The traceless Staudinger ligation with fluorine-18: A novel and versatile labeling technique for the synthesis of PET-radiotracers

Article abstract of DOI:10.1016/j.tetlet.2010.09.134

The development of rapid radiolabeling techniques under mild reaction conditions involving the short-lived positron emitter fluorine-18 remains a special challenge in organic PET chemistry. This work describes a novel and facile application of the traceless Staudinger ligation as a mild and versatile labeling method for preparation of various radiotracers labeled with fluorine-18.

Full text of DOI:10.1016/j.tetlet.2010.09.134

Tetrahedron Letters 51 (2010) 6410–6414
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
The traceless Staudinger ligation with fluorine-18: a novel and versatile
labeling technique for the synthesis of PET-radiotracers
Marc Pretze a, Frank Wuest b, Tim Peppel c, Martin Köckerling c, Constantin Mamat a,
⇑
a Institute of Radiopharmacy, Research Center Dresden-Rossendorf, PO Box 51 01 19, D-01314 Dresden, Germany
b Department of Oncologic Imaging, Cross Cancer Institute, University of Alberta, Edmonton, Alberta, Canada T6G 1Z2
c University of Rostock, Institute of Chemistry, Albert-Einstein-Str. 3a, D-18059 Rostock, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The development of rapid radiolabeling techniques under mild reaction conditions involving the short-
lived positron emitter fluorine-18 remains a special challenge in organic PET chemistry. This work
describes a novel and facile application of the traceless Staudinger ligation as a mild and versatile labeling
method for preparation of various radiotracers labeled with fluorine-18.
Received 13 August 2010
Revised 21 September 2010
Accepted 27 September 2010
Available online 7 October 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Radiolabeling
Traceless Staudinger ligation
Bioorthogonal
Fluorine-18
1. Introduction
copper(I)-catalyzed 1,2,3-triazole formation involving azides and
terminal acetylenes according to a 1,3-dipolar [3+2]cyclo-addition
Positron emission tomography (PET) and single photon emis-
sion computer tomography (SPECT) have extensively been used
for non-invasive diagnosis, staging, and therapy control of diseases
at the molecular and cellular level and have found numerous appli-
cations in the field of clinical oncology, cardiology, and neurology.1
The success of PET as the most sensitive molecular imaging
technique depends on the availability of suitable radiotracers,
which is limited by the speed and efficiency of synthetic methods.
In particular, the introduction of the short-lived positron emitter
18F (t1/2 = 109.8 min) into biopolymers like peptides, proteins,
oligonucleotides, and antibodies remains a special challenge. These
compounds usually cannot be labeled directly with 18F at high spe-
cific activity due to the required harsh reaction conditions. To cir-
cumvent this problem various prosthetic groups, also referred to as
bifunctional labeling agents, have been developed for 18F labeling
of peptides and proteins under mild conditions. Linkage of 18F-
labeled prosthetic groups to proteins can be accomplished via var-
ious bioconjugation techniques,2 including acylation, imidation,
photochemical conjugation, and thioether formation. However,
every method has advantages and limitations and further work
on the development of rapid, clean, and mild synthesis techniques
for 18F-labeled proteins is still needed.
has proved to be a particularly valuable tool for efficient 18F label-
ing of peptides. Moreover, copper salts as typically required for the
[3+2]cycloaddition between azides and alkynes proved to be cycto-
toxic4 and in contact with living systems the Cu catalyst must be
removed. Alongside other direct and indirect peptide or protein
radiolabeling methods with aluminum chelates5 or like the SiFa
technology6, the Staudinger ligation7 is another example of a pow-
erful bioconjugation approach to label molecules under mild con-
ditions and proceeds without Cu salts.
The bioorthogonal character8 of organic azides and phosphanes
as the reaction partners, and the mild reaction conditions have
made Staudinger ligations a valuable synthesis tool for the inter-
connection of molecular entities like carbohydrates, amino acids,
and proteins to give various hybrid-bioconjugates.9 Moreover,
incorporation of various labels into biomolecules, as frequently
exemplified by the use of fluorescence labels, has gained great
interest over the last years to study their behavior in complex bio-
logical systems.10
Recently, a radiolabeling method was pointed out using the
traceless Staudinger ligation where the bioactive part of the later
radiotracer is located on the phosphane moiety and the labeling
was done with 1-azido-2-[18F]fluoroethane.11 Our approach pre-
sents a facile and versatile labeling technique with a novel devel-
oped radiofluorinated phosphane which acts as labeling tool and
offers the possibility to label a wide variety of azide-functionalized
bioactive molecules as exemplified in Figure 1. Therefore, acyl-
functionalized phosphanes12 were prepared and tested as versatile
In recent years click chemistry has entered into many fields of
chemical sciences, including radiopharmaceutical chemistry.3 The
⇑
Corresponding author. Tel.: +49 351 260 2805; fax: +49 351 260 2915.
E-mail address: c.mamat@fzd.de (C. Mamat).
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.09.134

