An Efficient Bioorthogonal Strategy Using CuAAC Click Chemistry for Radiofluorinations of SNEW Peptides and the Role of Copper Depletion

Article abstract of DOI:10.1002/cmdc.201300053

The EphB2 receptor is known to be overexpressed in various types of cancer and is therefore a promising target for tumor cell imaging by positron emission tomography (PET). In this regard, imaging could facilitate the early detection of EphB2-overexpressing tumors, monitoring responses to therapy directed toward EphB2, and thus improvement in patient outcomes. We report the synthesis and evaluation of several fluorine-18-labeled peptides containing the SNEW amino acid motif, with high affinity for the EphB2 receptor, for their potential as radiotracers in the non-invasive imaging of cancer using PET. For the purposes of radiofluorination, EphB2-antagonistic SNEW peptides were varied at the Cterminus by the introduction of L-cysteine, and further by alkyne- or azide-modified amino acids. In addition, two novel bifunctional and bioorthogonal labeling building blocks [¹⁸F]AFP and [¹⁸F]BFP were applied, and their capacity to introduce fluorine-18 was compared with that of the established building block [¹⁸F]FBAM. Copper-assisted Huisgen 1,3-dipolar cycloaddition, which belongs to the set of bioorthogonal click chemistry reactions, was used to introduce both novel building blocks into azide- or alkyne-modified SNEW peptides under mild conditions. Finally, the depletion of copper immediately after radiolabeling is a highly important step of this novel methodology.

Full text of DOI:10.1002/cmdc.201300053

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DOI: 10.1002/cmdc.201300053
An Efficient Bioorthogonal Strategy Using CuAAC Click
Chemistry for Radiofluorinations of SNEW Peptides and
the Role of Copper Depletion**
Marc Pretze,[a, b] Manuela Kuchar,[a, b] Ralf Bergmann,[a] Jçrg Steinbach,[a, b] Jens Pietzsch,[a, b]
and Constantin Mamat*[a, b]
The EphB2 receptor is known to be overexpressed in various
types of cancer and is therefore a promising target for tumor
cell imaging by positron emission tomography (PET). In this
regard, imaging could facilitate the early detection of EphB2-
overexpressing tumors, monitoring responses to therapy di-
rected toward EphB2, and thus improvement in patient out-
comes. We report the synthesis and evaluation of several fluo-
rine-18-labeled peptides containing the SNEW amino acid
motif, with high affinity for the EphB2 receptor, for their poten-
tial as radiotracers in the non-invasive imaging of cancer using
PET. For the purposes of radiofluorination, EphB2-antagonistic
SNEW peptides were varied at the C terminus by the introduc-
tion of l-cysteine, and further by alkyne- or azide-modified
amino acids. In addition, two novel bifunctional and bioor-
thogonal labeling building blocks [18F]AFP and [18F]BFP were
applied, and their capacity to introduce fluorine-18 was com-
pared with that of the established building block [18F]FBAM.
Copper-assisted Huisgen 1,3-dipolar cycloaddition, which be-
longs to the set of bioorthogonal click chemistry reactions,
was used to introduce both novel building blocks into azide-
or alkyne-modified SNEW peptides under mild conditions.
Finally, the depletion of copper immediately after radiolabeling
is a highly important step of this novel methodology.
Introduction
Eph receptors have recently gained much attention in cancer
research owing to their dysregulation in various tumor enti-
ties.[1–3] Representing the largest family of receptor tyrosine
kinases (RTK), Eph receptors play important roles in carcino-
genesis, tumor angiogenesis, metastasis, and apoptosis.[4,5] In
particular, the EphB2 receptor is overexpressed in various solid
tumor entities including gastric,[6] colorectal,[7] ovarian,[8]
breast,[9] prostate,[10] and brain.[11] EphB2 receptor expression
levels differ depending on the tumor stage. Early-stage tumors
show a substantially higher expression of EphB2;[12] in contrast,
the expression of EphB2 is down-regulated in later stages.
Imaging of the EphB2 receptor is therefore considered to be
an important tool in the diagnosis of early-stage tumor entities
including metastasis as well as for monitoring anticancer thera-
py.
the use of small organic kinase inhibitors.[13–16] The other in-
volves an extracellular domain, which is available to bind
ephrin ligands (B1, B2, and A5 in the case of EphB2). In almost
the same manner, small organic molecules[17,18] as well as pep-
tides[19] (e.g., with the key amino acid sequence SNEW) have
been identified as high-affinity antagonist ligands.[20,21]
In principle, the radiolabeling of high-molecular-weight com-
pounds such as peptides, proteins, or antibodies with fluorine-
18 poses a considerable challenge. A radiolabeling strategy in-
volving 18F-labeled prosthetic groups, also known as 18F-label-
ing building blocks, is necessary owing to the harsh reaction
conditions required for the direct introduction of [18F]fluoride
into these biomacromolecules at high specific activity levels.
Because of this and the high number of functional groups
present in the peptides, a labeling strategy for nearly every
peptide must be developed. For this purpose, the conventional
building block N-(6-(4-[18F]fluorobenzylidene)aminooxyhexyl)-
maleimide ([18F]FBAM) was initially chosen.[22–24] [18F]FBAM is
available in high radiochemical yield using a remotely con-
trolled synthesis module. Based on radiolabeling results with
[18F]FBAM, the search for viable alternatives based on bio-
orthogonal reactions such as Staudinger ligation,[25,26] tetrazine
click,[27] or copper-mediated Huisgen click reaction[28–31] was the
main subject of the research effort reported herein.
There are two general approaches for targeting the EphB2
receptor. One involves an intracellular tyrosine kinase domain,
on which the receptor can be inhibited, particularly through
[a] M. Pretze, M. Kuchar, Dr. R. Bergmann, Prof. Dr. J. Steinbach,
Prof. Dr. J. Pietzsch, Dr. C. Mamat
Institut fꢀr Radiopharmazeutische Krebsforschung
Helmholtz-Zentrum Dresden-Rossendorf
Bautzner Landstraße 400, 01328 Dresden (Germany)
E-mail: c.mamat@hzdr.de
[b] M. Pretze, M. Kuchar, Prof. Dr. J. Steinbach, Prof. Dr. J. Pietzsch,
Dr. C. Mamat
Two novel and versatile labeling building blocks, 1-(but-3-
ynyl)-4-(3-[18F]fluoropropyl)piperazine ([18F]BFP) and 1-(3-azido-
propyl)-4-(3-fluoropropyl)piperazine ([18F]AFP),[32,33] were intro-
duced for our purposes, and subsequent radiofluorinations
were carried out with the 1,3-dipolar Huisgen cycloaddition
Fachrichtung Chemie und Lebensmittelchemie
Technische Universitꢁt Dresden, 01062 Dresden (Germany)
[**] Parts of this work were presented as a Highlight Presentation under the
topic “Radiochemie” at the 51st annual congress of the DGN 2013.
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(click approach).[34] It is mandatory for all subsequent labeling
methods that the peptide SNEWILPRLPQH be exclusively al-
tered at the C terminus to maintain high affinity for the EphB2
receptor, as the ligand–receptor interaction is associated with
the key amino acid sequence SNEW, located at the N terminus
of the peptide.[21]
The work reported herein comprises the preparation and
modification of new SNEW peptides as well as their radiolabel-
ing with fluorine-18 using two different labeling strategies, in-
cluding the application of novel fluorine-18-containing build-
ing blocks. Thus, the variation of peptides was achieved either
by introduction of l-cysteine or by a connection of amino
acids containing an alkyne or an azide residue. Further, the
broad adaptability of [18F]BFP and [18F]AFP as labeling building
blocks for radiofluorinations was analyzed. Importantly, a strat-
egy for the thorough removal of copper species is essential for
adjacent biological investigations. Finally, the feasibility of
these fluorine-18-labeled SNEW peptides as potent and versa-
tile imaging agents was evaluated by using small-animal PET in
a biological pilot study.
