemical yield of [18F]FPy5yne from 2 was 91% by radioTLC (15 min, 1101C, DMSO). From 3, best radiochemical yield
by radioTLC was 93% (15 min, 1101C, MeCN). HPLC-purified [18F]FPy5yne was ligated to model peptide N3–(CH2)4–CO–YK-
RI–OH by way of CuI-mediated Huisgen [3+2] cycloaddition in the presence of copper-stabilizing ligand tris(benzyl-
triazolylmethyl)amine (TBTA) and N,N-diisopropylethylamine (DIEA). Bioconjugate radiochemical yields were obtained in
average yields of 89%78.6% (n = 4), as judged by radioHPLC. Best non-decay-corrected, collected radiochemical yield of
modified peptide from end-of-bombardment was 5.8% (18.7% decay-corrected), with a total preparation time of 160 min
from start of synthesis.
Keywords: fluorine-18; peptide labeling; click; heteroaromatic substitution
are noteworthy in their capacity to both efficiently [18F]fluor-
Introduction
inate and ligate to biological molecules. [18F]FPyBrA was
originally used to radiolabel phosphorothiolate DNA;17 later it
The growing popularity of the Huisgen 1,3-dipolar cycloaddition
reaction1 in bioconjugate chemistry owes much to the discovery
that CuI accelerates the rate of this reaction and results in a
diasterospecific 1,4-disubstituted 1,2,3-triazole linkage.2,3 In this
fashion, a number of peptide- and protein-based bioconjugates
have been successfully prepared.4–6 Recently, this quintessential
‘click reaction’7 was applied to the 18F-labeling of model peptide
sequences with 5-[18F]fluoro-1-pentyne as a route to new PET
imaging agents.8 In addition, a number of small organic
compounds, including sugars, amino acids, and nucleosides
were efficiently coupled to aliphatic [18F]fluoroalkynes.9,10
[18F]Fluoroaromatic prosthetic group 4-[18F]fluoro-N-(prop-2-
ynyl)benzamide was synthesized by Ramenda et al. for the
was used to prepare 18F-modified Spiegelmers,18,19 short
interfering RNA,20 and polyamide nucleic acids.21,22 [18F]FPyME
was used to successfully label variety of thiol-containing
peptides and proteins.23
We sought to employ a 2-substituted pyridine [18F]fluorina-
tion in the preparation of a general labeling agent for use with
biomolecules of interest to PET. We reasoned that bifunctional
precursor molecules containing 2-nitro (2) and 2-trimethylam-
monium triflate (3) leaving groups and robust terminal alkyne
moieties could be used to prepare prosthetic label 2-[18F]fluoro-
3-(hex-5-ynyloxy)pyridine ([18F]FPy5yne , [18F]-1). It was antici-
pated that this compound could be synthesized in a single
radiochemical step (Scheme 1), purified, then conjugated to
biological molecules under chemospecific Huisgen [3 + 2]
cycloaddition ligation conditions. Herein, we report the success-
ful synthesis of [18F]FPy5yne and, as proof-of-principle, its
subsequent bioconjugation to an azide-modified peptide.
radiolabeling of the neurotensin [8–13] peptide (NT[8-13]).11
A
successful 18F-peptide preparation using 2-[18F]fluoroethylazide
has also been reported.12 Most recently, a [18F]fluorinated
triethylene glycol derivative was bioconjugated to RGD peptide
and subsequently used to image integrin aVb3-specific tu-
mours.13 The aforementioned study represents the first use of
1,2,3-triazole linkers for in vivo PET imaging.
Efficient [18F]fluorinations of 2-substituted pyridinyl moi-
eties14,15 have been utilized by the Orsay group and others
to prepare a number of 18F-based tracers and prosthetic
tags.16 Among the successful radiobioconjugations reported,
the synthesis of bifunctional molecules 2-bromo-N-3-(2-
[18F]fluoropyridin-3-yloxy)propyl]acetamide ([18F]FPyBrA) and 1-
aTRIUMF, 4004 Wesbrook Mall, Vancouver, BC, Canada V6T 2A3
b
´
´
´
´
Departement de Medecine Nucleaire et Radiobiologie, Universite de Sher-
brooke, 3001 12ieme Avenue Nord, Fleurimont, QC, Canada J1H 5N4
`
*Correspondence to: James A. H. Inkster, TRIUMF, 4004 Wesbrook Mall,
Vancouver, BC, Canada V6T 2A3.
