Automated preparation of [18F]AFP and [18F]BFP: Two novel bifunctional 18F-labeling building blocks for Huisgen-click

Article abstract of DOI:10.1016/j.jfluchem.2013.02.028

A bioorthogonal labeling approach based on the Huisgen-click reaction including the one-step radiosynthesis of two novel versatile and bifunctional labeling building blocks ([¹⁸F]AFP) [¹⁸F]12 and ([ ¹⁸F]BFP) [¹⁸F]6 with the PET radionuclide fluorine-18 is described. Optimized reaction conditions for the fully automated synthesis procedure using the TRACERlab Fx_FN module gave both piperazine derivatives [¹⁸F]6 and [¹⁸F]12 with radiochemical yields of 31 ± 9% (S.D., n = 8, d.c.) and 29 ± 5% (S.D., n = 19, d.c.), respectively, within 40 min synthesis time and high specific activities after convenient purification using silica gel cartdridges. First biological studies of both building blocks revealed a remarkable in vitro stability in rat blood as well as rat plasma over more than 60 min. Sample ligation reactions of [ ¹⁸F]6 and [¹⁸F]12 with azide and alkyne functionalized amino acid derivatives yielded the corresponding labeled triazoles in good to high RCYs. Moreover, the azide functionalized peptide 17, which is highly affine to the EphB2 receptor due to its SNEW sequence, was synthesized and successfully radiolabeled with [¹⁸F]BFP [¹⁸F]6 under relatively mild conditions yielding the corresponding triazolyl-peptide [ ¹⁸F]18.

Full text of DOI:10.1016/j.jfluchem.2013.02.028

Journal of Fluorine Chemistry 150 (2013) 25–35
Contents lists available at SciVerse ScienceDirect
Journal of Fluorine Chemistry
journal homepage: www.elsevier.com/locate/fluor
Automated preparation of [18F]AFP and [18F]BFP: Two novel
18
bifunctional F-labeling building blocks for Huisgen-click
Marc Pretze a,b, Constantin Mamat a,b,
*
a Helmholtz-Zentrum Dresden-Rossendorf, Institut fu¨r Radiopharmazeutische Krebsforschung, Bautzner Landstraße 400, D-01328 Dresden, Germany
b TU Dresden, Fachrichtung Chemie und Lebensmittelchemie, D-01062 Dresden, Germany
A R T I C L E I N F O
A B S T R A C T
Article history:
A bioorthogonal labeling approach based on the Huisgen-click reaction including the one-step
radiosynthesis of two novel versatile and bifunctional labeling building blocks ([18F]AFP) [18F]12 and
([18F]BFP) [18F]6 with the PET radionuclide fluorine-18 is described. Optimized reaction conditions for
the fully automated synthesis procedure using the TRACERlab FxFN module gave both piperazine
derivatives [18F]6 and [18F]12 with radiochemical yields of 31 ꢀ 9% (S.D., n = 8, d.c.) and 29 ꢀ 5% (S.D.,
n = 19, d.c.), respectively, within 40 min synthesis time and high specific activities after convenient
purification using silica gel cartdridges. First biological studies of both building blocks revealed a remarkable
in vitro stability in rat blood as well as rat plasma over more than 60 min. Sample ligation reactions of [18F]6
and [18F]12 with azide and alkyne functionalized amino acid derivatives yielded the corresponding labeled
triazoles in good to high RCYs. Moreover, the azide functionalized peptide 17, which is highly affine to the
EphB2 receptor due to its SNEW sequence, was synthesized and successfully radiolabeled with [18F]BFP
Received 15 January 2013
Received in revised form 25 February 2013
Accepted 28 February 2013
Available online 13 March 2013
Keywords:
Click chemistry
Bioorthogonal
Building block
Automated synthesis
Eph receptor
[
18F]6 under relatively mild conditions yielding the corresponding triazolyl-peptide [18F]18.
ß 2013 Elsevier B.V. All rights reserved.
1. Introduction
synthesis techniques for fluorine-18-labeled high molecular
weight derivatives like peptides and proteins is still needed.
To date, a wide range of small building blocks is accessible
(Fig. 1). These are specific for appropriate functional groups of the
biological active molecule in most of the cases. However, no
universally applicable and bioorthogonal building block exist, and
in this context, a labeling strategy for each peptide still needs to be
evaluated. Approaches for the radiofluorination of e.g. lysine
containing peptides with the well-established and most commonly
used building block [18F]SFB [2–5] and for cysteine containing
peptides using [18F]FBAM [6–8] as one of the maleimide based
radiolabeling building blocks were recently described in the
literature. Inappropriately, radiolabeling of peptides and proteins
in water or buffer sometimes remains sophisticated [7] due to the
high lipophilicity of aromatic building blocks like [18F]FB-CHO,
The chemoselective radiolabeling of biologically relevant high
molecular weight compounds with fluorine-18 still remains an
enormous challenge. The search and, in this regard, the develop-
ment of novel bifunctional building blocks for the mild and
convenient radiolabeling of biologically relevant biomacromole-
cules like peptides, proteins or antibodies is from considerable
interest. Normally, these high molecular weight molecules cannot
be labeled directly due to the harsh reaction conditions used for the
incorporation of the positron emitting radionuclide fluorine-18
(t1/2 = 109.8 min) with high specific activity (AS). To fix this
obstacle, various bifunctional labeling agents, also referred as
radiolabeling building blocks, have been developed for an introduc-
tion under mild conditions. Linkage of these fluorine-18 containing
building blocks to the respective molecules can be accomplished
via various bioconjugation techniques [1], including acylation,
imidation, photochemical conjugation, and thioether formation.
However, every method has its advantages and limitations,
and future work on the development of rapid, clean and mild
[
[
18F]SFB, [18F]FBnA or [18F]FBAM. Further, the selectivity of
18F]SFB is lacking because of pH dependent labeling conditions.
It is known that primary amines like lysine and arginine moieties
were labeled with [18F]SFB at the same time and, additionally,
[
18F]SFB leads to decomposition under physiological pH [9]. A
strategy for selective peptide labeling with [18F]SFB at only one of
the positions mentioned before was applied via solid phase peptide
synthesis [9].
* Corresponding author at: Helmholtz-Zentrum Dresden-Rossendorf, Institut fu¨r
Radiopharmazeutische Krebsforschung, Bautzner Landstraße 400, D-01328 Dres-
den, Germany. Tel.: +49 351 260 2805; fax: +49 351 260 3232.
E-mail address: c.mamat@hzdr.de (C. Mamat).
Besides, a single cysteine moiety in the biomolecule of interest
is mandatory for the labeling using
selectivity. Further, it is known that the Michael addition is a
[
18F]FBAM with high
0022-1139/$ – see front matter ß 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jfluchem.2013.02.028

26
M. Pretze, C. Mamat / Journal of Fluorine Chemistry 150 (2013) 25–35
O
Moreover, radiodefluorination processes of the resulting building
blocks were avoided as far as possible due to the use of the 3-
18F]fluoropropyl moiety [25] in contrast to e.g. the fluoromethyl
18F
N3
18F
N3
18F]FEA
H
[
18F]F-butyne
18F
[
[
18F
[
18F]FBnA
[
18F]FB-CHO
residue [26].
O
O
N
The access to these novel building blocks should be fast, facile
and reproducible. To the best of our knowledge, only four
publications were found describing remotely controlled prepara-
tions of building blocks for the Huisgen-click reaction [27–30]. All
these facts prompted us to evaluate an automated synthesis
procedure for [18F]AFP as well as [18F]BFP in a remotely controlled
system to provide reproducible yields and high specific activities.
Functionalized amino acids, which were aditionally applied for
the functionalization of biomolecules like peptides, were chosen as
proof of concept for radiolabeling of biologically relevant
molecules with [18F]AFP and [18F]BFP. Further, a peptide with
SNEW key sequence, which is known to be highly affine to the
EphB2 receptor, was selected and functionalized for this radi-
olabeling approach. This receptor is known to be dysregulated in
different tumor entities, and therefore, it represents a notable and
interesting target for specific tumor diagnosis [31].