Figure 1. HPLC traces of starting peptide 2, FBAM-labeled peptide 3, FBAM
1, and radiolabeled peptide [18F]3.
reference 3 and tracer [18F]3 is outlined in Scheme 1. Optimiza-
tions were achieved, with the result that ~500 mg peptide 2
are required for an almost complete conversion of [18F]FBAM
after 60 min.
Results and Discussion
The stability of radiolabeled peptide [18F]3 was determined
by treatment of [18F]3 with rat plasma, trypsin, and glycine·HCl
buffer (pH 2.0), respectively, at 378C for 1 h. The metabolites of
peptides are usually more hydrophilic than the parent peptide;
however, it was found that most of the activity (>85%) had
converted into a more lipophilic byproduct at tR =14.3 min rel-
ative to [18F]3 (tR =11.2 min) and [18F]FBAM (tR =14.7 min), as
determined by radio-HPLC analyses. Moreover, the same con-
version was observed in tests of the non-radioactive peptide 3.
A signal at m/z=638, which indicated twice the mass of FBAM
1, was observed in ESI-MS analyses. Moreover, the Michael ad-
dition has been reported to be a reversible process.[36] Owing
to the reversibility and instability of [18F]3, it was necessary to
evaluate an alternative, convenient and non-reversible strategy
for the radiofluorination of SNEW peptides.
Radiolabeling of cysteine-containing SNEW peptide 2 with
[18F]FBAM
In our first approach, the maleimide-based building block
[18F]FBAM was applied. [18F]FBAM ([18F]1) is well established,
readily accessible, and in current use for the regioselective
radiofluorination of cysteine-containing peptides. Cysteine-con-
taining SNEW peptide 2 was prepared, and it reacted readily
with non-radioactive FBAM (1) to yield reference peptide 3 in
high yield (72%, tR =11.1 min).
For radiofluorinations, [18F]FBAM was prepared by automat-
ed module synthesis.[35] Next, a solution of 2 in Sørensen phos-
phate buffer was added to the [18F]FBAM-containing solution,
and the mixture was incubated at 608C. After 60 min, a new
peak at tR =11.2 min was observed in analytical radio-HPLC, in-
dicating the formation of [18F]3 (Figure 1). The preparation of
In addition, cysteine-containing starting peptide 2 was
stored at ꢀ658C over a period of three months. Afterward,
Scheme 1. Synthetic pathway to 18/19F-labeled SNEW peptide 3/[18F]3 radiolabeled with [18/19F]FBAM 1/[18F]1. Reagents and conditions: a) Sørensen phosphate
buffer, CH3CN, 608C, 2 h; radiolabeling: b) Sørensen phosphate buffer, CH3CN, 608C, 1 h.
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a solution of this stored peptide was prepared and analyzed
by HPLC. Interestingly, one UV peak with a retention time
2 min later than the original peptide 2 was observed under
the same HPLC conditions. Furthermore, reaction of [18F]FBAM
with the stored precursor 2 showed no formation of [18F]3.
Peptide 2 appeared to dimerize due to oxidization of the cys-
teine residues. To circumvent this, the solution was treated
with tris(2-carboxyethyl)phosphine (TCEP) at ambient tempera-
ture for 1 h. After this procedure, the desired SNEW peptide 2
was observed by HPLC and was successfully applied as precur-
sor.
allows radiofluorinations of the chosen modified SNEW pep-
tides with high specific activities.
The following click labeling strategy was first evaluated with
the alkyne-containing building block [18/19F]BFP 4/[18F]4. A
SNEW peptide was functionalized with N-Fmoc-(S)-4-azido-l-
proline at the C terminus to obtain 6. Afterward, the non-radio-
active reference peptide 7 was prepared in 98% yield by the
click reaction of BFP 4 with SNEW peptide 6 in Sørensen phos-
phate buffer and ethanol for 30 min at ambient temperature
using sodium ascorbate and CuSO4. Purification was carried
out by preparative HPLC. For radiofluorination purposes, tris-
(benzyltriazolylmethyl)amine (TBTA), CuSO4, and sodium ascor-
bate (6:1:10 equiv) were added to a mixture of SNEW peptide
6 (1 equiv) in Sørensen phosphate buffer at pH 7.2 and
[18F]BFP. Optimized conditions were found to be 0.3–
0.5 mgmLꢀ1 peptide at 608C for 60 min. More insoluble resi-
dues were found in the reaction mixture at higher peptide
concentrations, which are thought to be an unknown triazole–
copper species.[38] The purification of [18F]7 from starting pep-
tide 6 was accomplished by semi-preparative HPLC followed
by SEC to separate [18F]4 from [18F]7 due to the similar tR
values. Finally, [18F]7 was obtained in 62% RCY with >95%
RCP (n=3).
Evaluation of an effective click radiolabeling strategy
The CuI-mediated azide–alkyne cycloaddition (CuAAC) reaction
belongs to the set of bioorthogonal labeling reactions. Recent-
ly, the radiolabeling of human serum albumin as well as func-
tionalized phosphopeptides and oligonucleotides was de-
scribed.[28,30] To further improve this approach, [18F]BFP ([18F]4)
and [18F]AFP ([18F]5)[33] were prepared as novel fluorine-18-con-
taining building blocks with favorable properties, based on the
piperazine skeleton. As an advantage, spiro-salts were used as
precursors,[37] facilitating separation from the desired radio-
labeled building block through the application of cartridges.
Moreover, the labeling process can be performed in water or
Metabolite analyses in rats indicated that most of the activi-
ty of [18F]7 resided in blood and furthermore substantially ac-
cumulated in liver and in lungs (PET data not shown). More-
over, the biodistribution showed that [18F]7 does not behave
like a “normal” peptide, e.g., by showing a more prominent
renal elimination. It was assumed that [18F]7, as well as refer-
ence 7, form aggregates with the copper species used for the
click reaction. In this regard, subsequent MS analyses (Figure 2)
of the respective reference peptide 7 gave evidence that the
copper species used for the click reaction was partly bound by
the peptide (m/z=943 [M+Cu]2+). Consequently, [18F]7 was
considered inappropriate for further biological investigations.
This could also explain the low retention time of 7/[18F]7.
Unfavorable strong copper complexes were formed with the
SNEW peptide due to such residues as serine, histidine, and ar-
ginine.[39] This implies that the copper was not removed by
buffer owing to the high hydrophilicity (logD=0.31 for [18F]4,
0.70 for [18F]5).[33] Separation of the radiolabeled product from
the starting peptide by size-exclusion chromatography (SEC)
was made amenable due to the medium molar mass of these
building blocks. An automated procedure was recently evaluat-
ed by using a remotely controlled synthesis module (TRACER-
lab FxFN) for the synthesis of [18F]BFP and [18F]AFP.[32,33] This
Scheme 2. Synthetic pathway to 18/19F-containing SNEW peptides 7/[18F]7 radiolabeled with [18/19F]BFP 4/[18F]4. Reagents and conditions: a) DMF, sodium ascor-
bate, CuSO4·5H2O, 408C, 16 h; radiolabeling: b) DMF/Sørensen phosphate buffer, TBTA, sodium ascorbate, CuSO4·5H2O, 50 W, 1 h.
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Table 1. Purification, HLPC, ESI-MS and ICP-MS analyses of synthesized
peptides 7, 9, 11, and 14.[a]
Peptide
Purification tR [min]
ESI-MS [m/z]
[Cu] [mggꢀ1
]
nCu/npept.