[3-(2-fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione ([18F]FPyME) E-mail: jamesi@triumf.ca
J. Label Compd. Radiopharm 2008, 51 444–452
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J. A. H. Inkster et al.
of 2-nitropyridine precursor 2 with commercial 1 M TBAF
solution (Scheme 2). As observed previously, fluorinations of
simple 2-substituted pyridines proceed smoothly with this
reagent, despite the fact that it contains as much as 5% water.26
Model peptide BG142, consisting of the azide-modified four
amino acid motif N3–(CH2)4–CO–Tyr–Lys–Arg–Ile–OH, was
synthesized using an automated system by way of standard
Fmoc protocols. Cold peptide standard 19F-BG142 was
prepared using a Huisgen 1,3-dipolar cycloaddition ligation
(Scheme 4). Acetonitrile-soluble complex Cu(CH3CN)4PF6 was
Results and discussion
Chemistry
An attempt was made to prepare 3-(hex-5-ynyloxy)-2-nitropyr-
idine (2) by way of Mistunobu condensation of commercially
available 2-nitro-3-hydroxypyridine and hex-5-yn-1-ol, in
a
protocol similar to those reported for the synthesis of FPyBrA17
and FPyME.23 Unfortunately, 2 was found to elute closely with
reaction by-product diisopropyl hydrazine-1,2-dicarboxylate,
and the two compounds could not be separated in our hands
using silica gel chromatography. Presumably, alternative Mitso-
nubu reagents with water-soluble by-products such as di-tert-
butyl azodicarboxylate (DBAD)24 and di-2-methoxyethyl azodi-
carboxylate (DMEAD)25 could be employed. In our case,
however, use of these expensive reagents proved unnecessary.
Compound 2 was prepared by coupling 3-hydroxy-2-nitropyr-
idine and 6-chlorohexy-1-yne under Wilkinson ether synthesis
conditions (NaH, 601C, DMF; Scheme 2). This approach resulted
in a clean reaction and straightforward purification. A nearly
identical procedure was used to prepare 2-dimethylamino-3-
(hex-5-ynyloxy)pyridine (5), which was subsequently methylated
to afford trimethylammonium triflate precursor 3 (Scheme 3).
Cold standard [19F]FPy5yne was obtained from the fluorination
37% CH O
2
aq
OH
N
NaCNBH , AcOH
OH
NH
3
52%
N
N
2
4
NaH
6-chloro-1-hexyne
DMF, 70oC, 73%
O
N
MeOTf
O
N
5
toluene, 0oC-RT
95%
N
N
OTf
O
3
Scheme 3. Synthesis of [3-(hex-5-ynyloxy)pyridin-2-yl]trimethylammonium triflur-
omethanesulfonate (3).
N
X
NH2
2: X = NO2
3: X = [NMe3][SO3CF3]
O
O
K[18F]F/K222
H
H
Table 1
N
N
OH
N
N
H
H
O
O
O
O
N3
OH
NH
NH2
HN
BG142
18/19F]-1
Cu(CH3CN)4PF6
TBTA
N
18F
[18F]FPy5yne, [18F]-1
[
Scheme 1. Radiosynthesis of [18F]Fpy5yne ([18F]-1).
NH2
NaH
OH
O
6-chloro-1-hexyne
DMF, 60oC, 75%
N
NO2
N
NO2
O
H
O
H
2
N
N
OH
N
H
N
H
1M TBAF
O
O
O
THF:DMF, 80oC
77%
N
N
OH
N
NH
HN
O
O
F
NH2
F
N
N
1, FPy5yne
18/19F-BG142
Scheme 2. Synthesis of 3-(hex-5-ynyloxy)-2-nitropyridine (2) and synthetic
standard [19F]-1.
Scheme 4. Preparation of fluorine-labeled peptides 18F-BG142 and 19F-BG142.
J. Label Compd. Radiopharm 2008, 51 444–452
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J. A. H. Inkster et al.
used as a source of CuI. Prior to contact with the peptide, the salt.28 Nevertheless, this impurity could be completely and
copper salt was complexed with CuI-chelating ligand tris- consistently separated from labeled [18F]-1 by HPLC and thus
(benzyltriazolylmethyl)amine (TBTA). TBTA has been shown to subsequent experiments were performed using precursor 3
preserve the oxidation state of the metal, while at the same time exclusively.
enhancing its catalytic role.27 The bioconjugate was purified by
Bioconjugation of [18F]-1 to model peptide sequence
semi-preparative HPLC and its identity confirmed by MALDI-TOF. N3–(CH2)4–CO–YKRI–OH (BG142) proceeded in a straightfor-
ward fashion. Radiochemical yields of 81–100% 18F-BG142
(89%78.6%, n = 4) as judged by radioHPLC were obtained in
Radiochemistry
10 min at 371C by mixing the azide-modified peptide with [18F]-
1 in the presence of tetrakis(acetonitrilo)copper(I) hexafluoro-
phosphate, TBTA, and N, N–diisopropylethylamine (DIEA). These
Bifunctional prosthetic group [18F]FPy5yne ([18F]-1) was synthe-
sized by way of 18F nucleophilic heteroaromatic substitution of
either 3-(hex-5-ynyloxy)-2-nitropyridine (2) or [3-(hex-5-ynyloxy)-
pyridin-2-yl]trimethylammonium trifluoromethanesulfonate (3;
Scheme 1). Standard KF[18F]-K222/potassium carbonate condi-
tions were used. Table 1 summarizes the various reaction
conditions employed in this study, where radiochemical yields
by radioTLC represent the percentage of the radioactive area
representing 18F-labeled product relative to the area of total
radioactivity measured. Both precursors 2 and 3 resulted in
satisfactory radiochemical yields of [18F]-1 at elevated tempera-
tures in DMSO over 15 min. In the case of 3, it was observed that
raising the temperature to 1201C or lowering the reaction time
to 10 min had a deleterious effect on radiochemical yield. In the
small number of synthesis reported here, MeCN (0.2 mL) was
comparable to that of DMSO (0.7 mL) at 1101C. However, while
assessing bioconjugation methodologies, we often chose to use
acetonitrile during [18F]-1 synthesis, since these reaction
mixtures could more reliably be diluted and injected directly
into an HPLC. In this way, we obviated the need for a manual
solid-phase extraction prior to LC purification. Specific activity of
[18F]-1 was determined to be 1650 mCi/mmol (61 GBq/mmol). A
representative radiochromatogram of a [18F]-1 reaction mixture
(MeCN, 1101C, 15 min) is shown in Figure 1. The chemical nature
of radioproduct [18F]FPy5yne was confirmed by way of HPLC co-
injection of collected material with its corresponding 19F
standard (Figure 2).