O
O
H
N
18F
H
O
O
( )6
N
18F]FBAM
O
18F
O
18F
18F]SFB
[
18F]FE-TCO
[
[
Fig. 1. Selected fluorine-18 containing building blocks.
reversible process even if using [18F]FBAM [10]. There is also
evidence of the formation of E- and Z-isomers due to the oxime
group of [18F]FBAM [8]. In addition, radiolabeling using [18F]FE-
TCO yielded two regioisomers [11] as well.
Bioorthogonal labeling strategies like the tetrazine-click [11],
the traceless Staudinger Ligation [12–15] or the Huisgen-click
reaction [16–19] were evaluated in the past. Azide or alkyne
functionalized small organic building blocks were successfully
applied [20,21] for the last two mentioned ligation reactions. Due
to small differences in molar mass (e.g. in the case of [18F]FEA), a
separation of the starting peptide from the labeled product is
sometimes intricate [20]. Moreover, the concomitant volatility of
these small fluorine-18 building blocks like [18F]FEA or [18F]F-
butyne frequently results in difficult handling during the radio-
fluorination process [22].
In this paper, we report the development and preparation of
two novel and potent piperazine based building blocks 1-(3-
azidopropyl)-4-(3-fluoropropyl)piperazine ([18F]AFP) and 1-(but-
3-ynyl)-4-(3-fluoropropyl)piperazine ([18F]BFP) with beneficial
properties like a high hydrophilicity or a medium molar mass.
Both were prepared for the versatile and irreversible introduction
of fluorine-18 into alkyne or azide functionalized bioactive
macromolecules in aqueous solutions (without the use of organic
solvents) using the Cu(I)-catalyzed Huisgen [3+2]cycloaddition.
Both novel building blocks are easily amenable for this purpose
due to the convenient two-step preparation of the non-radioactive
references as well as the facile access to the precursors which was
provided in a four-step synthesis from low priced starting material.
In preliminary publications, it was demonstrated, that the
nucleophilic introduction of fluorine-18 succeeded facile and
delivered high radiochemical yields with high specific activities in
most of the cases [23,24]. Further, the chemical nature of spiro salt
precursors allows the simple and adequate separation from the
respective radiofluorinated building block by the use of RP-18 or
silica gel cartridges, purifications via HPLC are not required.
2. Results and discussion
2.1. Synthesis of precursors and reference compounds
A successful and convenient approach for the preparation and
radiofluorination of piperazine containing compounds as samples
for the labeling of pyrido[2,3-d]pyrimidones was recently pub-
lished [24]. Herein, it was demonstrated that precursors which
contain an azoniaspirononane moiety are highly suitable and
efficient for the nucleophilic introduction of [18F]fluoride in
excellent radiochemical yields (RCY). Based on these findings,
two novel spiro precursors 5 and 11 with either azide or alkyne
function were easily available in a four-step synthesis procedure
whereas non-radioactive reference compounds 6 and 12 were
prepared in two steps each starting from piperazine.
First, piperazine derivative 2 was synthesized from but-3-ynyl
p-toluenesulfonate (1) [32] in a yield of 90%. Compound 2 was then
reacted with 1-fluoro-3-iodopropane to give the first building
block 1-(but-3-ynyl)-4-(3-fluoropropyl)piperazine – BFP (6) in 79%
yield. Further, 2 was treated with 3-bromopropanol to obtain
hydroxy derivative 3 (68%) which was next tosylated with p-
toluenesulfonyl chloride to yield the open-chained piperazine 4.
Subsequently, spiro salt 5 was formed in 43% yield when 4 was
dissolved in methanol and heated at 65 8C for 2 days. The synthesis
path of 6 and corresponding spiro precursor 5 was exhibited in
Scheme 1.
N
F
N
BFP 6
1-fluoro-3-iodopropane,
Et3N, THF, 50°C, 16 h
piperazine,
THF, 60°C, 3 h
3-bromopropanol, NaI,
Et3N, THF, 50°C, 16 h
N
N
TsO
HN
HO
N
1
2
3
p-TsCl, Et3N,
CH2Cl2, r.t., 1.5 h
methanol,
65°C, 2 d
OTs
N
N
N
TsO
N
4
spiro salt 5
Scheme 1. Synthesis of spiro precursor 5 and BFP 6.

M. Pretze, C. Mamat / Journal of Fluorine Chemistry 150 (2013) 25–35
27
N
N3
F
N
AFP 12
1-fluoro-3-iodopropane,
Et3N, THF, 50°C, 16 h
piperazine,
THF, 50°C, 16 h
3-bromopropanol, NaI,
N3
Et3N, THF, 50°C, 16 h
N
N
N3
TsO
N3
HN
HO
N
7
8
9
p-TsCl, Et3N,
CH2Cl2, r.t., 16 h
OTs
methanol,
N3
N
N
N3
60°C, 6 h
N
TsO
N
spiro salt 11
10
Scheme 2. Synthesis of precursor 11 and AFP 12.
The access to the second fluorine containing building block
1-(3-azidopropyl)-4-(3-fluoropropyl)piperazine – AFP (12) and the
appropriate precursor 11 is pursuant to the synthesis of 5 and 6. In
the first step, 3-azidopropyl tosylate (7) [33] was treated with
piperazine to yield 8 (73%). Next, derivative 8 was reacted with
1-fluoro-3-iodopropane to give the non-radioactive reference 12
(74%). Further, 8 was treated with 3-bromopropanol to give
compound 9 in nearly quantitative yield. Finally, 9 was tosylated
with p-TsCl. In this case, it was not possible to isolate 10. Thus, after
removal of the solvent, the crude 10 was subsequently dissolved in
methanol and heated at 60 8C for 6 h to give the desired spiro
precursor 11 in a yield of 43% after purification via RP-18 column
chromatography followed by lyophilization (Scheme 2).
Subsequently, the reaction mixture had to be purified by means
of cartridges. Initially, different RP-18 cartridges (CHROMAFIX1
C18, Waters Sep-Pak1 Light C18, Merck LiChrolut1 RP-18,
Phenomenex Strata1 C18-T) were tested, on which the crude
[
18F]6 was trapped, washed with water to remove unreacted spiro
precursor 5 and [18F]fluoride and eluted with ethanol to obtain
pure [18F]6. All RP-18 cartridges resulted in a radiochemical purity
(RCP) ranging from 84 to 99% but in a low AS (<2 GBq/mmol). In
addition, small amounts of unreacted [18F]fluoride were found in
the product fractions using Strata1 C18-T and Sep-Pak1 Light C18
cartridges. Thus, cartridges based on silica gel filling were tested.
The reaction mixture containing
[ a
18F]6 was trapped on
LiChrolut1 Si (Merck) cartridge, washed first with acetonitrile
(3 mL) and then eluted from the cartridge (Fig. 3) with a mixture of
water and acetonitrile (2 mL, v/v = 1:1). Unreacted spiro salt 5 and
traces of [18F]fluoride remained on the cartridge. Afterwards, the
solvent was removed at 90 8C in a gentle stream of nitrogen within
4–5 min to obtain [18F]6 which is then readily available for
following labeling procedures.
2.2. Automated module syntheses of [18F]AFP and [18F]BFP
First of all, ‘‘hand preparations’’ with precursor 5 according to
previous conditions ([18F]fluoride/K 2.2.2., acetonitrile, 100 8C,
15 min) [24] were accomplished for the synthesis of [18F]6. An
[
18F]fluoride conversion of >98% was determined by radio-TLC
Based on these findings, an automated procedure was evaluated
for the preparation of [18F]6. The following radiosyntheses were
analyses.
Fig. 2. Scheme of the synthesizer module for the automated radiosyntheses of [18F]6 and [18F]12.

