An efficient F-18 labeling method for PET study: Huisgen 1,3-dipolar cycloaddition of bioactive substances and F-18-labeled compounds

Article abstract of DOI:10.1016/j.tetlet.2007.04.048

The Cu(I)-catalyzed, 1,3-dipolar cycloaddition reaction was applied successfully to the synthesis of small, F-18-labeled biomolecules, and an optimal condition was developed for one-pot, two-step reaction without any interim purifications. This technique was employed in various F-18-labeled, 1,2,3-triazole syntheses with high radiochemical yield.

Full text of DOI:10.1016/j.tetlet.2007.04.048

Tetrahedron Letters 48 (2007) 3953–3957
An efficient F-18 labeling method for PET study: Huisgen
1,3-dipolar cycloaddition of bioactive substances and
F-18-labeled compounds
Uthaiwan Sirion,a, Hee Jun Kim,a, Jae Hak Lee,a Jai Woong Seo,a Byoung Se Lee,a,*
Sang Ju Lee,b Seung Jun Ohb and Dae Yoon Chia,*
aDepartment of Chemistry, Inha University, Incheon 402-751, South Korea
bDepartment of Nuclear Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul 138-736, South Korea
Received 5 February 2007; revised 10 April 2007; accepted 12 April 2007
Available online 19 April 2007
Abstract—The Cu(I)-catalyzed, 1,3-dipolar cycloaddition reaction was applied successfully to the synthesis of small, F-18-labeled
biomolecules, and an optimal condition was developed for one-pot, two-step reaction without any interim purifications. This tech-
nique was employed in various F-18-labeled, 1,2,3-triazole syntheses with high radiochemical yield.
Ó 2007 Elsevier Ltd. All rights reserved.
F-18-labeled compounds have been developed as the
most popular radiotracers for use in PET (positron
emission tomography) study, an emerging molecular
imaging technology for early-stage diagnosis of a variety
of human diseases.1 Among numerous radionuclides,
F-18 has not only superior properties such as an appro-
priate half-life (t1/2 = 110 min) and a small atomic size
similar to hydrogen, but it can also be readily produced
and incorporated into small organic compounds by
simple nucleophilic substitution reaction. However, in
contrast to small organic compounds, very mild reaction
conditions are essential for the F-18 labeling reaction of
biomolecules such as peptides, oligosaccharides, and
oligonucleotides to avoid undesired possible side
reactions.2 Typically, both [18F]fluorobenzaldehyde
([18F]FB-CHO)3 and N-succinimidyl-4-[18F]fluorobenz-
oate ([18F]SFB)4 have been utilized as prosthetic groups
for the preparation of bioconjugates by the reductive
amination or acylation with the amine functionality of
biomolecules, respectively. However, such reaction con-
ditions are susceptible to most biomolecules, affording
undesired hydrolytic bond-breaking and epimerization.
Furthermore, [18F]SFB requires multi-step synthesis
and an extended reaction time,2 which is critically disad-
vantageous for the F-18 labeling process because of its
short half-life of 110 min. Therefore, strong research
interest has focused on the development of more biolog-
ically friendly reaction conditions for the mild and rapid
preparation of F-18-incorporated biomolecules and bio-
mimetic molecules.
Recently, Sharpless et al. developed at room tempera-
ture, Huisgen 1,3-dipolar cycloaddition of azides and
alkynes using copper(I) salts,5 which is now recognized
as a representative ‘click reaction’. Since then, this reac-
tion has been widely applied to many bioorganic and
medicinal research fields,6 because it proceeds under
mild and tolerable conditions, in aqueous media, at neu-
tral pH, and at room temperature, all within a reason-
able reaction time.
Most recently, Marik and Sutcliffe adopted this reaction
for the preparation of F-18-labeled, short peptide frag-
ments, thereby demonstrating its potential use in PET
study, in which the conjugation reaction of unprotected
peptides was also tested to afford good yield and purity
based on the reaction tolerance of the click reaction to
other functional groups such as amines, alcohols, and
acids.7 As a Cu(I) catalyst, they used a CuI salt dissolved
only in acetonitrile, and not in other organic solvents or
water.8 However, considering both various substance-
dependent reaction conditions and the requirement of
the biomolecule friendly aqueous media reaction, an
*
Corresponding authors. Tel.: +82 32 876 0340; fax: +82 32 868 0340
(B.S.L.); e-mail addresses: flagon11d@inha.ac.kr; dychi@inha.ac.kr
U.S. and H.J.K. contributed equally to this work.
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.04.048