reagents were added in excess of the starting peptide. TBTA was
only partially soluble in the solvent mixture employed (20 mM
sodium phosphate buffer:DMF); thus, the reaction mixture was
clarified with the addition of 50% acetonitrile or centrifuged to
remove insolubles prior to HPLC purification. A representative
radiochromatogram of a 18F-BG142 reaction mixture is shown
in Figure 3. The chemical nature of 18F-BG142 was confirmed by
way of HPLC co-injection of collected material with its
corresponding 19F standard (Figure 4).
Experimental
Chemicals
Oxygen-18-enriched water ([18O]H2O, 497% pure) was
purchased from Rotem Industries (Beer Sheva, Israel). Fmoc-
Lys(Boc)-OH and 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetra-
methyluronium hexafluorophosphate (HATU) were obtained
from Chem Impex International, Inc. (Wood Dale, USA). Fmoc-
Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH and Fom-Ile-NovaSyns TGA
resin were purchased from Nova-Biochem. Succinimidyl
5-azidovalerate29–31 and TBTA27 were synthesized according to
published procedures. All other chemicals were purchased from
either Sigma-Aldrich or Alfa Aesar, and were classified ACS grade
or better.
We employed all three reverse phase HPLC sorbents
described in the experimental section below for the resolution
of [18F]-1 from 3-(hex-5-ynyloxy)-2-nitropyridine (2). Of these,
only the Luna PFP(2) column (Phenomenex, Inc.) afforded
baseline separations. In the case of [18F]fluorination of [3-(hex-5-
ynyloxy)pyridine-2-yl]trimethylammonium triflate (3), some
2-dimethylamino-3-(hex-5-ynyloxy)pyridine (5) was observed,
particularly in reactions employing DMSO. This is the result of
competitive demethylation of the trimethylammonium triflate
Analytical methods
TLC and radioTLC were performed on 60F254 silica gel plates
purchased from Sorbent Technoligies (Atlanta, USA). TLCs were
visualized using UV light or chemical stain (anisaldehyde or
ninhydrin). RadioTLCs were obtained using a Bioscan System
200 Imaging scanner.
All reverse phase HPLC reported herein utilized a Waters 600
Controller in combination with a Waters 2487 dual l absorbance
Table 1. Summary of reaction conditions for the preparation of [18F]FPy5ynes
Precursor
Solvent
Time (min)
Temp (1C)
Radiochemical yield (%)a
2
3
3
3
3
3
3
DMSOb
DMSOb
DMSOb
DMSOb
DMFb
MeCNd
MeCNd
15
15
10
15
15
15
15
120
110
110
80
110
75
91, 86
87, 88, 91 (89%72.1%)c
68
72
58
61, 59
110
90, 93, 88 (90%72.5%)c
aEach value is an average of two chromatograms and represents a unique radiochemical synthesis.
bReaction volume: 0.7 mL.
cAverage yield7SD.
dReaction volume: 0.2 mL.
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J. A. H. Inkster et al.
Figure 1. Preparative HPLC trace (HPLC B) of [18F]Fpy5yne reaction mixture (MeCN, 1101C, 15 min). Top: UV (260 nm). Impurity 5 Rt = 11.567 min. Bottom: Rad. [18F]-1
Rt = 13.846 min.
detector and a NaI detector. All HPLC solvents were filtered prior of azide-modified peptide sequence BG142 and bioconjugate
Column: Agilent Eclipse XDB-C18 19F-BG142 was carried out on a Micromass Tof Spec 2E
to use. HPLC
A
(250 mm  4.6 mm, 5 mm); Detector: 223 and 280 nm; Program: instrument and an Applied Biosystems Voyager System 4311
flow = 1 mL/min, 100% H2O for 5 min, then 0–100% MeCN in apparatus, respectively. In both cases, a-cyano-4-hydroxycin-
H2O over 30 min. HPLC B Column: Phenomenex Luna PFP(2) C18 namic acid was used as matrix.
(250 Â 10 mm, 5 mm); Detector: 214 and 260 nm; Program:
No-carrier-added [18F]fluoride was produced by 13 MeV
flow = 4 mL/min, 50:50 MeCN:0.1% TFA. HPLC C Column: Waters proton bombardment of [18O] water [18O(p,n)18F] on the TRIUMF
mBondapak C18 (100 Â 7.8 mm, 10 mm); Detector: 214 and TR13 cyclotron. Typical production was 65–155 mCi of [18F]FÀ at
280 nm; Program: linear gradient, flow = 4 mL/min, 0–100% end of bombardment for a 10 mA, 5 min irradiation.