M. Pretze et al. / Tetrahedron Letters 51 (2010) 6410–6414
6411
O
( )5
radio-
label
O
( )5
substrate
N3
radio-
label
O
substrate
N
H
DMF/H2O
PPh2
Figure 1. Labeling concept using the traceless Staudinger ligation.
[
18F]fluoride containing building blocks for radiolabeling purposes
with the traceless Staudinger ligation using various organic model
azides.
2. Results and discussion
Phosphane–borane adducts13 are frequently utilized in organic
chemistry and catalysis. Advantageous, BH3 as ‘protecting group’
stabilizes phosphorus in the oxidation state +3 and prevents side
reactions like the formation of phosphonium salts within the prep-
aration of functionalized phosphanes as starting material for Stau-
dinger ligations. The borane-protected phosphanol 114 functions as
Figure 3. Molecular structure of 4c in the crystalline state (thermal ellipsoids at the
50% probability level).
key compound for the preparation of various
alized phosphane–borane derivatives. For this purpose, compound
1 was treated with corresponding -haloacyl chlorides 2a–c in the
x-haloacyl function-
x
presence of t-BuOK as base to obtain 3a–c in yields between 59%
and 96% (Fig. 2).
Conversion of bromo and chloro derivatives 3a–c into the corre-
sponding iodo compounds 4a–c as suitable derivatives for the
introduction of [19F]fluoride and/or good leaving groups was
achieved via a Finkelstein substitution with NaI. Fluorination of
the iodo derivatives 4a–c was accomplished with AgF as fluoride
source in acetonitrile for 16 h at ambient temperature to afford
the fluoro compounds 6a–c in good to high yields of 54–73%,
whereas reaction of iodides 4a–c with AgOTs resulted in the for-
mation of tosylates 5a–c (23–79% yield) with good leaving group
capacity as suitable labeling precursors for nucleophilic radiofluo-
rination with [18F]fluoride (Fig. 2).
Figure 4. Molecular structure of 6a in the crystalline state (thermal ellipsoids at the
50% probability level).
O
( )5 O
NMR investigations of compounds 3a–c, 4a–c, 5a–c, and 6a–c
confirm the presence of the BH3 group while a broad multiplet
indicative of the borane protons was observed between d = 1–2
ppm in the 1H NMR spectra. Formation of the phosphane–borane
adducts was further confirmed by the multiplet at d = +20 ppm in
the 31P NMR spectra and a signal at d = À36 ppm in the 11B NMR
spectra. In addition, single crystals from 4c and 6a were obtained
and X-ray structures determined.15 Figures 3 and 4 depict the
molecular structures of compounds 4c and 6a.
Fluorination with AgF as well as tosylation with AgOTs gave the
best yields using 4c as starting material. Thus, we decided to apply
tosylate 5c as labeling precursor for subsequent radiolabeling
experiments with no-carrier-added (n.c.a.) [18F]fluoride and 6c as
respective reference compound. However, several attempts to
O
( )5
BH3
R
MeOH:toluene 1:1,
reflux, 2 h
R
O
PPh2
PPh2
7: R = OTs (99%)
8: R = F (99%)
5c: R = OTs
6c: R = F
Figure 5. Borane deprotection via methanolysis.
incorporate [18F]fluoride into the borane-containing tosylate 5c
failed due to the formation of stable [18F]fluoroborate adducts.
The formation of B-18F bonds by the reaction of n.c.a. [18F]fluoride
with organoboron compounds is well documented in radiophar-
maceutical chemistry.16 To avoid undesired B-18F bond formation,
AgOTs,
CH3CN,
r.t., 72 h
O
( )n
BH3
TsO
O
PPh2
O
5a: n = 3 (27%)
5b: n = 4 (23%)
5c: n = 5 (79%)
O
( )n
NaI, acetone,
r.t., 72 h
BH3
BH3
I
X
( )n O
BH3
O
O
( )n
t-BuOK, r.t., 16 h
OH
PPh2
+
PPh2
X
PPh2
Cl
AgF,
O
CH3CN,
r.t., 16 h
BH3
F
3a: n = 3, X = Cl (96%)
3b: n = 4, X = Cl (59%)
3c: n = 5, X = Br (90%)
4a: n = 3
4b: n = 4
4c: n = 5
2a: n = 3, X = Cl
2b: n = 4, X = Cl
2c: n = 5, X = Br
1
( )n
O
PPh2
6a: n = 3 (54%)
6b: n = 4 (56%)
6c: n = 5 (73%)
Figure 2. Preparation of the labeling precursors 5a–c and the non-radioactive fluorinated reference compounds 6a–c.