28
M. Pretze, C. Mamat / Journal of Fluorine Chemistry 150 (2013) 25–35
Scheme 3. Radiosyntheses of [18F]BFP [18F]6 and [18F]AFP [18F]12.
carried out in a remotely controlled synthesis module (TRACERlab
FxFN/GE Healthcare). Thus, spiro salt 5 was treated with dried
[
18F]fluoride/K 2.2.2. in anhydrous acetonitrile at 100 8C for 15 min
as demonstrated in Scheme 3.
TRACERlab FxFN was adapted in terms of program and
hardware. The scheme of the synthesizer with modifications in
the arrangement of tube lines and valves is outlined in Fig. 2. The
major modification involved the installation of a direct connection
between valve 14 and valve 17 for trapping of the reaction mixture
onto the silica gel cartridge. Noteworthy, the drying of [18F]fluoride
(approx. 20 min) is the most time consuming step. The following
reaction step proceeds within 15 min and the subsequent cleaning
step takes 3 min. All reagent solutions, solvents and reaction
mixtures within the module are transferred by nitrogen gas. The
tempering of the reactor vessel was realized by compressed air.
The remotely controlled preparation was performed in a total
synthesis time of approx. 40 min (starting from [18F]fluoride
drying). [18F]6 was obtained in 31 ꢀ 9% RCY (S.D., n = 8, d.c.) after
purification and drying and with a RCP (>98%) but with an increased
AS (7 ꢀ 4 GBq/mmol) as verified by radio-HPLC analyses (Fig. 4). The
sometimes low RCY of [18F]6 combined with the high [18F]fluoride
conversion can be explained with the loss of activity during washing,
elution and drying. Nevertheless, the setting for [18F]6 is comparable
with the synthesis of [18F]FBAM (40 min, 29% RCY, d.c.) [34]. In
contrast, [18F]SFB was prepared within 70 min with 25–38% RCY (d.c.)
[7]. Thus, a start activity of 2–6 GBq [18F]fluoride yielded 1 ꢀ 0.5 GBq
Fig. 4. Representative (radio-)HPLC chromatograms from the preparation of [18F]6
(dotted line, -trace, tR = 4.2 min), reference 6 (solid line, UV trace, tR = 4.1 min) and
g
precursor 5 (dashed line, UV trace, tR = 6.5 min).
v/v = 1:1) followed by the removal of the solvent (90 8C, N2 stream)
with a high radiochemical purity.
Using the remotely controlled synthesis procedure, [18F]12 was
obtained in a RCY of 29 ꢀ 5% (S.D., n = 19, d.c.) in a total synthesis
time of approx. 40 min (starting from [18F]fluoride drying) and with a
good RCP >97% after purification and drying as verified by radio-TLC
and radio-HPLC analyses (Fig. 6). A start activity of 1–12 GBq
[
18F]fluoride yielded 0.5–2.5 GBq [18F]12 (AS = 10 ꢀ 8 GBq/mmol).
2.3. Lipophilicity and in vitro stability tests of [18F]AFP and [18F]BFP
The liphophilicity of a pharmacologically active compound is a
fundamental physicochemical parameter and, in this regard, logP/
logD determinations are from high importance and relevance e.g.
to evaluate structure-activity relationships in medicinal chemistry
as well as in radiopharmacy [35]. Preferentially, peptide labeling
should be performed under physiological condition to avoid
decomposition, alteration or even loss of biological activity. In this
context, the liphophilicity can be directed when choosing the
respective labeling building block. For this purpose, the lipophi-
licity (logD) of [18F]6 and [18F]12 was determined at pH 7.47 using
PBS buffer and n-octanol according to the shake-flask method
[
18F]6.
The preparation and the settings of [18F]12 are similar to the
procedure of [18F]6. In this case, the reaction of spiro salt 11 with
18F]fluoride/K 2.2.2. was performed at 100 8C for 15 min in
[
anhydrous acetonitrile. Radio-TLC analyses of the reaction mixture
from ‘‘hand preparations’’ showed again more than 95% conversion
of
[ [
18F]fluoride into 18F]12 after 15 min, thus, no further
optimization was done. Subsequently, the reaction mixture was
purified using the approved LiChrolut1 Si cartridge (Fig. 5).
Unreacted spiro salt 11 and traces of [18F]fluoride remained on the
solid phase. After washing with acetonitrile (4 mL), [18F]12 was
simply eluted using a mixture of acetonitrile and water (1.5 mL,
Fig. 3. Radio-TLC (eluent: methanol) of [18F]6 after purification using a LiChrolut1
Fig. 5. Radio-TLC (eluent: methanol) of [18F]12 after purification using a LiChrolut1
Si cartridge.
Si cartridge.

M. Pretze, C. Mamat / Journal of Fluorine Chemistry 150 (2013) 25–35
29
Fig. 7. Results of in vitro stability determinations of [18F]6 in rat plasma & and in rat
blood after 20, 40 and
as well as [18F]12 in rat plasma & and in rat blood
60 min, respectively.
Fig. 6. Representative (radio-)HPLC chromatograms from the preparation of [18F]12
(dotted line, -trace, tR = 4.4 min), reference 12 (solid line, UV trace, tR = 4.2 min)
g
and precursor 11 (dashed line, UV trace, tR = 5.3 min).
temperatures (60 8C) and a longer reaction time (1 h) resulted in
a better RCY (59% d.c., starting from [18F]6) as outlined in Scheme
4. The conversion of [18F]6 into 18F-labeled amino acid [18F]14
(tR = 19.4 min) was controlled by radio-HPLC analyses (Fig. 8).
The second approach comprehends the functionalization of
biologically active molecules with an alkyne moiety. A simple way
to functionalize amino acid derivatives with alkyne residues
(OECD guideline) [36] being 0.31 ꢀ 0.03 (S.D., n = 3) for [18F]6 and
0.70 ꢀ 0.01 (S.D., n = 3) for [18F]12. On the basis of these values,
radiofluorinations are facilitated in water or buffer as well as in
organic solvents.
Next, the stability of both [18F]6 and [18F]12 was elaborated in
vitro. Therefore, a solution of [18F]6 in PBS buffer was treated with
rat plasma and rat blood (erythrocytes), respectively, and was
shaken for at least 60 min at 37 8C. Afterwards, radio-TLC as well as
radio-HPLC analyses showed no decomposition of [18F]6. The same
procedure was applied for the determination of the stability of
consists of the reaction of Fmoc-L-lysine with 4-pentynoic acid to
yield 15 [39]. As proof of concept, [18F]12 (tR = 4.4 min) was
applied for the radiofluorination of 15 (tR = 22 min) using
conditions (b). The click-labeled product [18F]16 (tR = 19.8 min)
was obtained in a RCY of 79% (d.c., starting from [18F]12) under
microwave conditions (50 watt, 1 h) (Scheme 5 and Fig. 9). For the
confirmation of [18F]16, the respective fluorine-19-containing
reference 16 was prepared by the reaction of 12 with compound 15
in a solution of DMF/THF with CuI and DIPEA for 16 h in 27% yield
according to conditions (a).
[
18F]12. Supplementary, no radiodefluorination was observed for
both novel building blocks as well. The results were outlined in
Fig. 7.