MeCN in H2O with 0.1% TFA over 25 min. HPLC D Column:
Phenomenex Luna PFP(2) C18 (250 Â 10 mm, 5 mm); Detector:
214 and 280 nm; Program: linear gradient, flow = 4 mL/min,
2-Dimethylamino-3-hydroxypyridine (4)
0–100% MeCN in H2O with 0.1% TFA over 35 min.
Into a round-bottomed flask containing 2-amino-3-hydroxypyr-
idine (0.50 g, 4.56 mmol) in acetonitrile (28 mL) was added
37% aqueous formaldehyde (3.4 mL), then sodium cyanobor-
ohydride (5.84 g, 92.9 mmol). Over the course of 4 h, the reaction
turned murky blue-black. Acetic acid (10 mL) was added and the
reaction immediately clarified. The reaction was poured into
100 mL of 15% aqueous ammonia and extracted three times into
CH2Cl2. The extract was dried over MgSO4, concentrated and
1H NMR and 13C NMR were recorded on a Bruker 400 MHz
apparatus. Chemical shifts are reported relative to the hydro-
genated residue of the deuterated solvents. 19F NMR was
collected on a Bruker 300 MHz apparatus using CFCl3 as internal
standard. High-resolution electron impact mass spectroscopy
was performed on a Kratos MS50 apparatus. High-resolution
electrospray mass spectroscopy was performed on a Micromass
LCT time-of-flight MS. MALDI time-of-flight mass spectroscopy
flash-chromatographed on
a
silica column with 1:1
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J. A. H. Inkster et al.
Figure 2. Co-injection of [18F]-1 with 19F synthetic standard (HPLC B). Top: UV (260 nm). Bottom: Rad.
hexanes:ethyl acetate to afford 326 mg (52%) of 2-dimethylamino- 147.1 [C]; 149.0 [C]. HRMS (EI) calcd. for C11H12N2O3: 220.08479
3-hydroxypyridine (4). Mp = 1281C [Lit.32 = 1281C]. 1H NMR [M+]. Found: 220.08437.
(CD3CN):d2.83 (s, 6H); 6.75 (dd, J= 7.7, 4.8Hz, 1H); 7.03 (dd,
J= 7.7, 1.5 Hz, 1H); 7.75 (dd, J= 4.8 Hz, 1.5, 1H). HRMS (EI) calcd. for 2-Fluoro-3-(hex-5-ynyloxy)pyridine (FPy5yne, [19F]-1)
C7H10N2O: 138.07931 [M+]. Found: 138.07966.
3-(Hex-5-ynyloxy)-2-nitropyridine (2, 326 mg, 1.48 mmol) in 1:1
THF:DMF (12 mL) was treated with 5.4 mL of 1 M tert-butylam-
3-(Hex-5-ynyloxy)-2-nitropyridine (2)
monium fluoride. The reaction was stirred for 24 h at 801C,
Into a dry flask containing NaH powder (107 mg, 4.46 mmol) was during which time the reaction turned dark red-orange. The
added DMF (12 mL) and the dispersion was cooled to 01C. 3- reaction was poured into water and extracted twice into ethyl
Hydroxy-2-nitropryridine (500 mg, 3.57 mmol) was added, fol- acetate. The organic portions were pooled, washed once with
lowed by 0.9 mL of 6-chloro-1-hexyne (8.02 mmol), dropwise. water and once with brine, and dried over MgSO4. Purification
The reaction was held 01C for 10 min, at room temperature for on a flash column of silica (3:2 hexanes:ethyl acetate) afforded
2 h, and finally heated at 601C for 45 h. The reaction was poured 221 mg (77%) of [19F]-1 as an oil. Bp = 69–701C. 1H NMR
into water and extracted three times into ethyl acetate. The (CDCl3):d1.72–1.78 (m, 2H); 1.92–2.00 (m, 3H); 2.29 (td, J = 6.9,
pooled organic portions were washed once with brine then 2.6 Hz, 2H); 4.07 (t, J = 6.2 Hz, 2H); 7.09 (dd, J = 7.2, 4.9 Hz, 1H);
dried over MgSO4. Purification on a flash column of silica (1:1 7.23–7.29 (m, 1H); 7.73 (d, J = 4.8 Hz, 1H). 13C NMR (CDCl3):d18.07
hexanes:ethyl acetate) afforded 589 mg (75%) of 2. 1H NMR [CH2]; 24.78 [CH2]; 27.98 [CH2]; 68.77 [CH2]; 68.81 [CH]; 83.80 [C];
(CDCl3):d1.69–1.76 (m, 2H); 1.94–2.01 (m, 3H); 2.28 (td, J = 6.9, 121.6 [JF-C = 4.3 Hz, CH]; 122.7 [JF-C = 4.4 Hz, CH]; 137.3 [JF-
2.6 Hz, 2H); 4.17 (t, J = 6.2, 2H); 7.49–7.54 (m, 2H); 8.08 (dd, J = 3.8, C = 13 Hz, CH]; 142.3 [JF-C = 26 Hz, C]; 153.9 [JF-C = 239 Hz, C]. 19F
2.0 Hz, 1H). 13C NMR (CDCl3):d17.92 [CH2]; 24.52 [CH2]; 27.67 NMR (CDCl3):d-84.38 (d). HRMS (EI) calcd. for C11H12NOF:
[CH2]; 68.93 [CH2]; 69.20 [CH]; 123.3 [CH]; 128.5 [CH]; 139.0 [CH]; 193.09029 [M+]. Found: 193.09061.