6412
M. Pretze et al. / Tetrahedron Letters 51 (2010) 6410–6414
the borane in compound 5c had to be removed prior to the radio-
fluorination with [18F]fluoride and subsequent traceless Staudinger
ligation. Borane deprotection succeeded in nearly quantitative
yields without side reactions through treatment of compounds
5c and 6c in a methanol/toluene mixture (v/v = 1:1) at 60 °C for
2 h (Fig. 5).17 31P NMR spectra showed a singlet at approx.
d = À15 ppm which indicates successful removal of the BH3 group
in compounds 7 and 8. Commonly used amines for borane depro-
tection like piperazine or morpholine led to aminolysis of the ester
residue in phosphane 5c and 6c.
of the reaction conditions. An influence of the solvent and used
base was investigated. No radiolabeled product was found when
DMF or DMSO was used. Furthermore, the amount of labeling pre-
cursor 7 was evaluated. Best results for the preparation of [18F]8
were obtained using [18F]fluoride in the presence of TBAOH as base
in a mixture of 500 lL acetonitrile/t-BuOH (v/v = 1:4) for 10 min at
100 °C.18 The results are summarized in Table 1. Due to different
retention times of tosylate 7 (tR = 11.3 min) and fluoro compound
[
18F]8 (tR = 8.5 min) a separation via HPLC proved to be successful
(Fig. 9).
Based on these results, the subsequent traceless Staudinger
Non-radioactive (fluorine-19) compounds as references for cor-
responding 18F radiotracers were prepared via the traceless
Staudinger ligation and could be obtained by the reaction of the
6-fluorohexanoyl-containing phosphane 8 with several organic
model azides like benzyl azide (9), azidoacetic acid (10) and a
6-azido-galactose derivative 11. The ligation reaction in a DMF/
water mixture (v/v = 10:1) at 90 °C afforded the desired amides
12, 13, and 14 in excellent chemical yields of 97–99% after chro-
matographic purification (Fig. 6).
ligations of 18F-labeled phosphane [18F]8 with several model
azides 9–11 were evaluated. In a first approach we used the reac-
tion conditions (except of the solvent: acetonitrile/t-BuOH,
v/v = 1:4) from the preparation of the non-radioactive reference
compounds. Therefore, water was added to the reaction vial to ob-
tain a 10:1 (v/v) solvent/water mixture. After the addition of the
respective azide the following reaction was carried out at 90 °C
for 10 min to achieve complete conversion of [18F]8. To test milder
conditions it was further shown that at 40 °C also a complete con-
version proceeded but with an elongation of 30 min for the ligation
process. The overall synthesis time was found to be 50 min at 90 °C
and 70 min at 40 °C (end of synthesis). The resulting products
Radiofluorination of tosylate 7 gave the 18F-labeled phosphane
[
18F]8 which acts as fluorine-18 containing building block for the
subsequent traceless Staudinger ligation as depicted in Figure 7.
The radiochemical yield of [18F]8 strongly depends on the choice
[
18F]12, [18F]13, and [18F]14 were obtained in radiochemical yields
ranging from 31% to 35% under both labeling conditions (overall
yield after a two step procedure starting from [18F]FÀ, decay cor-
rected) and the formation was verified by radio-HPLC and radio-
TLC analyses showing the same retention times and Rf values as
determined for corresponding non-radioactive reference com-
pounds 12, 13, and 14. An application of other solvents like DMF
or DMSO delivered no ligation product. Presumably, the radiola-
beled phosphane [18F]8 was oxidized or destroyed during the heat-
ing. Radiosyntheses of phosphane [18F]8 with subsequent traceless
Staudinger ligation are depicted in Figure 7.
N3
O
( )5 N
9
F
H
12 (97%)
H
N
O
( )5
N3
DMF/H2O 10:1,
90°C, 60 min
F
O
O
O
H
N
10
F
PPh2
( )5
N
H
O
13 (99%)
N3
O
O
O
8
Furthermore, a strategy for the radiolabeling of biotin as an
example for bioactive molecules was adopted. Therefore, (+)-biotin
O
O
H
O
F
( )5
N
O
O
11
O
O
Table 1
O
Selected reaction conditions for the preparation of [18F]8 from 7
14 (98%)
Entry Amount of 7 (mg) Solventa
Base
RCYb of [18F]8 (%)
Figure 6. Preparation of reference compounds 12, 13, and 14 via traceless
Staudinger ligation.
1
2
3
3
12
10
CH3CN
CH3CN
CH3CN:t-BuOH
3:7
K2CO3
K2C2O4
TBAOH
2
14
18
O
( )5
4
5
6
10
10
21
CH3CN:t-BuOH
1:9
CH3CN:t-BuOH
1:4
CH3CN:t-BuOH
1:4
TBAOH
TBAOH
19
32
TsO
O
PPh2
TBAOH 65c
7
[
18F]F-, TBAOH,
O
( )5 N
18F-labeling was carried out in 500
lL of the respective solvent (mixture) for
a
18F
CH3CN:t-BuOH 1:4,
100°C, 10 min
9
10 min.
H
Radiochemical yield (RCY) of [18F]8, determined by radio-TLC, decay corrected.
b
[
18F]12
(31%)
c
Mean value from 15 runs (range: 5%).
O
90°C, 10 min
18F
or
( )5
O
O
N
H
N
40°C, 30 min
10
18F
( )5
PPh2
(65%)
H
O
8/[18F]8
CH3CN:H2O 10:1,
[
18F]8
[
18F]13
(35%)
O
O
HN NH
HN NH
60°C
O
H
H
O
( )5
O
18F
O
H
( )4 N
H
H
18/19F
( )5
( )4 N
N
O
S
N
H
11
N3
S
H
O
O
H
[
18F]14
O
15
16: (21%)
O
(31%)
[
18F]16: (12%)
Figure 7. Radiofluorination of [18F]10 and one pot radiolabeling of [18F]14, [18F]15,
and [18F]16 using the traceless Staudinger ligation.
Figure 8. Functionalized biotin compound 15 and (radio)-fluorinated biotin
compound 16/[18F]16.