3954
U. Sirion et al. / Tetrahedron Letters 48 (2007) 3953–3957
alternative reaction system is required. Furthermore,
previous study was restricted to the use in peptides.
reaction media regardless of the water content. How-
ever, we hoped to achieve both [18F]fluorination and
1,2,3-triazole formation via a one-pot reaction without
the need for solvent change or any interim purifications.
Recently, Kim et al. reported that t-BuOH solvent
shows good reactivity and high selectivity in the
[18F]fluorination reaction, by reducing the competitive
decomposition reactions of mesylate precursors through
the formation of a weak F–H hydrogen bond.9
Therefore, we eventually chose aqueous t-BuOH as the
reaction media for [18F]fluorination, and t-BuOH/H2O
(4/1) for the second reaction.
Herein, we describe an alternative synthetic approach
for the preparation of F-18-labeled biomolecules using
‘click reaction’ with CuSO4/Na-ascorbate and several
model compounds such as small organic molecules,
sugar, amino acid, and nucleotide (Fig. 1).
Preliminary studies to find an optimal condition of 1,3-
dipolar cycloaddition for the two-step F-18 labeling pro-
cedure were performed using two CuI species in four
water-containing organic solvents, acetonitrile, DMF,
DMSO, and t-BuOH, in which 1,3-dipolar cycloaddi-
tion of 4-methoxybenzyl azide and phenyl acetylene
was employed as a model reaction (Fig. 2).
Further optimization reaction was performed by varying
the amount of CuSO4 from 2 to 50 mol % (Fig. 2) to find
the optimal concentration of catalyst in t-BuOH/H2O
(4/1) media. With regard to F-18 decay with a half-life
of 110 min, 20 mol % of CuSO4 showed a sufficient
catalysis effect within a short time.
Between two catalytic systems, CuSO4/Na-ascorbate
and CuI, the former showed better performance while
the latter yielded a 1,5-disubstituted, 1,2,3-triazole as a
by-product and the reaction proceeded slowly (for
detail, see Supplementary data). In the CuSO4/
Na-ascorbate system, aqueous DMSO was the best
We designed and synthesized four mesylates, two acetyl-
enes10 and two azides,11 as [18F]fluorination precursors
(Fig. 3). In addition, 10 counterpart acetylene or azide
compounds, including two glucoses,12 two prolines,
and two thymidines, were also prepared (for their syn-
thesis, see Supplementary data). These corresponding
fluorinated acetylene and azide compounds are ther-
mally stable and nearly nonvolatile, at the reaction tem-
perature under ambient pressure, and are therefore
suitable for one-pot, two-step synthesis.
Synthetic molecules
Oligosaccharides
Peptides
linker 2
linker 1
+
18F
N3
Oligonucleotides
Glycopeptides
lipids
CuI
18F
β+ emitter
N
N
N
Under optimal condition and with the acetylene/azide
precursors, the F-18-labeled, 1,2,3-triazole formation
reaction was examined to validate our reaction condi-
tion using acetylene mesylate 4a and O-trityl thymidinyl
azide 6 (Scheme 1).
Binding moiety
to target region
Administration
Real-time molecular imaging of living substance with PET
Figure 1. Schematic click conjugation strategy.
Aqueous [18F]fluoride ion was azeotropically heated
with TBAHCO3 (tetrabutylammonium bicarbonate)
and CH3CN to remove any water. Nucleophilic
[18F]fluorination of mesylate acetylene 4a was per-
formed in 0.4 mL of t-BuOH at 100 °C for 20 min in
the presence of TBAHCO3. The corresponding
[18F]fluorinated acetylene [18F]5 was obtained with an
yield of 90%, as determined by radio-TLC integration.
The reaction mixture was cooled to room temperature
in a water bath. Compound 6 (1.5 equiv), aqueous
Na-ascorbate (0.6 M, 50 lL), and aqueous CuSO4
(0.4 M, 50 lL) were added to the reaction mixture in
sequence to match the optimal condition (total solvent
volume and ratio, and concentration of catalysts) previ-
ously determined for the second reaction. 1,3-Dipolar
cycloaddition was monitored by radio-TLC every
minute until 5 min had elapsed. From the experimental
results, the cycloaddition reaction was almost completed
CuSO4/
Na-ascorbate
N3
+
t
-BuOH/H2O (4/1)
H3CO
1
2
rt, 1 h
N
N
3
N
H3CO
100
75
50
25
0
2 mol%
5 mol%
10 mol%
20 mol%
50 mol%
O
MsO
O
MsO
MsO
O
4a
4c
N3
O
0
10
20
30
Time (min)
40
50
60
O
MsO
N3
4b
Figure 3. Mesylate precursors.
4d
Figure 2. Time versus % conversion curve at different CuSO4/Na-
ascorbate concentrations.