7 (soln)
7 (resin)
7 (resin)
9 (resin)
11 (resin) HPLC+BIS
14 (resin) HPLC+BIS
HPLC
HPLC
HPLC+BIS
HPLC+BIS
4.1
4.1
15.0
15.2
15.4
13.9
943 [M+Cu]2+
1825 [M+H]+
1825 [M+H]+
1841 [M+H]+
1813 [M+H]+
1927 [M+H]+
ND[b]
ND[c]
611
ND
ND
1:57
1:50
1:70
1:77
692[d]
501
426
[a] ND: not determined. [b] Pale green. [c] Colorless. [d] Dissolved in H2O
c=1 gmLꢀ1
.
This prompted us to reconsider the position of the azide
moiety of the starting peptide. The azide residue of peptide
precursor 6 is very close to the Rink amide resin. Thus, the ster-
ically hindered 4-azidoproline moiety of 6 could be the reason
for the low yield of [18F]7. Therefore, two modified SNEW pep-
tide precursors 8 and 10 were prepared (Scheme 3). Peptide 8
contains an azidonorleucine moiety at the C terminus, whereas
peptide 10 has an azidopentanamido moiety at the N terminus
for an enhanced distance between the azide group and the
Rink amide resin, but certainly a decrease in affinity for the
EphB2 receptor. Unfortunately, both radiolabeled peptides
[18F]9 and [18F]11 showed no significantly higher decay-correct-
ed radiochemical yields ([18F]9: 0.5–1.4%, [18F]11: 1.8%). Results
of the radiolabeling are summarized in Scheme 3 and Figure 3.
Further investigations revealed an unknown byproduct in all
labeling reactions with [18F]BFP (Rf =0.58 in methanol) in addi-
tion to the desired peptides. In this case we found that Glaser
Figure 2. Part of the mass spectrum of 7 prepared in solution, indicating the
formation of the Cu–peptide species.
HPLC or SEC purifications. Therefore, it was important to devel-
op a new strategy that includes separation of the copper spe-
cies from the desired peptide. On that account, peptide 7 was
directly synthesized on solid support by incubating resin-
bound 6 with BFP 4 under standard conditions, followed by
cleavage from the resin at the end of the procedure. Unfortu-
nately, subsequent HPLC analysis showed no differences in the
retention time, still indicating the presence of copper species.
However, the lyophilized product was colorless, and no copper
impurities were found by ESI-MS analyses. Based on these find-
ings, resin-bound peptide 7 was treated and incubated with
strong chelating agents such as EDTA and bispidine (BIS),[40] re-
spectively, after the click reaction with BFP 4. Fortunately, pep-
tide 7 showed a higher retention time (tR =14.1 min) after in-
cubation with BIS and subsequent cleavage from resin; EDTA
had no effect. Synthesis of reference peptide 7 on solid sup-
port showed high-yielding conversion from precursor peptides
6 by using BFP/azide/sodium ascorbate/CuSO4 (1.5:1:55:36 v/v)
in DMF after stirring at 408C for 16 h (98% yield). Moreover,
the absence of copper was proven by ICP-MS analyses, deter-
mining the copper concentration in all reference peptide prep-
arations; the results are summarized in Scheme 2 and Table 1.
Based on these results, an adequate radiofluorination strat-
egy was developed, including the click labeling of resin-bound
peptide 6 with [18F]BFP, followed by a washing step with DMF
and H2O, incubation with BIS (2 equiv relative to CuSO4) in
methanol and DMF, a second washing step with DMF and H2O,
and final cleavage from the solid support with TFA/TIS (9:1
v/v). Nevertheless, radiosyntheses with [18F]BFP showed very
low yield for [18F]7 (0.4%, d.c.) in contrast to the non-radio-
active reference 4 (98% yield). A higher reaction temperature
(908C), microwave conditions (50, 90 W) for an electrostatic
fixed peptide backbone,[41] longer reaction times (60, 120 min),
and higher starting activities (3–6 GBq [18F]BFP) gave no signifi-
cant changes in the RCY.
Figure 3. HPLC traces of [18F]4, crude [18F]9, and crude [18F]11.
coupling[42] became the main reaction pathway instead of the
Huisgen click reaction at very low concentrations of the alkyne
[18F]BFP, which generally occur in these radiosyntheses. To
prove this assumption, non-radioactive BFP 4 was reacted in
the absence of azides and yielded diyne 12 (Scheme 4). Subse-
quently, compound 12 (Rf =0.27 in methanol) was compared
with the unknown byproduct from all labeling reactions by
means of (radio-)TLC to identify this byproduct as [18F]12. In
contrast, [18F]BFP has an Rf value of 0.58 in methanol. HPLC
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Scheme 3. Synthetic pathway to the 18F-labeled SNEW peptides [18F]9 and [18F]11 as well as references 9 and 11 using [18/19F]BFP 4/[18F]4. Reagents and condi-
tions: a) DMF/H2O, TBTA, sodium ascorbate, CuSO4·5H2O, 508C, 3 days; radiolabeling: b) DMF/Sørensen phosphate buffer, TBTA, sodium ascorbate,
CuSO4·5H2O, 908C, 1 h or 50 W, 1.5 h.
Reference peptide 14 was synthesized on solid support
using copper(I) iodide, DIPEA, and AFP (5) followed by incuba-
tion with BIS after click reaction. Radiolabeling of precursor
peptide 13 with [18F]AFP (Scheme 5 and Figure 4) was carried
out under optimized conditions: peptide/TBTA/CuSO4/sodium
ascorbate (1:8:7:10 v/v) at high concentrations (10 mg resin 13
in 270–300 mL solution). TBTA at a minimum of 1.1 equiv rela-
tive to CuSO4 was crucial for a high RCY, as the RCY decreases
significantly if the amount of TBTA is <1 equiv. Thus, the RCY
of [18F]14 was significantly higher and ranged from 46 to
Scheme 4. Glaser coupling of BFP 4 for the identification of byproduct 12.
164 MBq (8–13%, d.c.) with RCP >98% (after purification
using only semi-preparative HPLC) and an AS value of
Reagents and conditions: a) CuSO4·5H2O, DMF, 408C, 3 days.
~5 GBqmmolꢀ1
.
analyses of both compounds revealed tR values of 3.3 min for
the dimer 12 and 4.9 min for compound 4.[43]
Biological investigations of SNEW peptide [18F]14
All these results prompted us to switch the functionalities of
the peptide precursor and of the radiolabeling building block.
Therefore, [18F]AFP, similar to [18F]BFP, was applied, and peptide
precursor 13, with a pentyne moiety, was synthesized with
Rink amide resin preloaded with N-Fmoc-e-(pent-4-ynamido)-l-
lysine[44] under standard solid-phase peptide synthesis (SPPS)
conditions as described above. It has been pointed out that
such alkynylated peptides bound to a solid support are unable
to undergo the Glaser coupling.[45]
The stability of [18F]14 was investigated in vitro by incubation
with rat plasma and rat blood for 1 h at 378C (samples were
taken after 60 min). After this procedure, >80% of the activity
remained as the original compound as indicated by radio-
HPLC analyses. Additional hydrophilic metabolites were ob-
served, but were not determined to be [18F]fluoride by means
of radio-HPLC analyses. This result indicates that no radio-
defluorination of the 18F-propyl moiety occurs in vitro.