2.4. Radiolabeling using bioorthogonal Huisgen-Click
The Huisgen cycloaddition is highly attractive for the introduc-
tion of fluorine-18 into small organic compounds as well as in high
molecular weight molecules like peptides due to the bioorthogonal
character of this reaction. To investigate optimal conditions for the
radiolabeling of the azide functionalized peptides, the new
2.5. Application to peptides
The successful radiolabeling of sample amino acids prompted
us to label the azide functionalized SNEW peptide 17 as
pharmacologically relevant molecule with the alkyne containing
building block [18F]6. The SNEW key sequence is known to be
highly affine to the EphB2 receptor. In this context, it is known that
the EphB2 receptor is dysregulated in different tumor entities [31]
in correlation with e.g. tumor metastasis [40].
building block [18F]6 was first applied using N-Fmoc-
e-azido-L-
norleucine (13) [37]. Compound 13 was chosen as sample amino
acid due to its convenient availability. Furthermore, derivative 13
(and additionally 15) can be used for the functionalization of
respective SNEW peptides. Fluorine-19-containing reference 14
was prepared by the reaction of 6 with norleucine derivative 13 in a
solution of t-BuOH/H2O/acetonitrile with CuSO4 and sodium
ascorbate for 2 d in 55% yield. For the radiolabeling, solutions of
TBTA, sodium ascorbate and CuSO4 were added to a mixture of
amino acid 13 (tR = 22.4 min) in DMF and [18F]6 (tR = 4.2 min) in
Tris/HCl buffer at pH 8.0 (DMF was mandatory due to the high
lipophilicity of 13). In the beginning, the reaction was accom-
plished at ambient temperature and at 50 8C for 30 min according
to literature conditions [38]. But it was found that higher
Thus,
a
peptide with the sequence SNEWILPRLPQH was
-proline [41] at the C-terminus
functionalized with (4S)-4-azido-
L
to retain high affinity to its biological target, the EphB2 receptor,
which necessitates an unmodified N-terminus [42]. Next, the non-
radioactive reference peptide 18 was prepared by the click reaction
of 6 with peptide 17 in Sørensen phosphate buffer and ethanol for
30 min at ambient temperature using sodium ascorbate and
CuSO4ꢁ5 H2O with 98% yield. Purification was performed via
preparative HPLC and formation of 18 was confirmed using ESI-MS.
Afterwards, the conditions found for the labeling of azide
Scheme 4. Reaction of 6/[18F]6 with N-Fmoc-
e-azido-L-norleucine (13): (a) Normal conditions and (b) radiolabeling conditions.

30
M. Pretze, C. Mamat / Journal of Fluorine Chemistry 150 (2013) 25–35
Fig. 9. Representative radio-HPLC chromatograms of [18F]12 (dotted line,
g
g
-trace,
-trace,
Fig. 8. Representative radio-HPLC chromatograms of [18F]6 (dotted line,
g
g
-trace,
-trace,
[
18F]16 (solid line,
tR = 4.2 min) and of radiolabeled amino acid
tR = 19.4 min).
[
18F]14 (solid line,
tR = 4.4 min) and of radiolabeled amino acid
tR = 19.8 min).
containing amino acids were applied for the radiofluorination of
azide functionalized peptide 17. Thus, TBTA, CuSO4ꢁ5 H2O and
sodium ascorbate (6:1:10 eq) were added to a mixture of SNEW
peptide 17 (1 eq.) and [18F]6 in Sørensen phosphate buffer at pH
7.2. For optimizations, the reaction time (10, 30, 60 min) as well as
temperature (r.t., 40, 50, 60 8C) and amount of peptide precursor 17
conversion of
controlled by radio-HPLC (Figs. 10 and 11).
[ [
18F]6 into 18F-labeled peptide 18F]18 was
A separation of labeled peptide [18F]18 from starting peptide 17
was possible via HPLC, but the separation of [18F]18 from [18F]6
was not amenable due to the similar tR values (tR = 3.2 min for
[
18F]6 and 4.0 min for [18F]18) using various RP-18 columns and
(100–600
m
g) were varied. An optimal peptide amount was found
gradients. To overcome this problem, size-exclusion chromatog-
raphy (SEC) using a HighTrap1 desalting column with phosphate
buffer as eluent at a flow rate of 0.5 mL/min was applied, which
showed desirable separation properties due to the medium molar
mass (MW = 198.28 g/mol) of the introduced building block 6/
to be 0.3–0.5 mg/mL. More insoluble residues were found in the
reaction mixture when higher peptide concentrations were used,
which supposed to be an unknown triazole-copper species [43].
Best conditions were pointed out in (Scheme 6). The process of
Scheme 5. Reaction of 12/[18F]12 with lysine derivative 15: (a) Normal conditions and (b) radiolabeling conditions.
18/19F
N
N
6/[18F]6 ([18/19F]BFP)
(a) buffer/ethanol (1:1), r.t., 30 min,
CuSO4, Na-ascorbate
(b) buffer, 60°C, 1 h,
+ 17 (SNEWILPRLPQH-Azp)
TBTA, CuSO4, Na-ascorbate (6:1:10)
HN
NH2
O
O
OH
HN
O
O
NH2
HO
O
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
H2N
N
H
N
H
N
N
H
N
N
H
N
NH2
O
O
O
O
O
O
18/19F
HN
H2N
N
N
NH
18/[18F]18
N
N
N
N
Scheme 6. Synthesis pathway to the 18/19F-labeled peptide 18/[18F]18 with 6/[18F]6: (a) Normal conditions and (b) radiolabeling conditions.

M. Pretze, C. Mamat / Journal of Fluorine Chemistry 150 (2013) 25–35
31
revealed favorable properties and a high stability of both building
blocks, no radiodefluorination was observed. The 19F-references
and the appropriate precursors were easily available in two and
four preparation steps, respectively, starting from low cost starting
material. For radiofluorination purposes, Fmoc-protected amino
acid derivatives were modified and radiofluorinated using both
novel building blocks under milder reaction conditions in contrast
to harsh fluorine-18 labeling and with high RCY within the
click reaction. This procedure was successfully applied to the
radiolabeling of a SNEW peptide with high affinity to the EphB2
receptor. Due to the medium molar mass of both building blocks,
an easy separation of the 18F-peptide from the starting material
was accessible using SEC.
4. Experimental
4.1. General
Fig. 10. Representative HPLC chromatograms of SNEWILPRLPQH-Azp 17 (dotted
line, UV-trace, tR = 10.8 min) and of the reaction mixture of reference peptide 18
(solid line, UV-trace, tR = 4.1 min). Gradient: 0 min 10% ACN, 12 min 40% ACN (+0.1%
TFA).
All reagents were purchased from commercial sources and were
used without further purification. Anhydrous solvents (THF,
acetonitrile, dichloromethane) were purchased from Sigma-
Aldrich (over molecular sieves, 99.5%). 1-Fluoro-3-iodopropane
was purchased from Apollo Scientific. Starting materials 1 and 7
were prepared according to the literature [32,33]. Analytical TLCs
were performed on pre-coated Silica Gel 60 F254 plates or on F254
plates for RP-18 (Merck) and visualized under UV-light
= 254 nm) or detected by staining the plates with ninhydrin
s
(l
spray reagent (5% in ethanol) followed by heating. 1H NMR, 13C
NMR and 19F NMR spectra were recorded on a Varian Inova-400
spectrometer at 400, 101 and 376 MHz, respectively. Chemical
shifts are reported downfield from tetramethylsilane (TMS) and
trichlorofluoromethane as internal standard, respectively. MS
spectra were obtained on a Micromass Quattro-LC spectrometer
using electron spray as ionization method. Melting points were
recorded on a Cambridge Instruments Galen III apparatus and are
uncorrected. Microanalyses were carried out with a Hekatech
CHNS elemental analyser EuroEA 3000. Analytical HPLC was
performed on
a Elite LaChrom/VWR Hitachi HPLC system,
equipped with a reverse phase column (Phenomenex Aqua C18;
Fig. 11. Representative radio-HPLC chromatograms of [18F]6 (dotted line,
g-trace,
˚
250 ꢂ 4.6 mm; 100 A), a UV-diode array detector (220 nm) and a
tR = 3.2 min) and of radiolabeled peptide [18F]18 (solid line,
g-trace, tR = 4.0 min).
scintillation radiodetector (Raytest, Gabi Star). The elution was
done using a gradient of acetonitrile:water (containing 0.1% TFA)
(0–1 min: 10% ACN; 1–17 min: 10 ! 45% ACN; 17–18 min:
45 ! 90% ACN; 18–25 min: 90% ACN) unless otherwise stated at
a flow rate of 1 mL/min.
Gradient: 0 min 10% ACN, 12 min 40% ACN (+0.1% TFA).