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Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 444–452

J. A. H. Inkster et al.
Figure 3. Preparative HPLC trace (HPLC D) of 18F-BG142 reaction mixture [20 mM sodium phosphate (pH 8.5):DMF 2:1, 371C, 10 min]. Top: UV (280 nm) BG142
Rt = 17.455 min. Bottom: Rad. 18F-BG142 Rt = 19.545 min. [18F]-1 Rt = 29.977 min.
2-Dimethylamino-3-(hex-5-ynyloxy)pyridine (5)
[3-(Hex-5-ynyloxy)pyridin-2-yl]trimethylammonium trifluor-
omethanesulfonate (3)
To a slurry of 95% sodium hydride (81.2 mg, 3.38 mmol) in dry
DMF at 01C was added 2-dimethylamino-3-hydroxypyridine
(4, 286 mg, 2.07 mmol). The reaction was kept at this tempera-
ture for 20 min, then 6-chloro-1-hexyne (0.5 mL, 4.46 mmol)
was added dropwise via syringe. The reaction was allowed to
warm to room temperature over 30 min, then heated to 701C
for 22 h. The mixture was poured into water and extracted
three times into ethyl acetate, and then the organic portions
were combined and washed once with water and once
with brine. The extract was concentrated and purified on a
flash column of silica gel (1:1 hexanes:ethyl acetate) to afford
A solution of 5 (196 mg, 898 mmol) in dry toluene (3 mL) was cooled
to 01C and methyl trifluoromethanesulfonate (101 mL, 908 mmol) was
added via syringe. The reaction was stirred at 01C until fully mixed,
then allowed to warm to room temperature over 75 min. The
reaction was quenched with 5 mL of hexanes and quickly filtered.
The resulting off-white precipitate (3, 327mg, 95%) was washed
with two 5 mL portions of hexanes and dried en vacuo.
1
Mp = 66–671C. H NMR (CD2Cl2):d1.70–1.77 (m, 2H); 2.07–2.11 (m,
3H); 2.33 (td, J=6.8, 2.6Hz, 2H); 3.72 (s, 9H); 4.31 (t, J=6.5Hz, 2H)
7.62 (dd, J=8.3, 4.3Hz, 1H); 7.66 (dd, J= 8.3, 1.5 Hz, 1H); 8.11 (dd,
J=4.3, 1.6Hz, 1H). 13C NMR (CD2Cl2):d18.49 [CH2]; 25.56 [CH2]; 28.29
[CH2]; 54.78 [CH3]; 69.66 [CH2]; 70.48 [CH]; 83.99 [C]; 121.5 [q,
J=321Hz, CF3]; 124.8 [CH]; 129.2 [CH]; 139.4 [CH]; 142.9 [C]; 147.8 [C].
HRMS (ESI) calcd. for C14H21N2O: 233.1654 [M+]. Found: 233.1655.
330 mg (73%) of
5 as a
light yellow oil. 1H NMR
(CD2Cl2):d1.73–1.77 (m, 2H); 1.91–1.98 (m, 2H); 2.00 (t,
J = 2.7 Hz, 1H) 2.29 (td, J = 7.0, 2.6 Hz, 2H); 2.98 (s, 6 H); 3.98 (t,
J = 6.3 Hz, 2H); 6.70 (dd, J = 7.8, 4.9 Hz, 1H); 7.00 (dd, J = 7.8,
1.1 Hz, 1H); 7.77 (dd, J = 4.9, 1.4 Hz, 1H). 13C NMR (CD2Cl2):d18.45
[CH2]; 25.69 [CH2]; 28.70 [CH2]; 40.99 [CH3]; 68.13 [CH2]; 68.78
[CH]; 84.36 [C]; 115.5 [CH]; 118.5 [CH]; 138.7 [CH]; 146.0 [C]; 153.3
Preparation of azide-modified peptide N3–(CH2)4–CO–Tyr–
Lys–Arg–Ile–OH (BG142)
[C]. HRMS (EI) calcd. for C13H18N2O: 218.14191 [M+]. Found: The peptide BG142 was synthesized by continuous flow
method on a Pioneer Peptide Synthesis System using 1 g of
218.14217.
J. Label Compd. Radiopharm 2008, 51 444–452
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J. A. H. Inkster et al.
Figure 4. Co-injection of 18F-BG142 with 19F synthetic standard (HPLC C). Top: UV (280 nm). Bottom: Rad.
