One-step radiosynthesis of 4-[18F]flouro-3-nitro-N-2-propyn-1- yl-benzamide ([18F]FNPB): A new stable aromatic porosthetic group for efficient labeling of peptides with fluorine-18

Article abstract of DOI:10.1002/jlcr.2931

4-[¹⁸F]flouro-3-nitro-N-2-propyn-1-yl-benzamide ([ ¹⁸F]FNPB) was developed as a new stable aromatic prosthetic group for more efficient click labeling of peptides. A new aromatic precursor 3,4-dinitro-N-2-propyn-1-yl-benzamide was radiofluorinated using [ ¹⁸F]KF/K2.2.2 followed by HPLC purification to obtain the desired product [¹⁸F]FNPB. [¹⁸F]FNPB was synthesized with a 58% radiochemical yield, a specific activity > 350 GBq/μmol, and radiochemical purity was exceeded 98% in 40 min. The in vitro stability studies showed no detectable radiodefluorination over 2 h in mouse plasma. The click labeling yield of three different peptides with [¹⁸F]FNPB were all above 87%. The in vitro study suggests that [¹⁸F]FNPB may be stable in vivo and could have general application in labeling peptides with high radiochemical yield for positron emission tomography applications. Copyright

Full text of DOI:10.1002/jlcr.2931

Research Article
Received 8 February 2012,
Revised 10 April 2012,
Accepted 16 April 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.2931
One-step radiosynthesis of 4-[18F]flouro-3-
nitro-N-2-propyn-1-yl-benzamide ([18F]FNPB):
a new stable aromatic porosthetic group for
efficient labeling of peptides with fluorine-18
Yesen Li,a Yaqin Liu,a Lan Zhang,b† and Yuhong Xua,c
*
4-[18F]flouro-3-nitro-N-2-propyn-1-yl-benzamide ([18F]FNPB) was developed as a new stable aromatic prosthetic group for
more efficient click labeling of peptides. A new aromatic precursor 3,4-dinitro-N-2-propyn-1-yl-benzamide was radio-
fluorinated using [18F]KF/K2.2.2 followed by HPLC purification to obtain the desired product [18F]FNPB. [18F]FNPB was
synthesized with a 58% radiochemical yield, a specific activity > 350 GBq/mmol, and radiochemical purity was exceeded
98% in 40 min. The in vitro stability studies showed no detectable radiodefluorination over 2 h in mouse plasma. The click
labeling yield of three different peptides with [18F]FNPB were all above 87%. The in vitro study suggests that [18F]FNPB
may be stable in vivo and could have general application in labeling peptides with high radiochemical yield for positron
emission tomography applications.
Keywords: click chemistry; defluorination; peptides; [18F]FNPB
using click chemistry for labeling of radiopharmaceuticals.20 The
Copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition, also
Introduction
Positron emission tomography (PET) is one of the most
important techniques in the field of molecular imaging. With
high-resolution and sensitivity, PET can provide quantitative
referred as ‘the click reaction’, was the most commonly used
reaction aimed by those alkyne and azide prosthetic groups.21
However, in most of those designs, 18F is directly attached to an
aliphatic carbon.22–28 The structure-defluorination susceptibility
information of physiological, biochemical, and pharmacological
processes in living objects.1–4 Among all the available positron
emitting isotopes, 18F (t1/2 = 109.8 min) is the ideal and most
frequently used PET radiotracers because of its favorable nuclear
and chemical properties.5,6
relationship has not yet been fully understood,29,30 but some
aliphatic prosthetic groups were found to be susceptible to signif-
icant in vivo radiodefluorination.31,32 It is expected that prosthetic
groups on which fluorine is attached to an aromatic carbon would
be more stable in vivo than those with fluorine attached to alipha-
tic carbon because of the higher carbon-fluorine strength.33 Two
aromatic prosthetic groups for click labeling have been reported,
but the procedure involves multiple steps with radiolabeled
materials, or the radiochemical yield for the peptide labeling was
rather low.34,35 In this study, we described one-step radiosynthesis
of a new aromatic 18F prosthetic group for more efficient click
labeling of peptides with enhanced radiochemical yield.