[
18F]6. Peptide [18F]18 was obtained in 17–25% RCY (d.c., starting
from [18F]6) with >99% RCP and AS = 4 ꢀ 2 GBq/mmol (S.D., n = 3).
Additionally, the stability of [18F]18 for an application as
radiotracer was investigated in vitro by incubation using rat
plasma for 1 h at 37 8C (samples were taken after 20, 40 and
60 min). After 1 h, more than 81% of [18F]18 were found to be intact
as indicated by radio-HPLC analysis.
All radioactive compounds were identified using analytical
radio-HPLC by comparison of the retention time of the reference
compound. Decay-corrected RCYs were quantified by integration
of radioactive peaks on a radio-TLC using a radio-TLC scanner (Fuji,
BAS2000).
[
18F]Fluoride was produced utilizing the PET cyclotron Cyclone
18/9 (IBA, Belgium).
18O]H2O was irradiated with protons
(18 MeV, 30
A) exploiting the 18O(p,n)18F nuclear reaction.
3. Conclusions
[
m
The radiolabeling of peptides and proteins still remains an
outstanding challenge in radiopharmacy. Thus, the evaluation and
preparation of various labeling building blocks were found in the
literature. In general, their application highly depends on the
functional groups of the respective biomacromolecule.
4.2. General procedure for the peptide synthesis
Peptide 17 was prepared on rink amide-MBHA resin (extent of
labeling: 0.6 mmol/g loading) on a 0.05–0.10 M scale. The Fmoc
protecting group was removed with 20% piperidine in DMF for
15 min. The carboxyl group of the incoming amino acid was
activated with HBTU and HOBt. Fmoc-amino acid (5 eq), HBTU (5
eq), HOBt (5 eq), and DIPEA (10 eq) were dissolved in DMF and
NMP, respectively, and added to the resin. The coupling was carried
out twice for each residue in a coupling time of 2 h. The peptide
was deprotected and cleaved from the solid support with a mixture
of TFA/TIS/H2O 95:2.5:2.5 for 4 h at ambient temperature.
Herein, we demonstrated the evaluation of a fast and simple
automated synthesis procedures of two novel efficient and robust
building blocks 1-(3-azidopropyl)-4-(3-fluoropropyl)piperazine
([18F]AFP)
and
1-(but-3-ynyl)-4-(3-fluoropropyl)piperazine
([18F]BFP) with good RCYs and high RCPs suitable for convenient
radiofluorinations using the bioorthogonal Huisgen-click reaction.
Further, the subsequent purification from the precursor was
accomplished via cartridges. LogD determinations and in vitro tests

32
M. Pretze, C. Mamat / Journal of Fluorine Chemistry 150 (2013) 25–35
Afterwards, the resin was filtered, the crude peptide was
precipitated by adding cold diethyl ether and washed with ice-
cold diethyl ether. The residual ether was removed by evaporation
and the peptide was purified by preparative HPLC using a Varian
Dynamax Microsorb 60-8 C18 column (250 ꢂ 21.4 mm) at a flow
rate of 20 mL/min with gradients of water and acetonitrile with
0.1% TFA.
(OCH2CH2CH2), 30.0 (OCH2CH2), 24.5 (CH3), 16.9 (NCH2CH2); MS
(ESI+) m/z: 179 [M+ꢃOTs]; Anal. calcd for C18H26N2O3S (350.48): C,
61.69%; H, 7.48%; N, 7.99%; Found: C, 61.35%; H, 7.70%; N, 7.83%.
4.3.4. 7-(But-3-ynyl)-7-aza-4-azoniaspiro[3.5]nonane 4-
methylbenzene-sulfonate (5)
Compound 4 (240 mg, 0.68 mmol) dissolved in methanol
(5 mL) was stirred at 65 8C for 2 d. The solvent mixture was
concentrated and ice-cold toluene (3 mL) was added. The resulting
precipitate was filtered, washed with ice-cold toluene (10 mL) and
petroleum ether (10 mL), and the solid was dried in high vacuum to
obtain 5 as pale yellow solid (100 mg, 43%). M.p. 150–151 8C; 1H
4.3. Chemistry
4.3.1. 1-(But-3-yn-1-yl)piperazine (2)
Piperazine (7.21 g, 83.68 mmol) was dissolved in anhydrous
THF (70 mL), but-3-yn-1-yl 4-methylbenzenesulfonate (1) [32]
(5.38 g, 23.99 mmol) was added dropwise and the mixture was
stirred at 60 8C for 3 h. The resulting precipitate was filtered and
washed with THF (70 mL), the solvent was removed under reduced
pressure and the crude product was purified by column
chromatography (ethyl acetate ! methanol) to obtain compound
2 as pale yellow oil (2.98 g, 90%). Rf = 0.13 (methanol); 1H NMR
NMR (CD3CN):
d
7.60 (d, 3J = 8.1 Hz, 2H, H-o), 7.15 (d, 3J = 8.0 Hz,
2H, H-m), 4.14 (t, 3J = 8.3 Hz, 4H, H-1/3), 3.44–3.37 (m, 4H, Pip-H),
2.69–2.60 (m, 4H, Pip-H), 2.58 (t, 3J = 7.3 Hz, 2H, NCH2CH2), 2.56–
2.48 (m, 2H, H-2), 2.34 (dt, 3J = 7.5 Hz, 4J = 2.7 Hz, 2H, NCH2CH2),
2.33 (s, 3H, CH3), 2.20 (t, 3J = 2.7 Hz, 1H, CH); 13C NMR (CD3CN):
d
146.9 (C-p), 139.4 (C-i), 129.3 (C-m), 126.6 (C-o), 83.3 (Cq), 70.6
(CH), 60.2 (NCH2), 56.3 (C-1/3), 47.6 (C-5/6/8/9), 21.2 (CH3), 17.3
(NCH2CH2), 14.4 (C-2); MS (ESI+) m/z: 179 [M+ꢃOTs]; MS (ESIꢃ) m/
z: 171 [OTs]; Anal. calcd for C18H26N2O3S (350.48): C, 61.69%; H,
7.48%; N, 7.99%; Found: C 61.54%; H 7.55%; N 8.09%; Analytical
HPLC: tR = 6.5 min.
(CD3CN):
d
2.75–2.73 (m, 4H, Pip-H), 2.46 (t, 3J = 7.8 Hz, 2H,
NCH2CH2), 2.36–2.31 (m, 4H, Pip-H), 2.31 (dd, 3J = 7.8 Hz,
4J = 2.7 Hz, 2H, NCH2CH2), 2.15 (t, 4J = 2.7 Hz, 1H, CH); 13C NMR
(CD3CN):
d 84.1 (Cq), 70.1 (CH), 58.4 (NCH2CH2), 55.0 (Pip-C), 46.8
(Pip-C), 17.1 (NCH2CH2); MS (ESI+) m/z: 139 [M++H]; Anal. calcd for
C8H14N2 (138.20): C, 69.42%; H, 10.21%; N, 20.27%; Found: C,
69.30%; H, 10.17%; N, 20.83%.