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Scheme 5. Synthetic pathway to the 18F-labeled SNEW peptide [18F]14 using [18F]AFP [18F]5 and reference peptide 14. Reagents and conditions: a) THF, DIPEA,
CuI, RT, 3 days; radiolabeling: b) DMF/Sørensen phosphate buffer, TBTA, sodium ascorbate, CuSO4·5H2O, 50 W, 1 h.
urine. On the other hand, the activity concentration in the liver
was low (Figures 6 and 7). These results unambiguously indi-
cate that [18F]14 is unsuitable as a radiotracer for imaging of
the EphB2 receptor at this stage. Nevertheless, this new ap-
proach using SNEW peptides in fluorine-18 chemistry gives
access to labeled peptides that hold promise as radiolabeled
Eph ligands. Further improvement of the in vivo stability of the
peptide backbone is required, for example, through introduc-
tion of d-amino acids or methylation of peptide bonds.
Conclusions
Herein we present a successful radiofluorination approach
toward modified SNEW peptides that are potentially suitable
for noninvasive PET imaging of the EphB2 receptor. For this
purpose, l-cysteine-modified SNEW peptide 2 was first radio-
fluorinated using the conventional and well-known building
block [18F]FBAM. Unfortunately, 18F-labeled peptide [18F]3 as
well as the reference [19F]3 led to rapid decomposition due to
the reversibility of the Michael addition. Thus, [18F]BFP and
[18F]AFP were applied as novel and bioorthogonal radiolabel-
ing building blocks that have the beneficial properties of bio-
orthogonality, hydrophilicity (logD), in vitro stability, molecular
weight, and the irreversibility of ligation. These building blocks
allow the conjugation of azide- and alkyne-functionalized pep-
tides via CuI-mediated Huisgen [3+2] cycloaddition (click
chemistry approach). Unfortunately, strong complexes were
formed between all SNEW peptides and copper, which is re-
quired for click labeling. Thus, an alternative synthesis based
on radiolabeling with resin-bound peptides and subsequent
removal of the copper species by treatment with BIS as
a strong chelating agent was evaluated. As a result, azide-con-
taining peptides 6, 8, and 10 were successfully radiolabeled
with [18F]BFP, but with low RCYs. In this regard, a byproduct
was found which was mainly formed in the labeling step from
[18F]BFP following the mechanism of Glaser coupling. [18F]AFP
has characteristics similar to those of [18F]BFP, but was unable
to undergo this side reaction. Hence, the alkyne-containing
peptide 13 was successfully prepared and radiolabeled by the
click chemistry approach in sufficient radiochemical yield and
Figure 4. HPLC traces of starting peptide 13, reference peptide 14, [18F]AFP
[18F]5, and purified [18F]14.
The in vivo behavior of [18F]14 in Wistar rats was character-
ized by fast blood clearance and substantial renal elimination.
However, the rapid elimination was combined with degrada-
tion of the peptide. [18F]14 was metabolized with a biological
half-life of 4.3 min. At 5 min post-injection, only 17% of the
intact peptide was found in blood after a single intravenous in-
jection of [18F]14. After 1 h, no activity was detectable in the
blood. Three main metabolites (tR =5.4, 7.8, and 10.2 min)
were found in the urine after 1 h, but not identified structurally
(Figure 5). This finding can be explained by partial proteolysis
of the peptide at the position of arginine.[46] In addition, the ra-
diometabolite at tR =5.4 min seems to be a very small-molecu-
lar-weight 18F-labeled compound, but not [18F]fluoride, as no
bone accumulation was observed.
Most of the activity was found in kidneys and bladder (44
and 15% ID, respectively) 90 min after injection. This is consis-
tent with the assumed metabolism of peptide [18F]14. This
finding is also consistent with the common observation that
the kidneys frequently accumulate peptides in the cortical
tubuli after glomerular filtration. The PET experiments also re-
vealed very rapid blood clearance accompanied by continuous
accumulation in the kidneys, followed by elimination into the
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a result of the hydrophilicity of
[18F]AFP, a more polar ligation
product was obtained, and due
to the molecular weight, separa-
tion from the precursor peptide
was possible by SEC or HPLC.
Biological evaluations in vitro
demonstrated adequate stability
of the labeled peptide [18F]14 in
rat plasma in contrast to the
FBAM-labeled peptide [18F]3.
However, small-animal PET ex-
periments revealed a very brief
blood retention, accompanied
by metabolism and rapid renal
elimination of [18F]14 in rats. At
this stage, [18F]14 is not applica-
ble as a radiotracer for imaging
purposes. Based on these re-
sults, novel peptides with in-
creased in vivo stability must be
evaluated.
Experimental Section
Instrumentation and chemicals.
All reagents and solvents were
purchased from commercial sup-
pliers and were used without fur-
ther purification unless otherwise
specified. NMR spectra were re-
corded on a Varian Unity 400 MHz
spectrometer. Chemical shifts (d)
are given in ppm and are refer-
enced to the residual solvent reso-
nance signals relative to (CH3)4Si
(1H, 13C) and CFCl3 (19F). Mass spec-
tra were obtained on a Quattro/LC
mass spectrometer (Micromass) by
electrospray ionization. Preparative
column chromatography was per-
formed on Merck silica gel. Reac-
tions were monitored by thin-layer
chromatography (TLC) on Merck
silica gel F254 aluminum plates,
with visualization under UV (l=
254 nm) or by evaluation using
ninhydrin and heating. ICP-MS
data were obtained on an Elan
9000 Quadrupole instrument (Per-
kinElmer) using a Merck IV matrix.
Figure 5. Representative HPLC traces of a) peptide [18F]14 and determination of radioactive metabolites in b) arte-
rial blood plasma; c,d) kidneys; and e,f) urine following single intravenous injection into a Wistar rat, determined
5 min and 1 h post-injection, as indicated.
General procedure for peptide
syntheses. SNEW peptides 2, 6, 8,
and 13 were synthesized by an
Fmoc SPPS strategy on an auto-
mated multiple peptide synthesiz-
er (CEM Microwave Peptide Syn-
Figure 6. Maximum intensity projections of a representative PET experiment at after single intravenous injection
of [18F]14 into a male Wistar rat a) 1 min, b) 5 min, c) 60 min, d) 120 min.
purity. To remove the copper species for biological investiga-
tions, peptide [18F]14 was synthesized and radiolabeled on
resin. Subsequent incubation with BIS was necessary. As
thesizer). Fmoc-l-amino acid derivatives used in peptide syntheses
were purchased from MultiSynTech GmbH. The reactive side chains
of the Fmoc amino acids were protected as follows: amine: fluore-
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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a VWR Hitachi unit: System A: Nu-
cleosil Nautilus C18 column (250ꢁ
4.6 mm, 5 mm; eluent: CH3CN/
H2O+0.1% TFA; gradient: 20–
50% CH3CN+0.1% TFA: 0!
10 min, 50–80% CH3CN+0.1%
TFA: 10!11 min). System B: Phe-
nomenex Aqua C18 column (250ꢁ
4.6 mm, 5 mm, 100 ꢂ; eluent:
CH3CN/H2O+0.1% TFA; flow rate:
1.0 mLminꢀ1
; gradient: 10–45%
CH3CN+0.1% TFA: 0!17 min,
45–95% CH3CN+0.1% TFA: 17!
18 min).
Peptide syntheses
SNEWILPRLPQHC (2). SNEW pep-
tide 2 was prepared according to
the general procedure using
83 mg of unloaded resin and was
obtained as a colorless powder
after purification by preparative
HPLC (20–50% CH3CN+0.1% TFA
within 10 min, tR =7.1 min) and
lyophilization. Analytical HPLC
(System A): tR =7.4 min; yield:
17.3 mg (22%); MS (ESI+): m/z
calcd: 1591 [M]+, found: 1593
[M+2H]+, 808 [M+H+Na]2+
797 [M+2H]2+
,
.