An important application of PET imaging is in the field of
cancer diagnosis. Because many cancer cells were found to have
over expression of certain receptors that regulate their life cycles,
peptides-based ligands that target to these receptors were
developed and have become a new, promising class of
radiopharmaceuticals.
tivity with the targeting properties of peptides was extensively
described for highly specific and early tumor diagnostic and
imaging applications. But peptides are not readily amenable to
direct fluorination with no-carrier-added [18F]fluoride, direct
18F-labeling of peptides is not a commonly used method.9 Thus,
there have been many 18F-labeling strategies developed including
various prosthetic groups10 and conjugation via acylation,11,12
amidation,13 oxime formation,14,15 alkylation,16 photochemical
reaction, and so on.17 However, there are still many rooms for
improvement because most of these strategies either had poor
site-specificity or low radiochemical yield.
7,8
The combining of 18F purposive sensi-
aZhejiang-California International NanoSystems Institute, Zhejiang University,
268 Kaixuan road, Hangzhou, China
bChinese Academy of Sciences, Shanghai Institute of Applied Physics, 2019 Jialuo
Road, Shanghai, China
cMed-X Research Institute of Shanghai Jiaotong University, Shanghai Jiaotong
University, 1954 Huashan road, Shanghai, China
Because Sharpless, K. B. first coined of the term ‘click chemistry’
*Correspondence to: Xu, Yuhong, Zhejiang-California International NanoSystems
in 2001,18 the Cu (I)-catalyzed Huisgen reaction has made a signifi- Institute, Zhejiang University, 268 Kaixuan road, Hangzhou, China.
E-mail: molimg@zju.edu.cn
cant impact on bioconjugation processes; it can be performed with
†Correspondence to: Lan Zhang, Chinese Academy of Sciences, Shanghai Institute
defined site-specificity, under mild condition without protective
groups, and in high yields.19 There have been many reports on of Applied Physics, 2019 Jialuo Road, Shanghai, China.
J. Label Compd. Radiopharm 2012
Copyright © 2012 John Wiley & Sons, Ltd.

Y. Li et al.
m/z = 249.04; HRMS (high resolution mass spectrometry) calcu-
lated for C10H8N3O5 250.0464,found 250.0461.1H NMR
(400 MHz, DMSO) d 9.50 (t, J = 5.2 Hz, 1H), 8.64 (d, J = 1.5 Hz,
1H), 8.39 (dd, J = 8.4, 1.6 Hz, 1H), 8.35 (d, J = 8.4 Hz, 1H), 4.13
(dd, J = 5.4, 2.5 Hz, 2H), 3.32 (s, 1H). 13C NMR (101 MHz, DMSO)
d 162.39 (s), 143.43 (s), 141.58 (s), 138.44 (s), 133.39 (s), 126.01
(s), 124.60 (s), 80.33 (s), 73.59 (s), 28.94 (s) (IR spectra see support-
ing information Figure 1).
Materials and methods
All reagents and solvents were purchased from Acros Organics,
Sigma-Aldrich, Merck, or Sinopharm Chemical Reagent Co. All che-
micals were used as supplied unless stated otherwise. [18F]fluorine
was purchased from ShangHai Atom Kexing Pharmaceuticals Co.,
Ltd.. Nuclear magnetic resonance spectra were recorded on Bruker
Avance III 400MHz (Bruker Corporation, USA) with the correspond-
ing solvent signals as an internal standard. Chemical shift (d) are
reported in parts per million (ppm) relative to tetramethylsilane
(d = 0). Coupling constants (J) are given in Hertz (Hz). Splitting pat-
terns are defined by s (singlet), d (doublet), dd (doublet doublet), t
(triplet), m (multiplet). Mass spectra were taken on a Q-Tof Premier
Mass Spectrometer (Waters Corporation, USA). IR (infrared) spectra
were measured with a Bruker Equinox 55 FT-IR Spectrometer (Bruker
Corporation, USA). Analytical as well as semipreparative reversed-
phase high-performance liquid chromatography was performed
on an agilent 1100 system with VWD detector and Flow-Count
radio-HPLC detector system (Bioscan. INC.). Semipreparative
radio-PHLC was performed using Beckman Coulter Ods C18
Semi-Preparative Column (5 mm, 10 ꢀ 250 mm). The flow rate was
set at 3mL/min, with the mobile phase solvent A (0.1% trifluoroa-
cetic acid [TFA] in water) and solvent B (0.1% TFA in acetonitrile).