4.3.5. 1-(But-3-ynyl)-4-(3-fluoropropyl)piperazine–BFP (6)
1-Fluoro-3-iodopropane (0.6 mL, 7.20 mmol) and triethyla-
mine (1.0 mL, 7.20 mmol) were added to a solution of compound
2 (1.00 g, 7.23 mmol) in anhydrous THF (10 mL). The mixture was
stirred at 50 8C for 16 h. Afterwards, the solvent was removed
under reduced pressure and the residue was purified by column
chromatography (ethyl acetate) to obtain 6 as a yellow oil (1.13 g,
4.3.2. 3-(4-(But-3-ynyl)piperazin-1-yl)propan-1-ol (3)
3-Bromopropanol (3 mL, 35.92 mmol) was added to a solution
of compound 2 (2.92 g, 21.13 mmol), NaI (320 mg, 2.11 mmol) and
triethylamine (3.5 mL, 25.36 mmol) in anhydrous THF (20 mL) and
the mixture was stirred at 50 8C for 16 h. Afterwards, the solvent
was removed under reduced pressure and the residue was
dissolved in water. The aqueous layer was extracted with
dichloromethane (3 ꢂ 15 mL), the organic layers were dried over
Na2SO4, the solvent was removed, and the residue was purified by
column chromatography (methanol) to obtain compound 3 as a
colorless solid (2.84 g, 68%). Rf = 0.42 (methanol); M.p. 94–97 8C;
79%). Rf = 0.58 (methanol); 1H NMR (CDCl3):
d 4.49 (dt,
2JH,F = 47.2 Hz, 3J = 6.0 Hz, 2H, FCH2), 2.60 (t, 3J = 5.5 Hz, 2H,
NCH2), 2.57–2.38 (m, 8H, Pip-H), 2.44 (t, 3J = 7.2 Hz, 2H, NCH2),
2.38 (dt, 3J = 7.0 Hz, 4J = 2.6 Hz, 2H, CH2), 1.97 (t, 3J = 2.7 Hz, 1H,
CH), 1.94–1.81 (m, 2H, FCH2CH2); 13C NMR (CDCl3):
d 82.9 (Cq),
1
82.7 (d, JC,F = 164.3 Hz, FCH2), 69.1 (CH), 57.1 (NCH2), 54.4 (d,
3JC,F = 5.5 Hz, NCH2), 53.2, 53.0 (2 ꢂ Pip-C), 28.8 (d, 2JC,F = 19.8 Hz,
1H NMR (CD3OD):
d
3.62 (t, 3J = 5.2 Hz, 2H, OCH2), 2.59 (t,
FCH2CH2), 16.9 (CH2); 19F NMR (CDCl3):
d–220.4; MS (ESI+) m/z:
3J = 5.8 Hz, 2H, NCH2CH2), 2.52 (t, 3J = 7.2 Hz, 2H, OCH2CH2CH2),
2.67–2.45 (m, 8H, Pip-H), 2.39 (dt, 3J = 7.5 Hz, 4J = 2.7 Hz, 2H,
NCH2CH2), 2.28 (t, 3J = 2.7 Hz, 1H, CH), 1.77–1.71 (m, 2H,
221 [M++Na]; Anal. calcd for C11H19FN2 (198.28): C, 66.63%; H,
9.66%; N, 14.13%; Found: C, 66.51%; H, 9.70%; N, 13.83%; Analytical
HPLC: tR = 4.1 min.
OCH2CH2); 13C NMR (CD3OD):
d 106.2 (Cq), 70.6 (CH), 61.7
(OCH2), 58.0 (NCH2), 56.8 (OCH2CH2CH2), 53.8 (Pip-C), 53.4 (Pip-
C), 30.0 (OCH2CH2), 17.2 (NCH2CH2); MS (ESI+) m/z: 197 [M++H];
Anal. calcd for C11H20N2O (196.29): C, 67.31%; H, 10.27%; N,
14.27%; Found: C 67.30%; H 10.01%; N 14.33%.
4.3.6. 1-(3-Azidopropyl)piperazine (8)
Piperazine (670 mg, 7.78 mmol) was dissolved in anhydrous
THF (10 mL), 3-azidopropyl 4-methylbenzenesulfonate (7) [33]
(904 mg, 3.54 mmol) was added dropwise and the mixture was
stirred at 50 8C for 16 h. The resulting precipitate was filtered and
washed with THF (10 mL), the solvent was removed under reduced
pressure and the crude product was purified either by bulb tube
distillation (100 8C, 5 ꢂ 10ꢃ2 mbar) or by column chromatography
(ethyl acetate:ethanol 1:1) to yield compound 8 as pale yellow oil
4.3.3. 3-(4-(But-3-ynyl)piperazin-1-yl)propyl 4-
methylbenzenesulfonate (4)
4-Methylbenzenesulfonyl chloride (330 mg, 1.74 mmol) was
added to a solution of compound 3 (310 mg, 1.58 mmol) and
triethylamine (0.9 mL, 6.32 mmol) dissolved in anhydrous CH2Cl2
(5 mL). The mixture was stirred at ambient temperature for 1.5 h.
Afterwards, the solvent was removed under reduced pressure at
25 8C and the residue was purified by column chromatography
(ethyl acetate:methanol 10:1 ! 6:1) to obtain 4 as pale yellow oil
(240 mg, 43%). Rf = 0.65 (methanol). Little amounts of 5 were
(325 mg, 54%). Rf = 0.14 (methanol); 1H NMR (CDCl3):
d 3.33 (t,
3J = 6.8 Hz, 3H, CH2N3), 2.88 (t, 3J = 4.9 Hz, 4H, NCH2), 2.35–2.45 (m,
6H, NCH2), 1.71–1.80 (m, 2H, NCH2CH2), 1.61 (s, 1H, NH); 13C NMR
(CDCl3):
d 56.0 (NCH2), 54.6 (Pip-NCH2), 49.8 (CH2N3), 46.2 (Pip-
NCH2), 26.2 (NCH2CH2); MS (ESI+) m/z: 170 (100) [M++H]; Anal.
calcd for C7H15N5 (169.23): C, 49.68%; H, 8.93%; N, 41.38%. Found:
C 49.77%; H 9.19%; N 41.33%.
formed during the purification; 1H NMR (CDCl3):
d 7.79 (d,
3J = 8.3 Hz, 2H, H-o), 7.34 (d, 3J = 8.0 Hz, 2H, H-m), 4.09 (t,
3J = 6.4 Hz, 2H, OCH2), 2.60 (t, 3J = 7.4 Hz, 2H, NCH2CH2), 2.72–
2.57 (m, 8H, Pip-H), 2.48 (t, 3J = 7.0 Hz, 2H, OCH2CH2CH2), 2.45 (s,
3H, CH3), 2.38 (dt, 3J = 7.8 Hz, 4J = 2.6 Hz, 2H, NCH2CH2), 1.97 (t,
3J = 2.6 Hz, 1H, CH), 1.96–1.91 (m, 2H, OCH2CH2); 13C NMR (CDCl3):
4.3.7. 3-(4-(3-Azidopropyl)piperazin-1-yl)propan-1-ol (9)
NaI (10 mg, 0.06 mmol) and triethylamine (77 mg, 0.77 mmol)
were added to a solution of compound 8 (108 mg, 0.64 mmol) in
anhydrous THF (4 mL). 3-Bromopropanol (133 mg, 0.96 mmol)
was added and the mixture was stirred at 50 8C for 16 h.
Afterwards, the solvent was removed under reduced pressure
d
130.1 (C-p), 129.7 (C-i), 128.1 (C-m), 127.2 (C-o), 82.9 (Cq), 69.2
(CH), 57.1 (OCH2), 55.6 (NCH2), 53.3 (Pip-C), 53.0 (Pip-C), 43.4

M. Pretze, C. Mamat / Journal of Fluorine Chemistry 150 (2013) 25–35
33
and the residue was dissolved in water. The aqueous layer was
extracted three times with dichloromethane and the organic layer
was dried over Na2SO4, the solvent was removed, and the residue
was purified by column chromatography (ethanol) to yield
and washed with ethyl acetate and the filtrate was purified via
column chromatography (ethyl acetate ! ethyl acetate:methanol
2:1 ! 1:2) to yield 14 as an orange oil. Yield: 310 mg (55%).