SNEWILPRLPQHC-FBAM (3). A so-
lution of FBAM (1; 1.50 mg,
4.71 mmol) in CH3CN (100 mL) was
Figure 7. Time–activity curves in a) blood, b) heart, c) liver, and d) kidney given as SUVmean, as well as in e) kidney
and f) urine given as percent ID in a male Wistar rat after single intravenous injection of [18F]14 and followed up
to 90 min post-injection.
added to
WILPRLPQHC
2.95 mmol) in Sørensen phosphate
a
solution of SNE-
(2; 4.70 mg,
nylmethyloxycarbonyl (Fmoc); Arg: 2,2,4,6,7-pentamethyldihydro-
benzofuran-5-sulfonyl (Pbf); Trp: tert-butyloxycarbonyl (Boc); Glu
and Ser: O-tert-butyl (O-tBu); Asn, Cys, Gln, and His: triphenylmeth-
yl (Trt). Resin was purchased from Bachem. N,N-Diisopropylethyla-
mine (DIPEA), trifluoroacetic acid (TFA), N,N-dimethylformamide
(DMF), piperidine, and N-methyl-2-pyrrolidone (NMP) were pur-
chased from Biosolve Ltd. HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate) and HOBt (N-hydroxy-
benzotriazole) were purchased from Calbiochem–Novabiochem. All
peptides were prepared on Rink amide MBHA resin (extent of la-
beling: 0.6 mmolgꢀ1 loading). Syntheses were performed on
a 0.05–0.10mm 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 equiv), HBTU (5 equiv), HOBt (5 equiv), and DIPEA
(10 equiv) were dissolved in DMF and NMP, respectively, and
added to the resin. The coupling time was 2 h, and the coupling
was carried out twice for each residue. All peptides were depro-
tected and cleaved from the solid support with TFA/TIS/H2O
(95:2.5:2.5 v/v/v) at ambient temperature for 4 h.[47] Afterward, the
resin was filtered, the crude peptide was precipitated by adding
cold Et2O and washed with ice-cold Et2O. Residual ether was re-
moved by evaporation, and the peptides were purified by prepara-
tive HPLC onto a Varian Dynamax Microsorb 60-8 C18 column
(250ꢁ21.4 mm) at a flow rate of 20 mLminꢀ1 with gradients of H2O
and CH3CN with 0.1% TFA. Analytical HPLC was performed on
buffer (100 mL, pH 7.2, 47.9mm Na2HPO4, 18.1mm NaH2PO4) into
a vial and was stirred at 608C for 2 h. SNEW peptide 3 was ob-
tained as a colorless powder after purification by preparative HPLC
(10–40% CH3CN+0.1% TFA within 12 min, tR =11.1 min) and lyo-
philization. Analytical HPLC (System A): tR =11.1 min; yield: 4.05 mg
(72%); MS (ESI+): m/z calcd: 1910 [M]+, found: 1911 [M+H]+, 956
[M+2H]2+
.
SNEWILPRLPQH-Azp (6). SNEW peptide 6 was prepared according
to the general procedure for peptides using 275 mg of resin pre-
loaded with N-Fmoc-(S)-4-azido-l-proline. SNEW peptide 6 was ob-
tained as a colorless powder after purification by preparative HPLC
(10–40% CH3CN+0.1% TFA within 12 min, tR =10.7 min) and lyo-
philization. Analytical HPLC (System B): tR =16.3 min; yield: 14.3 mg
(9%); MS (ESI+): m/z calcd: 1626 [M]+, found: 814 [M+2H]2+
.
SNEWILPRLPQH-triazolyl-FP (7). BFP 4 (164 mg, 0.83 mmol) was
added to a suspension of SNEWILPRLPQH-Azp (6) bound to resin
(900 mg, 0.55 mmol) in DMF (500 mL) into a frit syringe. Sodium as-
corbate (50 mL, 0.6m, 30 mmol) and CuSO4·5H2O (50 mL, 0.4m,
20 mmol) were added, and the solution was stirred at 408C for
16 h. The resin was washed with DMF (5 mL), CH3OH (5 mL) and in-
cubated with bispidine (BIS; 24.5 mg, 40 mmol) in DMF/CH3OH
(1 mL, 1:1 v/v) at 408C for an additional 30 min. Afterward, the
peptide was cleaved using the standard procedure described
above. SNEW peptide 7 was obtained as a colorless powder after
purification by preparative HPLC (10–45% CH3CN+0.1% TFA
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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within 17 min, tR =14.1 min) and lyophilization. Analytical HPLC
(System B): tR =15.0 min; yield: 1.02 mg (98%); MS (ESI+): m/z
calcd: 1824 [M]+, found: 1825 [M+H]+, 913 [M+2H]2+, 609
1,8-Bis(4-(3-fluoropropyl)piperazin-1-yl)octa-3,5-diyne
(12).
CuSO4·5H2O (6 mg, 20 mmol) was added to a solution of 1-(but-3-
ynyl)-4-(3-fluoropropyl)piperazine (4; 45 mg, 230 mmol) and DMF
(4 mL), and was stirred at 408C for 3 days. The solvent was re-
moved in vacuo, and the crude product was purified by column
chromatography (EtOAc!EtOAc/CH3OH 5:1) to yield compound
12 as an orange solid (10 mg, 11%): Rf =0.27 (CH3OH); mp: 68–
708C; 1H NMR (400 MHz, CDCl3): d=4.48 (dt, 3JH,F =47.2 Hz, 3J=
5.9 Hz, 4H, FCH2), 2.61–2.41 (m, 24H, Pip-H/FCH2CH2CH2/NCH2),
1.95–1.82 (m, 4H, NCH2CH2), 1.47–1.44 ppm (m, 4H, FCH2CH2);
13C NMR (CDCl3, 101 MHz): d=82.7 (d, 1JC,F =164.3 Hz, FCH2), 76.0
[M+3H]3+
.
SNEWILPRLPQH-Anl (8). SNEW peptide 8 was prepared according
to the general procedure for peptides using 86 mg of resin pre-
loaded with N-Fmoc-e-azido-l-norleucine. Afterward, the peptide
was cleaved using the standard procedure described above. SNEW
peptide 8 was obtained as a colorless powder after purification by
preparative HPLC (10–45% CH3CN+0.1% TFA within 17 min, tR =
15.4 min) and lyophilization. Analytical HPLC (System B): tR =
17,2 min; yield: 6.5 mg (26%); MS (ESI+): m/z calcd: 1642 [M]+,
found: 1643 [M+H]+, 822 [M+2H]2+, 549 [M+3H]+.
(CH2CꢁC), 66.1 (CH2CꢁC), 56.8 (NCH2), 54.4 (d, 3JC,F =5.5 Hz,
2
FCH2CH2CH2), 53.2 (Pip-C-2,6), 52.9 (Pip-C-3,5), 28.1 (d, JC,F
=
19.7 Hz, FCH2CH2), 16.9 ppm (NCH2CH2); 19F NMR (CDCl3, 376 MHz):
d=ꢀ220.6 ppm; MS (ESI+): m/z 395 [M+H]+, 198 [M+2H]2+
.
SNEWILPRLPQH-e-(4-(2-(4-(3-fluoropropyl)piperazin-1-yl)ethyl)-
1H-1,2,3-triazol-1-yl)Nle (9). BFP 4 (9.57 mg, 48.27 mmol) was
added to a suspension of SNEWILPRLPQH-Anl (8) bound to resin
(100 mg, 9.65 mmol) in DMF (1 mL) into a frit syringe. Then, TBTA
(12.8 mg, 24.13 mmol), CuSO4·5H2O (12.1 mg, 48.27 mmol) in H2O
(500 mL), and sodium ascorbate (8.8 mg, 96.50 mmol) in H2O
(500 mL) were added in this order, and the reaction was stirred at
508C for 3 days. The resin was washed with DMF (5 mL), CH3OH
(5 mL), and incubated with BIS (41.41 mg, 67.55 mmol) in DMF/
CH3OH/H2O (6 mL, 1:1:1 v/v/v) at 408C for an additional 30 min.