The analytical HPLC was performed using the same gradient system
but with a Beckman Coulter Ultrasphere Reversed Phase Column
(5 mm, 4.6 ꢀ 250 mm). The flow rate was set at 1 mL/min, with the
mobile phase solvent A (0.1% TFA in water) and solvent B (0.1%
TFA in acetonitrile). The UV absorbance was monitored at 214 nm.
Synthesis of 4-[19F]flouro-3-nitro-N-2-propyn-1-yl-
benzamide ([19F]FNPB, 5)
A mixture of potassium fluoride dehydrate (15 mg) and Kryptofix
[222] (30 mg) in anhydrate acetonitrile (2 mL) was heated at
100ꢂC under a steam of nitrogen for 5 min, subsequently, 2 mL
of anhydrate acetonitrile was added three times and evaporated
to dryness. A solution of 3,4-dinitro-N-2-propyn-1-yl-benzamide
(20 mg) in anhydrate acetonitrile was added to the dried mixture,
ꢂ
and the reaction mixture was heated at 100 C for 30 min. After
the reaction, the crude product was purified by semipreparative
HPLC and the solvent was removed in vacuo. The final
product 4-flouro-3-nitro-N-2-propyn-1-yl-benzamide (5.4 mg)
was obtained in 30% yield. ESI-MS: m/z = 221.1 (M-H)- for
C10H7FN2O3 calcd m/z = 222.04; HRMS calculated for C10H8N2O3F
223.0519, found 223.0514,1H NMR (400 MHz, DMSO) d 9.32
(t, J = 5.0 Hz, 1H), 8.65 (dd, J = 7.3, 2.2 Hz, 1H), 8.40–8.20 (m, 1H),
7.68 (dt, J = 84.6, 42.3 Hz, 1H), 4.10 (dd, J = 5.4, 2.5 Hz, 2H), 3.18
(t, J = 2.5 Hz, 1H). 13C NMR (101 MHz, DMSO) d 162.97 (s),
156.32 (d, J = 266.3 Hz), 135.25 (s), 130.53 (d, J = 3.8 Hz), 125.41
(s), 118.87 (d, J = 21.4 Hz), 80.69 (s), 73.28 (s), 28.75 (s). 19F NMR
(376 MHz, DMSO) d ꢃ114.74 (s).
Synthesis of 3,4-dinitro-N-2-propyn-1-yl-benzamide (4)
3,4-Dinitrobenzoic acid (212 mg, 1 mmol) was dissolved in dry
dimethylformamide (DMF), 2-propynylamine (55 mg, 1.1 mmol)
was added to the solution followed by HOBt (120 mg, 0.88 mmol)
and EDCꢁHCl (316 mg, 1.66 mmol). The resulting solution was
General procedure for the synthesis of azido acids
Synthesis of triflyl azide
stirred at room temperature for 24 h. Ethyl acetate (EA) (40 ml) Sodium azide (4 g, 61.6 mmol) was dissolved in distilled water
was added to the reaction mixture after the reaction and washed (10 mL) and then DCM (10 mL) was added. The mixture was
twice with water. The organic layer was dried with Mg2SO4, and cooled on ice bath for 20 min. Triflyl anhydride (2 mL, 12 mmol)
the solvent was removed under reduced pressure. The crude was added slowly over 5 min and the mixture was stirred for
product was purified by column chromatography on silica gel 2 h. The mixture was extracted with DCM (2 ꢀ 8 mL). The organic
(dichloromethane [DCM]/MeOH = 95:5) to give 135 mg of portions, containing the triflyl azide, were pooled, washed
the desired 3,4-dinitro-N-2-propyn-1-yl-benzamide in the form once with saturated Na2CO3 (40 mL), and used without
of yellow solid. ESI-MS: m/z = 248 (M-H)- for C10H7N3O5 calcd. further purification.
Figure 1. Radio-chromatograms and UV-chromatograms of [18F]FNPB, co-injected with the standard [19F]FNPB(column and conditions see Materials and Methods).