U. Sirion et al. / Tetrahedron Letters 48 (2007) 3953–3957
3955
18F-
TBAHCO3,
100 oC, 20 min
O
4a
O
18F
O
OMs
O
18F]5
t-BuOH
[
O
N
HN
O
TrtO
O
O
O
aq. CuSO4
aq. Na-ascorbate
HN
TrtO
[
18F]7
O
N
+
O
rt, 10 min
N
N
N
18F
N3
6
O
Scheme 1. Synthesis of F-18-labeled, 1,2,3-triazole compound [18F]7.
at 5 min and was entirely so at 10 min (pseudo first-
order reaction, kobs = 0.607 sÀ1, t1/2 = 1.14 min). In terms
of radio-TLC determination, the overall conversion
yield was 90%, and the two-step reaction was completed
within 40 min from the end of bombardment. As shown
in Figure 4, the resulting F-18-labeled, O-trityl thymid-
inyl 1,2,3-triazole was very clearly isolated by HPLC
separation. More interestingly, no significant unlabeled
thymidinyl by-products were observed on UV detector
(254 nm) except unreacted mesylate triazole compound
(retention time = 23.0 min). This indicated that other
side reactions during the [18F]fluorination of acetylene
mesylate 4a were suppressed by the remarkable reduc-
soluble, 1,2,3-triazole compounds and copper catalyst
formed soon after the mixing of the aqueous CuSO4
and Na-ascorbate. These resulting copper species were
mostly removed by filtration with a membrane filter.
The product residue was dissolved by washing twice
with CH3CN solvent.
Following the determination of these optimal reaction
and purification conditions, we further applied this pro-
cedure to various other compounds in both protected
and unprotected forms. F-19-incorporated, 1,2,3-tri-
azole compounds were prepared in advance (for detail,
see Supplementary data) under the same reaction condi-
tion, at a yield of 66–91%. The obtained cold authentic
molecules were used to identify the F-18-labeled, 1,2,3-
triazole compounds by HPLC coinjection. As shown in
Table 1, [18F]fluorinated azides and acetylenes were pre-
pared from the corresponding mesylate precursors 4a–d
in good radio-TLC yields (85–95%) under the same
reaction conditions. However, in the second step, small,
non-polar, organic compounds [18F]8–11 needed longer
reaction times (up to 30 min) than did the other simple,
polar biomolecules [18F]7,12–16, probably due to their
low solubility in the polar solvent system. Conversion
yields were limited to 71%, even with the longer reaction
times.
tion of the basicity of the [18F]fluoride and HCO3
À
anion through a hydrogen bond with the t-BuOH
solvent. This result is consistent with our own recent
report.9 In addition, one of the goals of this research
was to perform two-step, F-18 labeling reaction in
one-pot without the need for interim work-up and puri-
fication. Our procedure enabled both [18F]fluorination
and Huisgen 1,3-dipolar cycloaddition to be conducted
in the same reaction vessel and solvent system.
Also noteworthy were the remains of the insoluble sub-
stances in the reaction mixture after the reaction com-
pletion. These remains presumably consisted of poorly
Figure 4. HPLC profile of [18F]7.