The resulting blue solution was removed, and the resin was
washed with DMF (5 mL), CH3OH (5 mL), and Et2O (5 mL). After-
ward, the peptide was cleaved using the standard procedure de-
scribed above. SNEW peptide 9 was obtained as a colorless
powder after purification by preparative HPLC (10–45% CH3CN+
0.1% TFA within 17 min, tR =13.3 min) and lyophilization. Analytical
HPLC (System B): tR =15.2 min; yield: 5.4 mg (86%); MS (ESI+): m/z
SNEWILPRLPQH-e-(pent-4-ynamido)Lys (13). SNEW peptide 13
was prepared according to the general procedure for peptides
using 430 mg of resin preloaded with N-Fmoc-e-(pent-4-ynamido)-
l-lysine. Afterward, the peptide was cleaved using the standard
procedure described above. SNEW peptide 13 was obtained as
a colorless powder after purification by preparative HPLC (10–45%
CH3CN+0.1% TFA within 17 min, tR =15.6 min) and lyophilization.
Analytical HPLC (System B): tR =15.2 min; yield: 3.5 mg (11%);
MS (ESI+): m/z calcd: 1697 [M]+, found: 1698 [M+H]+, 850
[M+2H]2+, 566 [M+3H]+.
SNEWILPRLPQH-e-(3-(1-(3-fluoropropyl)-1H-1,2,3-triazol-4-yl)pro-
panamido)Lys (14). AFP 5 (5.2 mg, 22.8 mmol) was added to a sus-
pension of SNEWILPRLPQH-e-(pent-4-ynamido)Lys (13) bound to
resin (33 mg, 11.4 mmol) in THF (1.2 mL) into a frit syringe. Then,
DIPEA (99 mg, 570 mmol) and CuI (4.3 mg, 22.8 mmol) were added,
and the reaction was stirred at ambient temperature for 3 days.
The resin was washed with DMF (5 mL), CH3OH (5 mL), and incu-
bated with BIS 7 (27.9 mg, 45.6 mmol) in DMF/CH3OH/H2O (3 mL,
1:1:1 v/v/v) at 408C for an additional 30 min. The resulting blue so-
lution was removed, and the resin was washed with DMF (5 mL),
CH3OH (5 mL), and Et2O (5 mL). Afterward, the peptide was cleaved
using the standard procedure described above. SNEW peptide 14
was obtained as a colorless powder after purification by prepara-
tive HPLC (10–45% CH3CN+0.1% TFA within 17 min, tR =14.6 min)
and lyophilization. Analytical HPLC (System B): tR =13.9 min; yield:
5.4 mg (77%); MS (ESI+): m/z calcd: 1926 [M]+, found: 1927
[M+H]+, 964 [M+2H]2+, 643 [M+3H]+.
calcd: 1840 [M]+, found: 1841 [M+H]+, 921 [M+2H]2+
.
N-(5-Azidopentanamido)-SNEWILPRLPQH (10). Succinimidyl 5-azi-
dopentanoate (24 mg, 100 mmol) was added to a suspension of
SNEWILPRLPQH bound to resin (112 mg, 25 mmol) in DMF (1 mL)
and was stirred at 408C for 3 days. Afterward, the peptide was
cleaved using the standard procedure described above. SNEW pep-
tide 10 was obtained as a colorless powder after purification by
preparative HPLC (10–45% CH3CN+0.1% TFA within 17 min, tR =
16.4 min) and lyophilization. Analytical HPLC (System B): tR =
18,4 min; yield: 3.6 mg (90%); MS (ESI+): m/z calcd: 1613 [M]+,
found: 1614 [M+H]+, 808 [M+2H]2+, 546 [M+3H]+.
N-(5-(4-(2-(4-(3-Fluoropropyl)piperazin-1-yl)ethyl)-1H-1,2,3-tria-
Radiochemical syntheses
zol-1-yl)pentanamido)-SNEWILPRLPQH (11). BFP
4 (16.6 mg,
84.1 mmol) was added to a suspension of N-(5-azidopentanamido)-
SNEWILPRLPQH (10) bound to resin (23.6 mg, 8.41 mmol) in DMF
No-carrier-added aqueous [18F]fluoride was produced using an IBA
Cyclone 18/9 cyclotron by irradiation of [18O]H2O via the 18O(p,n)18F
nuclear reaction. Synthesis of [18F]FBAM ([18F]1) was performed in
a remotely controlled synthesis module (Nuclear Interface, Mꢃnster,
Germany) as previously described.[31] Analytical radio-HPLC was
performed with either System A or B as described above. All prod-
ucts were monitored by a UV detector at l=220 nm and by g de-
tection with a scintillation detector GABI (Raytest). A difference of
0.2 min between UV and g-trace occurs as a function of the HPLC
setup. Semi-preparative HPLC was performed on a Jasco unit with
a Nucleosilꢄ Standard C18 column (250ꢁ16 mm, 7 mm, 100 ꢂ;
eluent: CH3CN/H2O+0.1% TFA; flow rate: 4.0 mLminꢀ1; gradient:
10–45% CH3CN+0.1% TFA: 0!17 min, 45–95% CH3CN+0.1%
TFA: 17!18 min). Size-exclusion chromatography (SEC) was per-
formed on an ꢅKTA Prime Plus unit using a HighTrap desalting
column (2.5ꢁ1.6 cm, 5 mL) with a calcium buffer (150mm NaCl,
20mm HEPES, 1.2mm MgCl2, 1.3mm CaCl2, pH 7.5) as eluent, at
(1 mL) into
a frit syringe. Then, TBTA (4.5 mg, 8.41 mmol),
CuSO4·5H2O (2.1 mg, 8.41 mmol) in H2O (200 mL), and sodium ascor-
bate (16.6 mg, 84.10 mmol) in H2O (200 mL) were added in this
order, and the reaction was stirred at 508C for 3 days. The resin
was washed with DMF (5 mL), CH3OH (5 mL), and incubated with
BIS (10.6 mg, 16.82 mmol) in DMF/CH3OH/H2O (6 mL, 1:1:1 v/v/v) at
408C for an additional 30 min. The resulting blue solution was re-
moved, and the resin was washed with DMF (5 mL), CH3OH (5 mL),
and Et2O (5 mL). Afterward, the peptide was cleaved using the
standard procedure described above. SNEW peptide 11 was ob-
tained as a colorless powder after purification by preparative HPLC
(10–45% CH3CN+0.1% TFA within 17 min, tR =13.3 min) and lyo-
philization. Analytical HPLC (System B): tR =15.4 min; yield: 5.4 mg
(77%); MS (ESI+): m/z calcd: 1812 [M]+, found: 1813 [M+H]+, 907
[M+2H]2+, 605 [M+3H]+.
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a flow rate of 0.5 mLminꢀ1. Radio-TLC analyses of coupling reac-
tions with [18F]BFP, [18F]AFP, and [18F]FBAM were performed with
Merck RP18 F254 aluminum plates.
HPLC (System B): tR =15.6 min; RCY: 12.5 MBq (1.8%, d.c.); RCP:
>98%; AS: ~5 GBqmmolꢀ1
.
SNEWILPRLPQH-e-(3-(1-(3-[18F]fluoropropyl)-1H-1,2,3-triazol-4-
yl)propanamido)Lys ([18F]14). TBTA (2.9 mg, 5.7 mmol) was added
SNEWILPRLPQHC-[18F]FBAM ([18F]3).