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J. Label Compd. Radiopharm 2012

Y. Li et al.
Synthesis of azido-Leu
temperature for 20 min, and the reaction was then quenched
and purified by semipreparative HPLC.
Leucine (786 mg, 6 mmol) was combined with K2CO3 (1189 mg,
9 mmol) and CuSO4ꢁ5H2O (15.6 mg, 60 mmol) in a distilled water
(20 mL) and methanol (MeOH, 40 mL). The triflyl azide in CH2Cl2
(DCM, 25 mL) was added and the mixture was stirred at ambient
temperature and pressure for 36 h. Subsequently, the organic
solvents were removed under reduced pressure and the
Synthesis of [19F]fluoro-triazole-7
The [19F]fluoro-triazole-7 was obtained as white powder
(9 mg, 69%). MS m/z: [M + H] + = 934.9 for C41H64FN13O11 calcd
m/z = 933.48.
aqueous slurry was diluted with water (100 mL). This was Synthesis of [19F]fluoro-triazole-8
acidified to pH 6 with conc. HCl and diluted with phosphate buffer
The [19F]fluoro-triazole-8 was obtained as white powder
(250mM, pH 6.2, 100 mL) and extracted with EA (4ꢀ 20 mL) to
remove sulfonamide by-product. The aqueous phase was then
acidified to pH 2 with conc. HCl. The product was obtained from
another round of EA extractions (3ꢀ 20 mL). These EA extracts
were combined, dried (on MgSO4), and evaporated to dryness
giving 753-mg pale yellow oils in 80% yield with no need for
further purification.36
(9 mg, 71%). MS m/z: [M + H] + = 1047.7 for C41H64FN13O11
calcd m/z = 1046.57.
Synthesis of [19F]fluoro-triazole-9
The [19F]fluoro-triazole-9 was obtained as white powder
(10 mg, 76%). MS m/z: [M + H] + = 965.5 for C41H64FN13O11
calcd m/z = 965.00.
Synthesis of 6-azidohexanoic acid
Radiochemistry
6-Azidohexanoic acid was synthesized according to the literature
procedure with slight modification37; briefly, sodium azide was
added to a solution of 6-bromohexanoic acid (1.5 g, 7.7 mmol)
in DMF (5 mL), and the mixture was stirred and heated at 85ꢂC
for 18 h. The crude reaction mixture was diluted in DCM, and this
solution washed with 0.1 N aq HCl. The organic layer was dried
over anhydrous MgSO4, filtered, and concentrated under
reduced pressure to give 6-azidohexanoic acid (1.0 g, 82%) as
an oil, which was used without further purification.
Synthesis of [18]FNPB
No-carrier-added [18F]fluoride was produced by proton
bombardment of enriched 18O-water via the 18O(p, n)18F
reaction, and it was then trapped on an SEP-PAK LIGHT QMA
(Waters). The [18F]fluoride was eluted from the cartridge into a
reaction vessel using a solution of K2.2.2./K2CO3 solution (2-mL
96/4 MeCN/water, 9.5-mg K2.2.2., 1.7-mg K2CO3). The elute was
dried at 100ꢂC under a gentle stream of nitrogen gas. Then
compound 4 in anhydrate acetonitrile (1 mL) was added,
and the mixture was heated at 90ꢂC for 10 min. The reaction
was then quenched and purified by semipreparative HPLC
using a gradient of 10 ! 60% solvent B over 20 min. The
retention time of 18.6 min peak was collected and evaporated
to dryness for the next click labeling step. Co-injection was
performed by analytical HPLC using a gradient of 10 ! 50%
solvent B over 10 min.
General procedure for the synthesis of peptides
Peptides were synthesized using standard solid-phase peptide
synthesis strategy on resin (standard coupling procedure: 5
equiv amino acid, 5 equiv HBTU, 6 equiv HOBt, 6 equiv DIPEA;
Fmoc-cleavage: 20% piperidine in DMF). The peptide
was cleaved from the resin by incubation with a mixture of
TFA/triisopropylsilane/H2O (94:3:3) for 2 h, precipitated in diethyl
ether, and purified by semipreparative HPLC.