3956
U. Sirion et al. / Tetrahedron Letters 48 (2007) 3953–3957
Table 1. Various one-pot, two-step, F-18 incorporations
18F-
B
aq. CuSO4
MsO A
18F
A
C
t
-BuOH
aq. Na-ascorbate
4a-d
100 oC
Y1
rt
Y2
Compounds
Time (min) (Y1/Y2)
20/30
Radio-TLC (%) (Y1/Y2)
92/71
N
N
18F
N
O
[
18F]8
N
N
N
[
18F]9
20/30
20/30
20/30
20/10
94/73
89/100
95/72
18F
O
[
18F]10
MeO
N
N
N
O
18F
O
N
N
[
18F]11
N
MeO
O
18F
AcO
AcO
AcO
18F]12
O
[
N
89/100
N
N
O
18F
AcO
O
O
CO2Me
NBoc
18F
O
20/10
20/10
88/100
85/97
N
N
[
18F]13
N
O
HN
[
18F]7
O
O
N
TrtO
O
N
N
N
18F
HO
O
HO
O
[
18F]14
N
20/10
20/10
87/100
93/100
N
HO
N
18F
HO
O
O
O
CO2Me
NH
18F
O
N
[
18F]15
N
N
O
N
HN
[
18F]16
O
HO
O
20/10
92/100
O
N
N
N
18F
O
In contrast, 1,2,3-triazole syntheses of [18F]fluorinated
acetylene [18F]5 and polar azide compounds were quan-
titatively completed within 10 min. [18F]Fluorinated tetra-
acetyl glucosyl 1,2,3-triazole [18F]12, N-Boc-protected
prolinyl 1,2,3-triazole [18F]13, and O-trityl thymidinyl
1,2,3-triazole [18F]7 were synthesized in excellent yields
of 97–100%. The corresponding unprotected 1,2,3-tri-
azole compounds [18F]14–16 were also quantitatively
prepared under the same condition. This simple prepa-
ration of [18F]14–16 confirmed the absence of any need
to protect the heteroatom functionality in this conjuga-
tion reaction. Consequently, small biomolecules can be
covalently bonded to F-18-labeled, prosthetic molecules
in the naked form, thereby eliminating the protection
step and reducing the reaction time. This result is consis-
tent with a previous report.7

U. Sirion et al. / Tetrahedron Letters 48 (2007) 3953–3957
3957
In conclusion, the CuI-catalyzed, 1,3-dipolar cycloaddi-
tion reaction was applied successfully to the synthesis of
small, F-18-labeled, biomolecule-like compounds, and
an optimal reaction condition was developed for one-
pot, two-step reaction without interim purification. This
condition was employed in various 1,2,3-triazole synthe-
ses of F-18-labeled azides or acetylenes and their corre-
sponding azide or acetylene compounds, including
biomolecules. We expect that the reaction condition pre-
sented here will widen the application of the click reac-
tion for the preparation of F-18-labeled peptides to
various types of biomolecules.
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Acknowledgments
This work was supported by the National Research and
Development Program of MOST, Korea (2005-03184).
Supplementary data
Experimental procedures and spectral data for all com-
pounds are given in the Supplementary data. Supple-
mentary data associated with this article can be found,
in the online version, at doi:10.1016/j.tetlet.2007.04.048.
7. Marik, J.; Sutcliffe, J. L. Tetrahedron Lett. 2006, 47, 6681–
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