A
solution of [18F]FBAM
to
a suspension of SNEWILPRLPQH-e-(pent-4-ynamido)Lys (13)
([18F]1; 200–350 MBq) in CH3CN (100 mL) was added to a solution of
SNEWILPRLPQHC 2 (470 mg, 295 nmol) in Sørensen phosphate
buffer (100 mL, pH 7.2, 47.9mm Na2HPO4, 18.1mm NaH2PO4) in
a vial and was stirred at 608C for 1 h. The rate of conversion of
[18F]1 into [18F]3 was >94% within 60 min. Analytical radio-HPLC
(System A): tR =11.2 min; RCY: 30–52 MBq (15%, d.c.); RCP: >94%;
bound to resin (12 mg, 0.71 mmol) in DMF (150 mL) into a frit sy-
ringe. Then, CuSO4·5H2O (1.2 mg, 4.95 mmol) in H2O (10 mL) and
sodium ascorbate (1.4 mg, 7.07 mmol) in H2O (10 mL) were added.
Subsequently, [18F]AFP ([18F]5; 2.30–3.34 GBq) in Tris·HCl buffer
(100 mL, pH 8.0) was added, and the solution was irradiated at
50 W for 1 h. The resin was washed with DMF (2 mL), H2O (2 mL),
and incubated with BIS 7 (7.8 mg, 10 mmol) in DMF/CH3OH/H2O
(1 mL, 1:1:0.1 v/v) at 30 W for an additional 20 min. The resin was
washed with DMF (2 mL), H2O (2 mL), and the peptide was cleaved
under microwave irradiation (30 W) for 20 min using TFA/TIS
(700 mL, 95:5 v/v). SNEW peptide [18F]14 was obtained after purifi-
cation by semi-preparative HPLC (tR =20.5 min) and removal of the
solvent under reduced pressure at 508C. Analytical radio-HPLC
(System B): tR =14.1 min; RCY: 46–164 MBq (8–13%, d.c.); RCP:
AS: ~2.6 GBqmmolꢀ1
.
SNEWILPRLPQH-triazolyl-[18F]FP
([18F]7).
TBTA
(0.51 mg,
0.96 mmol) was added to a suspension of SNEWILPRLPQH-Azp (6)
bound to resin (5 mg, 0.31 mmol) in DMF (100 mL) into a frit sy-
ringe. Then, CuSO4·5H2O (0.48 mg, 1.92 mmol) and sodium ascor-
bate (5.61 mg, 28.3 mmol) in Tris·HCl buffer (100 mL, pH 8.0) were
added. Subsequently, [18F]BFP ([18F]4; ~770 MBq) in DMF/H2O
(150 mL, 1:1 v/v) was added, and the solution was microwave irradi-
ated at 50 W for 1 h. The resin was washed with DMF (2 mL), H2O
(2 mL), and incubated with BIS (2.4 mg, 4 mmol) in DMF/CH3OH/
H2O (150 mL, 1:1:0.1 v/v/v) at 30 W for an additional 20 min. The
resin was washed with DMF (2 mL), H2O (2 mL), and the peptide
was cleaved at 30 W for 60 min using TFA/TIS (400 mL, 95:5 v/v).
SNEW peptide [18F]7 was obtained after purification by semi-prepa-
rative HPLC (tR =20.5 min) and removal of the solvent under re-
duced pressure at 508C. Analytical radio-HPLC (System B): tR =
15.1 min; RCY: 1.4 MBq (0.4%, d.c.); RCP: >98%; AS:
>98%; AS: ~5 GBqmmolꢀ1
.
Radiopharmacological studies
Animal experiments were carried out according to the guidelines
of the German Regulations for Animal Welfare. The protocol was
approved by the local Ethical Committee for Animal Experiments.
To investigate in vitro stability, either blood or plasma (400 mL) ob-
tained from male Wistar rats (aged 5–8 weeks) was added to a solu-
tion of all purified radiolabeled peptides [18F]3, [18F]7, and [18F]14
(dissolved in 100 mL PBS, pH 7.5). The rat blood was diluted 1:1
with Ficoll Paque buffer (pH 7.5). The solutions were stirred at
378C for 1 h. Both in vivo stability studies and small-animal PET ex-
periments were also performed in male Wistar rats. Therefore, rats
were positioned and immobilized prone with their medial axis par-
allel to the axial axis of the scanner (microPET P4, Siemens Medical
Solutions). The radiotracer was administered intravenously into
one tail vein (34 MBq, in 30% albumin/E-153 solution, 1 mL) under
desflurane (oxygen/air 7:30%) anesthesia. Transmission scans and
PET acquisition were performed according to a protocol reported
by us in detail previously.[48] The standardized uptake values (SUV)
were calculated over the ROI as the ratio of activity concentration
at time t and injected dose at the time of injection divided by
body weight. The in vivo stability of the radiofluorinated peptide
[18F]14 was determined using arterial blood samples obtained from
rats at various time points during the PET scan. Therefore, blood
samples (400 mL) were withdrawn via catheter from the right femo-
ral artery at 3, 5, 10, 20, 30, 60, and 90 min post-injection. Plasma
was separated by centrifugation (3 min, 16200 g, 48C) followed by
precipitation of the plasma proteins with ice-cold CH3OH (1.5 parts
per 1 part plasma) and centrifugation (3 min, 16200 g, 48C). The
supernatants were analyzed by radio-HPLC on a Zorbax 300SB-C18
(250ꢁ9.4 mm; 5 mm) column with H2O/0.05% TFA (A) and CH3CN/
0.04% TFA (B) as eluents at a flow rate of 2 mLminꢀ1. The follow-
ing gradient was used: 0 min 20% B, at 15 min 50% B, at 16–
20 min 90% B.
~1.0 GBqmmolꢀ1
.
SNEWILPRLPQH-e-(4-(2-(4-(3-[18F]fluoropropyl)piperazin-1-yl)eth-
yl)-1H-1,2,3-triazol-1-yl)Nle ([18F]9). TBTA (1.1 mg, 2 mmol) was
added to a suspension of SNEWILPRLPQH-Anl (8) bound to resin
(12.8 mg, 0.79 mmol) in DMF (200 mL) into a frit syringe. Then,
CuSO4·5H2O (0.13 mg, 0.5 mmol) in H2O (25 mL) and sodium ascor-
bate (1.98 mg, 10 mmol) in H2O (37 mL) were added. Subsequently,
[18F]BFP (460–960 MBq) in Tris·HCl buffer (100 mL, pH 8.0) was
added, and the solution was stirred at 908C for 1 h. The resin was
washed with DMF (2 mL), H2O (2 mL), and incubated with BIS 7
(3.06 mg, 5 mmol) in DMF/CH3OH/H2O (300 mL, 1:1:0.1 v/v) at 30 W
for an additional 20 min. The resin was washed with DMF (2 mL),
H2O (2 mL), and the peptide was cleaved at 30 W for 20 min using
TFA/TIS (600 mL, 95:5 v/v). SNEW peptide [18F]9 was obtained after
purification by semi-preparative HPLC (tR =20.5 min) and removal
of the solvent under reduced pressure at 508C. Analytical radio-
HPLC (System B): tR =14.8 min; RCY: 1.3–4.0 MBq (0.5–1.4%, d.c.);
RCP: >98%; AS: 1.2 GBqmmolꢀ1
.