Rf = 0.35 (methanol). 1H NMR (acetone-D6, 400 MHz):
d 7.86 (d,
compound
9
as
a
colorless syrup (139 mg, 96%). Rf = 0.34
4.00 (br. s, 1H, OH), 3.67 (t,
3J = 7.5 Hz, 2H, Fmoc-H-4,5), 7.82 (s, 1H, H-triazole), 7.71 (t,
3J = 6.6 Hz, 2H, Fmoc-H-1/8), 7.41 (t, 3J = 7.4 Hz, 2H, Fmoc-H-3/6),
7.33 (t, 3J = 7.4 Hz, 2H, Fmoc-H-2/7), 6.94–6.90 (m, 1H, NH), 4.55
(dt, 3JH,F = 47.4 Hz, 3J = 5.6 Hz, 2H, FCH2), 4.42–4.33 (m, 3H, CH2O,
(methanol); 1H NMR (CDCl3):
d
3J = 5.4 Hz, 2H, OCH2), 3.30 (t, 3J = 6.9 Hz, 2H, CH2N3), 2.79–2.25 (m,
12H, Pip-H, 2ꢂ NCH2), 1.79–1.64 (m, 4H, CH2); 13C NMR (CDCl3):
d
64.7 (OCH2), 58.9 (NCH2), 55.3 (NCH2), 53.8, 53.4 (2ꢂ Pip-C), 49.7
(CH2N3), 27.2, 26.4 (2ꢂ CH2); MS (ESI+): m/z (%) 228 (100) [M++H],
143 (20) [M+ꢃC3H3N3]. Anal. calcd for C10H21N5O (227.31): C,
52.84%; H, 9.31%; N, 30.81%; Found: C, 52.55%; H, 9.33%; N, 30.54%.
H-
3.46–3.21 (m, 8H, Pip-H, CH2CH2C55), 3.13–3.03 (m, 4H, NCH2,
CH2C55), 1.95–1.84 (m, 6H, FCH2CH2, H- ), 1.42–1.37 (m, 2H, H-
). 13C NMR (acetone-D6, 101 MHz):
161.6 (O(C55O)N), 145.1
a e),
), 4.23 (t, 3J = 6.9 Hz, 1H, Fmoc-C-9), 3.80–3.76 (m, 2H, H-
b,d
g
d
(Fmoc-C-10/80), 144.2 (Fmoc-C-40/50), 142.1 (CH2C55C), 128.5
(Fmoc-C-3/4/5/6), 128.0 (Fmoc-C-2,7), 126.2 (CH2C55C), 120.8
4.3.8. 3-(4-(3-Azidopropyl)piperazin-1-yl)propyl 4-
methylbenzenesulfonate (10) and 7-(3-azidopropyl)-7-aza-4-
azoniaspiro[3.5]nonane 4-methylbenzenesulfonate (11)
(Fmoc-C-1/8), 82.2 (d, 1JC,F = 163.6 Hz, FCH2), 67.1 (CH2O), 56.6 (C-
a
3
), 53.9 (d, JC,F = 5.7 Hz, FCH2CH2CH2), 50.7 (Pip-C), 50.3 (NCH2),
4-Methylbenzenesulfonyl chloride (1.13 g, 5.94 mmol) was
added to a solution of compound 9 (900 mg, 3.96 mmol) and
Et3N (802 mg, 7.92 mmol) dissolved in anhydrous CH2Cl2 (20 mL).
The mixture was stirred at ambient temperature for 16 h.
Afterwards, the solvent was replaced by methanol (20 mL) and
the mixture was heated at 60 8C for 6 h. After cooling to r.t., the
solvent was removed under reduced pressure and the residue was
purified via RP-18 column chromatography (LiChroprep: 5–
48.1 (C-
2JC,F = 20.4 Hz, FCH2CH2), 23.3 (C-
D6, 376 MHz):
-222.0. MS (ESI+): m/z (%) 593 (40) [M++H].
e
), 32.6 (Fmoc-C-9), 29.1 (C-
b
), 28.9 (CH2C55C), 26.7 (d,
d), 22.2 (C-
g
). 19F NMR (acetone-
d
Analytical HPLC: tR = 19.3 min.
4.3.11. (S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-6-(3-(1-
(3-(4-(3-fluoropropyl)piperazin-1-yl)propyl)-1H-1,2,3-triazol-4-
yl)propanamido)hexanoic acid (16)
20
11 as colorless solid (240 mg, 43%). Rf = 0.10 (RP-TLC, methanol).
7.64 (d, 3J = 8.1 Hz, 2H, H-o),
m
m; water:methanol 8:2 ! 1:1) and lyophilization to yield
To a solution of N-Fmoc-
0.11 mmol) in a mixture of DMF (1 mL) and THF (1 mL) CuI (4.2 mg,
0.01 mmol), 12 (12.8 mg, 0.06 mmol), and DIPEA (95 L,
e-pentyneamido-L-lysine 15 (52 mg,
M.p. 215–216 8C. 1H NMR (CD3CN):
d
m
7.21 (d, 3J = 7.9 Hz, 2H, H-m), 4.26 (t, 3J = 8.2 Hz, 4H, H-1/3), 3.78–
3.67 (m, 4H, Pip-H), 3.62 (t, 3J = 6.5 Hz, 2H, CH2N3), 3.39–3.34 (m,
4H, Pip-H), 3.11–3.07 (m, 2H, NCH2), 2.55 (dt, 2J = 17.0 Hz,
3J = 8.3 Hz, 2H, H-2), 2.35 (s, 3H, CH3), 1.90–1.88 (m, 2H, NCH2CH2).
0.56 mmol) were added and the mixture was stirred 16 h at
37 8C. Then water (5 mL) was added and the product was extracted
with ethyl acetate (5 mL) twice. The organic phase was concen-
trated and purified via preparative HPLC. The product was obtained
via lyophilization as pale yellow, hygroscopic solid. Yield: 10 mg
13C NMR (CD3CN):
d 145.0 (C-p), 140.6 (C-i), 129.6 (C-m), 126.6 (C-
o), 66.3 (NCH2), 54.7 (C-1/3), 49.3 (CH2N3), 47.6 (C-5/6/8/9), 25.0
(NCH2CH2), 21.3 (CH3), 14.4 (C-2). MS (ESI+): m/z (%) 210 (100)
[M+ꢃOTs]. MS (ESIꢃ): m/z (%) 171 (100) [OTs]. Anal. calcd for
(27%). Rf = 0.51 (methanol). 1H NMR (acetone-D6, 400 MHz):
d 7.86
(d, 3J = 7.5 Hz, 2H, Fmoc-H-4/5), 7.73 (dd, 3J = 7.1 Hz, 3J = 4.0 Hz,
2H, Fmoc-H-1/8), 7.68 (s, 1H, H-triazole), 7.41 (t, 3J = 7.4 Hz, 2H,
Fmoc-H-3/6), 7.32 (t, 3J = 7.4 Hz, 2H, Fmoc-H-2/7), 7.22 (s, 1H,
C17H27N5O3S (381.49): C 53.52%; H 7.13%; N 18.36%. Found: C
3
53.50%; H 7.42%; N 18.18%. Analytical HPLC: tR = 5.3 min.
NHCO), 6.84 (d, 3J = 7.7 Hz, 1H, NHCOO), 4.54 (dt, JH,F = 47.4 Hz,
3J = 5.8 Hz, 2H, FCH2), 4.46 (t, 3J = 7.0 Hz, 2H, CH2O), 4.34 (d,
4.3.9. 1-(3-Azidopropyl)-4-(3-fluoropropyl)piperazine – AFP (12)
1-Fluoro-3-iodopropane (471 mg, 2.50 mmol) was added to a
solution of compound 8 (424 mg, 2.50 mmol) and Et3N (380 mg,
3.76 mmol) in anhydrous THF (8 mL). The mixture was stirred at
50 8C for 16 h. Thereafter, the solvent was removed under reduced
pressure and the residue was purified by column chromatography
J = 7.0 Hz, 1H, H-a), 4.26–4.19 (m, 3H, CH2N3, Fmoc-H-9), 3.26–
3.05 (m, 12H, Pip-H, 2ꢂ NCH2), 2.95 (t, 3J = 6.8 Hz, 2H, H-
e), 2.80 (t,
3J = 6.0 Hz, 2H, CH2C55), 2.51 (t, 3J = 6.9 Hz, 2H, CH2CO), 1.89–1.77
(m, 4H, H-
b
, CH2CH2F), 1.49–1.40 (m, 6H, H-
g
/
d
/CH2CH2N3). 13C
NMR (acetone-D6, 101 MHz):
d
157.2 (O(C55O)N), 145.1 (Fmoc-C-
10/80), 145.0 (Fmoc-C-40/50), 142.1 (CH2C55C), 128.5 (Fmoc-C-3/4/5/
(ethyl acetate) to yield 12 as a pale yellow syrup (424 mg, 74%).