N-a-Fmoc-Ile-NovaSyns TGA resin with a substitution rate of was removed by filtration and washed with TFA. Combined
0.27 mmol/g. Fmoc deprotection was performed in 20% filtrates were added dropwise to ethyl ether (1 mL of TFA/10 mL
piperidine in DMF and monitored through UV detection at of ether). The precipitated peptide was centrifuged at 1200 rpm
364 nm. A two-fold excess of Fmoc-protected amino acids over for 15 min and the ether solution was decanted. The crude
resin substitution rate was used for coupling. Synthesis was peptide was dissolved in water and purified by Flash chromato-
performed using amine-free DMF. Fmoc-protected amino acids graphy on a Biotage SP4 system (Charlottesville, VA) using a
are activated for coupling with an equimolar amount of HATU, FLASH+s C18 Cartridge 25+M eluting with a linear gradient of
and 2 equivalents of DIEA. After tyrosine Fmoc deprotection, a 0–60% of acetonitrile in water for ten volumes of column. The
two-fold excess of 5-azidovalerate and 6-chloro-1-hydroxyben- peak corresponding to the desired peptide was collected,
zotriazole in solution in DMSO were added to the partially checked by HPLC (HPLC A) for purity prior to combination of
protected peptide on resin. Mechanical agitation was main- collected fractions and lyophilization to give 58 mg (31%) of
tained for 24 h at room temperature. The Fmoc deprotection BG142 as a white solid. Purity by HPLC was 97%; MALDI-TOF: m/
and the coupling steps were followed by a Kaiser’s test on resin; z 705.4 ([MH+H]+, 100%).
and the resulting colorimetric reaction between resin and
ninhydrin indicated the presence of free primary amines after
Fmoc deprotection (blue beads) and their absence after
Cold peptide bioconjugation (19F-BG142)
coupling (yellow beads). After coupling, the resin was washed
with DMF (3 Â 10 mL), MeOH (3 Â 10 mL), DMF (3 Â 10 mL),
MeOH (3 Â 10 mL), and DCM (3 Â 10 mL). The azido peptide
BG142 was deprotected and cleaved from the polymer support
by treatment with a cocktail of TFA:H2O:thioanisole (92:2:6 v/v)
for 4 h at room temperature with mechanical agitation. The resin
Peptide BG142 (1.0 mg, 1.42 mmol) was dissolved in 200 mL of
phosphate buffered saline (PBS, 75 mM, pH 7.2) and added to a
screw-cap plastic vial (2 mL) containing [19F]-1 (2.7 mg,
14.0 mmol, 10 equiv.) in 50 mL DMF. Into a separate vial
Cu(CH3CN)4PF6 (5.3 mg, 14.2 mmol, 10 equiv.) and TBTA
(7.9 mg, 14.9 mmol, 10.5 equiv.) were mixed together as powders
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Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 444–452

J. A. H. Inkster et al.
Preparation of 18F-labeled peptide [18F]-BG142
and dissolved in 50 mL DMF. This freshly prepared solution was
added to the peptide and the reaction mixture was shaken
vigorously for 3½ h. After this time, the reaction was centrifuged
to remove undissolved materials and the decanted solution
containing 19F-BG142 was purified by semi-preparative HPLC
(HPLC C) and concentrated in vacuo. Yield by HPLC was 96%.
MALDI-TOF: m/z 1211.5 ([M]+, 100%).
A solution containing 30.0 mCi of prosthetic label [18F]-1 in
methylene chloride was placed under a stream of helium and
carefully dried down at 501C. To this residue was added a 50 mL
aliquot of CuI-TBTA complex (5.3 equiv.) in DMF. This solution
was prepared immediately before addition to the reaction
mixture and had a concentration of 56 mg/mL Cu(CH3CN)4PF6
and 80 mg/mL TBTA. DIEA was then added (2 mL, 8.1 equiv.),
followed by peptide BG142 (1.0 mg, 1.4 mmol, 1 equiv.) in 100 mL
of sodium phosphate buffer (20 mM, pH 8.5). The reaction was
heated at 371C for 10 min, then diluted with 400 mL of 1:1
acetonitrile:water and purified by semi-preparative HPLC (HPLC
D). The collected eluent was evaporated azeotropically following
the addition of 2 Â 1 mL of acetonitrile, then formulated in
600 mL PBS (150 mM, pH 7.2). Collected material (5.65 mCi)
represents a non-decay-corrected yield of 5.8% from end-of-
bombardment (18.7% decay-corrected).
Radiosynthesis
2-[18F]Fluoro-3-(hex-5-ynyloxy)pyridine ([18F]FPy5yne, [18F]-1)
From precursor
3
in DMSO: [18F]fluoride (65.9 mCi) was
immobilized on an ion exchange 18F trap and release column
(ORTG, Inc.), then eluted into a 5 mL conical vial with a
1 mL:0.3 mL MeCN:H2O solution of Kryptofix 222 (14.1 mg,
37.5 mmol) and potassium carbonate (3.4 mg, 24.6 mmol). The
solution was evaporated to dryness under a stream of helium
(180 mL/min) at 1101C. Residual water was removed by
azeotropic distillation with acetonitrile (2 Â 1 mL). To this
residue was added precursor 3 (3.5 mg, 9.2 mmol) in 0.7 mL
DMSO, and the reaction was heated at 1101C for 15 min. Extent
of 18F incorporation was assayed by means of thin layer
radiochromatography (SiO2, ethyl acetate as eluent). Rf: 0.72.