In vitro stability studies
Synthesis of azido-LARLLT (7)
The stability of [18]FNPB was investigated in mouse plasma at var-
ious incubation times (0 min, 30 min, 60 min, and 120 min) and
37oC. PBS was used as a control. After incubation, plasma proteins
were precipitated with acetonitrile and centrifuged for 10 min at
16500 rpm and 4oC. The PBS (Phosphate buffered saline) control
was diluted with the same volume of acetonitrile. The superna-
tants and the PBS control were analyzed by analytic HPLC.
Azido-leucine-alanine-arginine-leucine-leucine-threonine
(azido-LARLLT) was obtained as white powder. ESI-MS: m/z = 712.3
(M + H) + for C31H57N11O8 calcd m/z = 711.4.
acid
Synthesis of 6-azidohexanoic acid-LARLLT (8)
6-Azidohexanoic acid conjugated-leucine-alanine-arginine-leucine-
leucine-threonine acid (6-azidohexanoic acid-LARLLT) was obtained
as white powder. ESI-MS: m/z =825.8 (M+H)+for C31H57N11O8 calcd
m/z =825.0.
General procedure for the synthesis of
[
18F]fluoro-triazole-peptides
A total of 0.5-mg peptide was added to the reactor vial, followed
by a solution of [18F]FNPB in MeOH/H2O (50:50, 1 mL). Copper
sulfate and sodium ascorbate were added to the aforemen-
tioned solution, and the mixture was stirred vigorously at room
temperature for 15 min. The reaction was then quenched and
analyzed by HPLC.
Synthesis of 6-azidohexanoic acid-cRGDfK (9)
6-Azidohexanoic acid conjugated-cyclic arginine-glycine-aspartic-
phenylalanine-lysine acid (6-azidohexanoic acid-cRGDfK) was
obtained as white powder. ESI-MS: m/z = 743.8 (M+ H)+ for
C31H57N11O8 calcd m/z = 742.8.
General procedure for the synthesis of
[
Result and discussion
19F]fluoro-triazole-peptide
We first tried to use 4-fluorophenylacetylene as a click labeling pros-
To a solution of 10-mg peptide and 3.4-mg [19]FNPB in MeOH/ thetic group, but our attempts to convert 4-nitrophenylacetylene
H2O(1:1, 1600 mL), 1.05 mg CuSO4 and sodium 2.52 mg ascorbate into 4-[18F]fluorophenylacetylene using [18F]KF/K2.2.2. resulted in
was added. The mixture was stirred vigorously at room no radiolabeled product. In order to improve the fluoride
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Y. Li et al.
substitution efficiency, we studied the reports by Sun and DiMagno; condition of 90ꢂC and 10 min. It should be noted that there
they suggested that the aromatic ring should bear at least one would be some lose during the HPLC purification, in a typical
electron-withdrawing group that is more potent than the ester experiment, 166 MBq of [18F]FNPB with a specific activity > 350
group in order for the substitution of nitro group by fluoride.38,39 GBq/mmol could be obtained from 370 MBq [18F]fluoride in
Therefore, we designed compound 4 as a precursor for 18F-labeling. about 40 min. The stability of [18F]FNPB was investigated in
As shown in Scheme 1, compound 4 was obtained by using acyla- mouse plasma at 37ꢂC and analyzed by radio-HPLC. Plasma
tion of 3,4-dinitrobenzoic acid with 2-propynylamine in 63% yield.
studies with [18F]FNPB revealed no detectable radiodefluorination
Cold labeling experiments by using [19F]KF/K2.2.2 indicated over 2 h.
that there would be two reaction products: [19F]FNPB and
The [18F]FNPB precursor obtained was then used to label pep-
3-[19F]fluoro-4-nitro-N-2-propyn-1-yl-benzamide (6). But we can tide probes containing the azide group. We here explored the
control the yield of the undesirable byproduct compound 6 by radiolabeling of the D4 peptide (Leu-Ala-Arg-Leu-Leu-Thr) and
lowering the reaction temperature.
For the radiolabeling experiment according to Scheme 1, ligand, and cRGDfK is a widely used ligand for anb3 integrin.40,41
the resulted [18F]FNPB was purified by semipreparative HPLC.