6), 128.0 (Fmoc-C-2/7), 126.2 (CH2C55C), 120.8 (Fmoc-C-1/8), 81.4
2
1
Rf = 0.46 (methanol). 1H NMR (CDCl3):
d
4.50 (dt, JH,F = 47.3 Hz,
(d, JC,F = 163.9 Hz, FCH2), 67.2 (CH2O), 54.5 (C-
a
), 53.2 (d,
3J = 5.9 Hz, 2H, FCH2), 3.33 (t, 3J = 6.7 Hz, 2H, CH2N3), 2.63–2.37 (m,
3JC,F = 6.0 Hz, FCH2CH2CH2), 51.2 (CH2CH2CH2N3), 50.7 (CH2N3),
48.0 (Pip-C), 39.1 (C- ), 35.4 (Fmoc-C-9), 31.9 (CH2CO), 30.5 (C-
), 29.1 (CH2CH2N3), 26.6 (CH2C55C), 26.5 (d, JC,F = 20.9 Hz,
12H, Pip-H, 2ꢂ NCH2), 1.95–1.72 (m, 4H, CH2). 13C NMR (CDCl3):
d
e
1
3
2
82.7 (d, JC,F = 164.6 Hz, FCH2), 55.4 (NCH2), 54.4 (d, JC,F = 5.5 Hz,
b,d
2
NCH2), 53.3, 53.2 (Pip-C), 49.7 (CH2N3), 28.0 (d, JC,F = 19.8 Hz,
FCH2CH2), 23.5 (C-g d -222.2.
). 19F NMR (acetone-D6, 376 MHz):
FCH2CH2), 26.4 (CH2). 19F NMR (CDCl3):
d
–220.4. MS (ESI+): m/z (%)
MS (ESI+): m/z (%) 678 (100) [M++H]. Analytical HPLC:
tR = 19.7 min.
252 (10) [M++Na], 230 (100) [M++H], 159 (25) [M+ꢃC2H4N3]. Anal.
calcd for C10H20FN5 (229.30): C, 52.38%; H, 8.79%; N, 30.54%;
Found: C, 52.33%; H, 8.89%; N, 30.66%; Analytical HPLC:
tR = 4.2 min.
4.3.12. SNEWILPRLPQH-Azp (17)
Peptide 17 was prepared according to the general procedure for
peptides using 275 mg of resin preloaded with N-Fmoc-(S)-4-
4.3.10. (S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)-6-(4-(2-
(4-(3-fluoropropyl)piperazin-1-yl)ethyl)-1H-1,2,3-triazol-1-
yl)hexanoic acid (14)
azido-L-proline. Peptide 17 was obtained as a white powder after
purification by preparative HPLC (10–40% ACN+ 0.1% TFA within
12 min, tR = 14.4 min; Analytical HPLC: 10–45% ACN+ 0.1% TFA
within 17 min, tR = 16.3 min) and lyophilization. Yield: 14.3 mg,
To a solution of N-Fmoc-e-azido-L-norleucine (13) (370 mg,
0.97 mmol) in a mixture of t-butanol, water and acetonitrile
(15 mL, v/v/v = 1:1:1) 6 (192 mg, 0.97 mmol), CuSO4 (24 mg,
0.09 mmol) and sodium ascorbate (192 mg, 0.97 mmol) were
added and the mixture was stirred at ambient temperature for
48 h. The solvent was removed under reduced pressure and the
residue was triturated with ethyl acetate. The solid was filtered
9%, MW C73H111N25O18 calculated 1626 [M]+, found 814 [M+2H]2+
.
4.3.13. SNEWILPRLPQH-triazolyl-FP (18)
Compound 6 (164
solution of SNEWILPRLPQH-Azp (17) (900
Sørensen phosphate buffer (400 L, 0.1 M, pH 7.2) into a vial.
m
g, 0.83
m
mol) in ethanol was added to a
m
g, 0.55 mol) in
m
m

34
M. Pretze, C. Mamat / Journal of Fluorine Chemistry 150 (2013) 25–35
Na-ascorbate (50
m
L, 0.6 M, 30
m
mol) and CuSO4ꢁ5 H2O (50
m
L,
(ꢄ1 GBq) dissolved in Tris/HCl buffer (200
mL, pH 8.0) was added
0.4 M, 20
m
mol) were added and the solution was stirred at
and the reaction was irradiated at 50 W for 1 h. [18F]16 was
obtained in a RCY of 79% (d.c.) with a RCP >91% by means of HPLC.
Radio-HPLC: tR = 19.8 min. Radio-TLC: Rf = 0.57 (methanol).
ambient temperature for 30 min. Peptide 18 was obtained as a
powder after purification by preparative HPLC (10–40% ACN+ 0.1%
TFA within 12 min, tR = 5.4 min; Analytical HPLC: 10–45% ACN+
0.1% TFA within 17 min, tR = 4.3 min) and lyophilization. Yield:
4.4.5. SNEWILPRLPQH-triazolyl-[18F]FP ([18F]18)
983
m
g (98%). MW C84H130FN27O18 calculated 1824 [M]+, found
A solution of [18F]6 (250–850 MBq) in Sørensen phosphate
913 [M+2H]2+
.
buffer (500
mol) and TBTA (900
WILPRLPQH-Azp 17 (120–170
(1 mg, 4 mol) was added immediately and the solution was
m
L, 0.1 M, pH 7.2) with Na-ascorbate (1.25 mg,
g, mol) was added to SNE-
g, 74–105 nmol). CuSO4ꢁ5 H2O
6
m
m
2 m
m
4.4. Radiochemistry
m
4.4.1. 1-(But-3-ynyl)-4-(3-[18F]fluoropropyl)piperazine–[18F]BFP
([18F]6)
stirred at 60 8C for 1 h. Purification of the reaction mixture was
accomplished by size-exclusion chromatography (SEC). The
solvent was evaporated in vacuo at 50 8C to afford 30–150 MBq
(RCY: 17–25%, d.c.) of [18F]18 with RCP >99% within 120 min
(starting from [18F]fluoride drying). Radio-HPLC: tR = 4.3 min.
[
18F]Fluoride (4 ꢀ 2 GBq) was trapped on a Waters QMA cartridge
in the remotely controlled synthesis apparatus TRACERlab FxFN
18F]Fluoride was eluted with a Kryptofix1 K 2.2.2./K2CO3 solution
.
[
(1.5 mL) into the reaction vessel and dried via azeotropic distillation
with anhydrous acetonitrile (3 mL) for approx. 20 min. Afterwards, 5
(3 mg, 8.56 mmol) dissolved in acetonitrile (500 mL) was added, and
the reaction mixture was stirred at 100 8C for 15 min. Acetonitrile
(2 mL) was added, the solution was passed through a LiChrolut1 Si
cartridge, the cartridge was washed with acetonitrile (3 mL) and
Acknowledgments
The authors are grateful to Waldemar Herzog, Stephan
Preusche, and Tilow Krauss for their excellent technical assistance
and we are very thankful for the preparation of 4-azido-L-proline
done by Peggy Wecke. This work was financially supported by the
Fonds der Chemischen Industrie (FCI).
[
18F]6 was eluted with ACN/H2O (2 mL, v/v = 1:1). The solvent was
evaporated at 90 8C in a gentle stream of nitrogen for 4–5 min to
afford 1 ꢀ 0.5 GBq (RCY = 31 ꢀ 9, d.c.) [18F]6 with a RCP >98% within
approx. 40 min and a AS = 7 ꢀ 4 GBq/mmol (S.D., n = 8). Radio-HPLC:
tR = 4.2 min. Radio-TLC: Rf = 0.56 (methanol).
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