M. Pretze et al. / Tetrahedron Letters 51 (2010) 6410–6414
6413
Figure 9. Radio-HPLC diagram (top) of [18F]8 (
c
-trace) and HPLC diagrams of compounds 7 and 8 (both UV trace).
was converted into an amide under Steglich conditions with
3-azidopropanol that leads to 15. The subsequent traceless
Staudinger ligation delivered the non-radioactive fluoro compound
16 in 21% yield. Radiolabeling of 15 with [18F]8 was carried out at
60 °C for 1 h in a mixture of acetonitrile/water (v/v = 10:1) and
gave [18F]16 in 12% (decay corrected) conversion over a two step
procedure (Fig. 8). Verification was done using radio-HPLC and
radio-TLC analyses.
Acknowledgments
The authors thank the Fonds der Chemischen Industrie (FCI,
Germany) for the financial support.
Supplementary data
Supplementary data (general experimental details of (radio-)
chemistry and characterization data) associated with this article
can be found, in the online version, at doi:10.1016/j.tetlet.2010.
09.134.
3. Conclusion
We developed a straightforward and convenient synthesis route
for the introduction of fluorine-18 into small organic and bioactive
molecules. Therefore, functionalized phosphanes were synthesized
and labeled with [18F]fluoride which act as starting material for the
traceless Staudinger ligation. Due to their functionalization with
good leaving groups like tosylate these phosphanes still offer the
opportunity to introduce chemical as well as radioactive labels into
various azide-functionalized (bioactive) compounds. A protection
of the central phosphorus with the BH3 group is essential for a pre-
vention of side reactions like the generation of phosphonium salts
or oxidation processes. To test the practical utility, ligation reac-
tions were carried out with good yields to introduce the 6-fluoro-
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