Afterwards, the solution was cooled to room temperature in a
water bath, then diluted with 17 mL of H2O and loaded onto a
Waters Sep-Pak tC18 cartridge (400 mg, activated previously
with 3 mL MeOH and 10 mL water). The column was washed
with 2 Â 5 mL of water and partially dried with a syringe filled
with 5 mL of air. [18F]-1 (30.0 mCi) was eluted off of the column
with 1 mL of acetonitrile. This solution was purified by
semi-preparative HPLC (HPLC B). The collected eluent was
poured into 20 mL of H2O above a tC18 Sep-pak cartridge
(activated as above) and the mixture was diluted to 50 mL total
with H2O, then loaded onto the column. The cartridge was then
washed with 5 mL H2O and partially dried using He flow
(180 mL/min) for 3 min. Purified [18F]-1 (15.8 mCi) was eluted off
of the column with 1 mL of acetonitrile. Non-decay-corrected,
collected yield was 24% (42% decay-corrected) from start of
synthesis
Conclusion
We prepared bifunctional 18F-bearing prosthetic molecule
[18F]FPy5yne ([18F]-1) from precursor molecules 3-(hex-5-yny-
loxy)-2-nitropyridine (2) and [3-(hex-5-ynyloxy)pyridin-2-yl]tri-
methylammonium trifluoromethanesulfonate (3) by way of
nucleophilic heteroaromatic [18F]fluorination at the 2-position
of the pyridine ring. Both precursors afforded the desired
molecule in a single, high yielding radiochemical step, but
reaction mixtures starting from 3 proved easier to purify by
HPLC. Excellent radiochemical yields of [18F]FPy5yne from 3
(87–93%) were recorded over 15 min in either DMSO or MeCN at
1101C. As for the bioconjugation step, we incorporated a
terminal alkyne into our precursors for use in diastereospecific
Huisgen 1,3-dipolar cycloaddition ligations. Of particular im-
portance to our strategy was the chemical inertness of the
alkyne, which is tolerant of the high temperatures and basic
environments typically used to facilitate nucleophilic aromatic
[18F]fluorinations. After HPLC purification, [18F]FPy5yne was
efficiently conjugated to an azide-modified (though otherwise
unprotected) peptide sequence.
It should be noted that the advantages of this new 18F-based
prosthetic group (e.g. excellent 18F incorporation and ease of
synthesis) are mitigated somewhat by product losses suffered
during evaporation of the solution containing purified
[18F]FPy5yne. Careful drydown from a low boiling point solvent
such as methylene chloride or diethyl ether is essential.
Presumably, the volatility of [18F]-1 (bp = 69–701C) would allow
for a modified protocol where [18F]FPy5yne is concentrated by
way of distillation. However, one disadvantage of this approach
is that radiochemical distillations are often difficult to automate
and reproduce. Out of the five currently published protocols
known to us for preparing 18F-based bifunctional molecules for
use in Huisgen [3+2] cycloaddition ligations, two include
distillations,8,12 while three (including ours) do not.11,13
From
precursor
2
in
DMSO:
A
radiosynthesis
starting from 3-(hex-5-ynyloxy)-2-nitropyridine (2; 4.2 mg,
19.1 mmol) were performed in a manner very similar to the
above protocol, except the reaction temperature was set to
1201C and HPLC-purified [18F]F-1 (12.9 mCi) was eluted off a
tC18 Sep-Pak with methylene chloride (2 mL). Non-decay-
corrected, collected yield was 22% (50% decay-corrected) from
start of synthesis.
From precursor 3 in acetonitrile: Starting from 71.8 mCi,
K[18F]F-K222 complex was prepared as above. To this ‘dried’
[18F]fluoride residue, precursor 3 (3.3 mg, 8.6 mmol) was added in
0.2 mL acetonitrile, and the reaction was heated at 1101C for
15 min. The solution containing [18F]-1 was diluted with 0.5 mL
of acetonitrile and injected directly into the HPLC (HPLC B). The
collected eluent was diluted to 50 mL with H2O and trapped on
a tC18 Sep-pak cartridge (activated as above). The cartridge was
then washed with 5 mL H2O and partially dried with a flow of
helium (180 mL/min). Purified [18F]-1 (16.8 mCi) was eluted off
the column with diethyl ether (2 mL). Non-decay-corrected,
collected yield was 23% (34% decay-corrected) from start of
synthesis.
Many pharmaceuticals bearing aliphatic fluorines can be
stripped of fluorine in vivo by way of a-hydroxylation and
subsequent elimination of hydrofluoric acid. Thus, they are
typically considered more susceptible to metabolism than
fluoroaromatics.33 To our knowledge, the only other reported
azide/alkyne-based prosthetic group bearing an aromatic
[18F]fluorine is 4-[18F]fluoro-N-(prop-2-ynyl)benzamide.11 This
compound was prepared by coupling N-succinimidyl
J. Label Compd. Radiopharm 2008, 51 444–452
Copyright r 2008 John Wiley & Sons, Ltd.