We modified the peptides with azido-Leu or 6-Azidohexanoic
cRGDfK. The D4 peptide was identified as an EGFR targeting
The fraction that peaked at around 18.6 min was identified as acid to facilitate click labeling. The azido-peptides were synthe-
the desirable product based on its co-injection with the 19F sized by standard solid-phase peptide synthesis strategy on resin
reference compound (Figure 1). They were collected and dried (Scheme 2).
for the next click labeling step. The radiochemical yields as well
as the radiochemical purity were reported in Table 1.
The click labeling of the peptides with [19F]FNPB was evalu-
ated using Cu2+/sodium ascorbate as catalysts, and the resulted
With the increase of reaction temperature, the yield of 18F- 19F-peptides were purified by HPLC and confirmed by MS. For
labeling would improve (Table 1). But because we found that the radiolabeling experiments using [18F]FNPB (Table 2), the
at temperatures above 100ꢂC, the yield of the byproduct 6 reaction was performed in methanol and water (50:50) at room
would also increase dramatically. So we select the reaction temperature for 15 min under vigorous mixing. The reaction
products were again purified by the semipreparative HPLC to
obtain 18F-labeled peptide probes.
The quantity of catalysts was optimized. We observed that
the reaction time could be dramatically shortened and the
yield increased in the presence of higher concentration of
Cu2+. Figure 2 plotted the effect of Cu2+ concentration on
the reaction yield. At low Cu2+ concentrations (≤0.1 equiv.),
there was hardly any click labeling product found. At high
Cu2+ concentrations (~0.6 equiv of CuSO4), the radiolabeling
yield was 88% within 15 min.
The radiolabeling yield of different peptide structures (8 and 9)
using the same procedure all resulted in high yield of close to
90% (Table 2). The high radiolabeling efficiency was quite signifi-
cant as compared with about 37% reported by Vaidyanathan
G.35 In addition, we did not observed the crucial effect of peptide
concentration as reported by many other studies.4,35 Although a
concentration of 12.5mg/mL is in the range typically used for
peptide labeling reactions with [18F]SFB as the most commonly
employed prosthetic group, we could obtain as high as 80%
radiochemical yield using a relatively low peptide concentration
of 0.5mg/mL. The final 18F-labeled peptides can be obtained after
semipreparative HPLC (see supporting information Figure 2, 3, 4).
Scheme 1. Synthesis of FNPB. Reagents and conditions: (a) [18/19F]KF, Kryptofix,
90ꢂC, 10 min.; (b) 2-Propynylamine, HOBt, EDC.HCl, rt., 24 h.
Table 1. Different reaction temperatures of 4-[18F]flouro-3-
nitro-N-2-propyn-1-yl-benzamide were studied
Temperature
Yield (RCY)1 (%)
RCP2 (%)
85
90
95
100
67.3
82.5
83.7
88.6
98
98
98
98
N ≥ 3
1The radiochemical yield was determined on the basis of the
HPLC analysis.
2Radiochemical purity was analyzed by radio-HPLC.
Scheme 2. The structure of the different peptides
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J. Label Compd. Radiopharm 2012

Y. Li et al.
Table 2. Click labeling of different azido-peptides with [18F]FNPB
Entry
Azido-peptide
Product
Yield (%)2
1
2
3
7
8
9
[18F]fluoro-triazole-7
[18F]fluoro-triazole-8
[18F]fluoro-triazole-9
93
92
87
1Reaction conditions: 0.5-mg peptide, [18F]FNPB, CuSO4ꢁ5H2O (0.6 equiv), sodium ascorbate (1.8 equiv), Methanol/
H2O = 50:50, rt., 15 min.
2The yield was determined on the basis of the HPLC analysis.
Conflict of Interest
The authors declare that they have no conflict of interest.
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We will be looking into the in vivo stability and PET imaging
properties of these peptide probes in further studies.
The current process required HPLC purification twice, which
was somewhat time-consuming. We are trying to utilize the
quaternary ammonium salt as the prosthetic group in order to
separate the [18F]FNPB by use of SEP-PAK C18 cartridges (Waters
Corporation, USA).
Conclusions
We have developed a one-step procedure for radiosynthesis of [16] M. Glaser, H. Karlsen, M. Solbakken, J. Arukwe, F. Brady, S. K. Luthra,
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peptides via click chemistry with high radiochemical yield. With
18F attached to an aryl sp2 carbon, the structure was shown to
be stable in plasma. We hope it will have general applications
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