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J. A. H. Inkster et al.
4-[18F]fluorobenzoate ([18F]SFB) with an excess of propargyl
amine. Bioconjugation to NT[8-13] (yield by radioHPLC = 66%)
[7] H. C. Kolb, K. B. Sharpless, Drug Discovery Today 2003, 8,
1128–1137.
resulted in
a
radiopeptide whose 19F standard showed
[8] J. Marik, J. L. Sutcliffe, Tetrahedron Lett. 2006, 47, 6681–6684.
[9] D. H. Kim, Y. S. Choe, K. H. Jung, K. H. Lee, J. Y. Choi, Y. Choi,
B. T. Kim, Arch. Pharmacal Res. 2008, 31, 587–593.
[10] U. Sirion, H. J. Kim, J. H. Lee, J. W. Seo, B. S. Lee, S. J. Lee, S. J. Oh,
D. Y. Chi, Tetrahedron Lett. 2007, 48, 3953–3957.
[11] T. Ramenda, R. Bergmann, F. Wuest, Lett. Drug Des. Discovery 2007,
4, 279–285.
[12] M. Glaser, E. Arstad, Bioconjugate Chem. 2007, 18, 989–993.
[13] Z. B. Li, Z. Wu, K. Chen, F. T. Chin, X. Chen, Bioconjugate Chem.
2007, 18, 1987–1994.
[14] L. Dolci, F. Dolle, S. Jubeau, F. Vaufrey, C. Crouzel, J. Labelled
Compd. Radiopharmaceuticals 1999, 42, 975–985.
[15] M. Karramkam, F. Hinnen, F. Vaufrey, F. Dolle, J. Labelled Compd.
Radiopharmaceuticals 2003, 46, 979–992.
[16] F. Dolle, Curr. Pharm. Des. 2005, 11, 3221–3235.
[17] B. Kuhnast, A. de Bruin, F. Hinnen, B. Tavitian, F. Dolle, Bioconjugate
Chem. 2004, 15, 617–627.
[18] R. Boisgard, B. Kuhnast, S. Vonhoff, C. Younes, F. Hinnen,
J. M. Verbavatz, B. Rousseau, J. Furste, B. Wlotzka, F. Dolle,
S. Klussmann, B. Tavitian, Eur. J. Nucl. Med. Mol. Imaging 2005, 32,
470–477.
nanomolar receptor binding in vitro. As an alternative,
[18F]FPy5yne can be prepared in a single radiochemical step
(versus four for [18F]fluoro-N-(prop-2-ynyl)benzamide) and cou-
ples efficiently (89%78.6%, n = 4) in 10 min at 371C.
The ultimate value of this new 18F prosthetic group depends
on further enquiries designed to test the effect of
a
[18F]FPy5yne-based side chain on the distribution and targeting
efficiency of biological imaging agents. To this end, experiments
designed to expand the utility of [18F]FPy5yne for the labeling
and in vivo assessment of a novel peptide sequence targeting
breast cancer are currently underway.
Acknowledgement
We are grateful for the assistance of Sarah Doran, Janet Lee,
Rosealie Moorlag, and Eric Price during radiochemical prepara-
[19] B. Kuhnast, S. Klussmann, F. Hinnen, R. Boisgard, B. Rousseau,
J. P. Furste, B. Tavitian, F. Dolle, J. Labelled Compd. Radio-
pharmaceuticals 2003, 46, 1205–1219.
[20] T. Viel, B. Kuhnast, F. Hinnen, R. Boisgard, B. Tavitian, F. Dolle,
J. Labelled Compd. Radiopharmaceuticals 2007, 50, 1159–1168.
[21] B. Kuhnast, F. Hinnen, R. Hamzavi, R. Boisgard, B. Tavitian,
P. E. Nielsen, F. Dolle, J. Labelled Compd. Radiopharmaceuticals
2005, 48, 51–61.
[22] B. Kuhnast, F. Dolle, B. Tavitian, J. Labelled Compd. Radio-
pharmaceuticals 2002, 45, 1–11.
[23] B. de Bruin, B. Kuhnast, F. Hinnen, L. Yaouancq, M. Amessou,
L. Johannes, A. Samson, R. Boisgard, B. Tavitian, F. Dolle,
Bioconjugate Chem. 2005, 16, 406–420.
¨
tions. We would like to thank Ken Buckley, Cornelia Hohr, and
the rest at TRIUMF Life Sciences Cyclotron Operations for
providing us with [18F]fluorine. We would also like to thank
Suzanne Perry (Micheal Smith Proteomics Unit, UBC) and
Morgan Hughes (SAMS Centre, University of Calgary) for
performing the MALDI-TOF cited herein, as well as the UBC
Mass Spec and NMR Facilities for their contributions. This work
was supported by a Canadian Institutes of Health Research
Operating Grant and a TRIUMF Life Science Grant.
[24] M. Kiankarimi, R. Lowe, J. R. McCarthy, J. P. Whitten, Tetrahedron
Lett. 1999, 40, 4497–4500.
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[28] H. R. Sun, S. G. DiMagno, J. Fluorine Chem. 2007, 128, 806–812.
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J. Label Compd. Radiopharm 2008, 51 444–452