N-(5-(4-(2-(4-(3-[18F]Fluoropropyl)piperazin-1-yl)ethyl)-1H-1,2,3-
triazol-1-yl)pentanamido)-SNEWILPRLPQH ([18F]11). TBTA (1.7 mg,
3.2 mmol) was added to a suspension of N-(5-azidopentanamido)-
SNEWILPRLPQH (10) bound to resin (5 mg, 0.31 mmol) in DMF
(80 mL) into a frit syringe. Then, CuSO4·5H2O (0.2 mg, 0.8 mmol) and
sodium ascorbate (1.6 mg, 8 mmol) were added. Subsequently,
[18F]BFP (~1.76 GBq) in Tris·HCl buffer (100 mL, pH 8.0) was added,
and the solution was irradiated at 50 W for 1.5 h. The resin was
washed with DMF (2 mL), H2O (2 mL), and incubated with BIS 7
(7.8 mg, 10 mmol) in DMF/CH3OH/H2O (1 mL, 1:1:0.1 v/v) at 30 W
for an additional 20 min. The resin was washed with DMF (2 mL),
H2O (2 mL), and the peptide was cleaved at 30 W for 30 min using
TFA/TIS (600 mL, 95:5 v/v). SNEW peptide [18F]11 was obtained after
purification by semi-preparative HPLC (tR =20.5 min) and removal
of the solvent under reduced pressure at 508C. Analytical radio-
Acknowledgements
The authors are grateful to Waldemar Herzog, Stephan Preusche,
and Tilow Krauss for their excellent technical assistance. We are
very thankful to Peggy Wecke for the preparation of N-Fmoc-(S)-
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 935 – 945 944

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
4-azido-l-proline, Uta Lenkeit for the synthesis of [18F]FBAM, Silke
Fꢁhnemann for the synthesis and supply of bispidine, Andrea
Suhr and Regina Herrlich for in vivo experiments, and Aline Ritter
for ICP-MS measurements. This work was supported in part by
a grant (to C.M.) from the Fonds der Chemischen Industrie (FCI).
This work is part of a research initiative within the Helmholtz-
Portfoliothema “Technologie und Medizin—Multimodale Bildge-
bung zur Aufklꢁrung des In-vivo-Verhaltens von polymeren Bio-
materialien”.
[19] C. Xiong, M. Huang, R. Zhang, S. Song, W. Lu, L. Flores II, J. Gelovani, C.
Li, J. Nucl. Med. 2011, 52, 241–248.
[20] B. Wu, Z. Zhang, R. Noberini, E. Barile, M. Giulianotti, C. Pinilla, R. A.
Houghten, E. B. Pasquale, M. Pellecchia, Chem. Biol. 2013, 20, 19–33.
[21] J. E. Chrencik, A. Brooun, M. I. Recht, G. Nicola, L. K. Davis, R. Abagyan,
H. Widmer, E. B. Pasquale, P. Kuhn, J. Biol. Chem. 2007, 282, 36505–
36513.
[22] M. Berndt, J. Pietzsch, F. Wuest, Nucl. Med. Biol. 2007, 34, 5–15.
[23] F. Wuest, L. Koehler, M. Berndt, J. Pietzsch, Amino Acids 2009, 36, 283–
295.
[24] J. Kapty, T. Kniess, F. Wuest, J. R. Mercer, Appl. Radiat. Isot. 2011, 69,
1218–1225.
[25] M. Pretze, F. Wuest, T. Peppel, M. Kçckerling, C. Mamat, Tetrahedron Lett.
2010, 51, 6410–6414.
[26] C. Mamat, M. Franke, T. Peppel, M. Kçckerling, J. Steinbach, Tetrahedron
2011, 67, 4521–4529.
Keywords: click chemistry · EphB2 · imaging agents · kinases ·
peptides · receptors
[27] E. J. Keliher, T. Reiner, G. M. Thurber, R. Upadhyay, R. Weissleder, Chem.
Open 2012, 1, 177–183.
[28] T. Ramenda, T. Knieß, R. Bergmann, J. Steinbach, F. Wꢃst, Chem.
Commun. 2009, 7521–7523.
[29] M. Glaser, M. Solbakken, D. R. Turton, R. Pettitt, J. Barnett, J. Arukwe, H.
Karlsen, A. Cuthbertson, S. K. Luthra, E. ꢂrstad, Amino Acids 2009, 37,
717–724.
[30] T. Ramenda, J. Steinbach, F. Wuest, Amino Acids 2013, 44, 1167–1180.
[31] J. Marik, J. L. Sutcliffe, Tetrahedron Lett. 2006, 47, 6681–6684.
[32] M. Pretze, C. Mamat, J. Steinbach, J. Labelled Compd. Radiopharm. 2011,
54, S460.
[1] H. Surawska, P. C. Ma, R. Salgia, Cytokine Growth Factor Rev. 2004, 15,
419–433.
[2] R. Noberini, I. Lamberto, E. B. Pasquale, Semin. Cell Dev. Biol. 2012, 23,
51–57.
[3] S. Wang, W. J. Placzek, J. L. Stebbins, S. Mitra, R. Noberini, M. Koolpe, Z.
Zhang, R. Dahl, E. B. Pasquale, M. Pellecchia, J. Med. Chem. 2012, 55,
2427–2436.
[4] E. B. Pasquale, Nat. Rev. Cancer 2010, 10, 165–180.
[5] B. Mosch, B. Reissenweber, C. Neuber, J. Pietzsch, J. Oncol. 2010,
135285.
[6] V. Davalos, H. Dopeso, S. Velho, A. M. Ferreira, L. Cirnes, N. Dꢆaz-Chico,
C. Bilbao, R. Ramꢆrez, G. Rodrꢆguez, O. Falcꢇn, L. Leꢇn, R. C. Niessen, G.
Keller, G. Dallenbach-Hellweg, E. Espꢆn, M. Armengol, A. Plaja, M. Peru-
cho, K. Imai, H. Yamamoto, J. F. Gebert, J. C. Dꢆaz-Chico, R. M. Hofstra,
S. M. Woerner, R. Seruca, S. Schwartz, Jr., D. Arango, Oncogene 2007, 26,
308–311.
[7] E. Batlle, J. T. Henderson, H. Beghtel, M. M. W. van den Born, E. Sancho,
G. Huls, J. Meeldijk, J. Robertson, M. van de Wetering, T. Pawson, H.
Clevers, Cell 2002, 111, 251–263.
[8] Q. Wu, Z. Suo, G. B. Kristensen, M. Baekelandt, J. M. Nesland, Gynecol.
Oncol. 2006, 102, 15–21.
[9] Q. Wu, Z. Suo, B. Risberg, M. G. Karlsson, K. Villman, J. M. Nesland,
Pathol. Oncol. Res. 2004, 10, 26–33.
[10] J. T. Dong, J. Cell. Biochem. 2006, 97, 433–447.
[11] M. Nakada, J. A. Niska, H. Miyamori, W. S. McDonough, J. Wu, H. Sato,
M. E. Berens, Cancer Res. 2004, 64, 3179–3185.
[12] M. Kandouz, K. Haidara, J. Zhao, M. L. Brisson, G. Batist, Cell Cycle 2010,
9, 398–407.
[13] C. Bardelle, T. Coleman, D. Cross, S. Davenport, J. G. Kettle, E. J. Ko,
Bioorg. Med. Chem. Lett. 2008, 18, 5717–5721.
[14] R. Noberini, M. Koolpe, S. Peddibhotla, R. Dahl, Y. Su, N. D. P. Cosford,
G. P. Roth, E. B. Pasquale, J. Biol. Chem. 2008, 283, 29461–29472.
[15] K. Lafleur, D. Huang, T. Zhou, A. Caflisch, C. Nevado, J. Med. Chem. 2009,
52, 6433–6446.
[33] M. Pretze, C. Mamat, J. Fluorine. Chem. 2013, DOI: 10.1016/j.jflu-
chem.2013.02.028.
[34] V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem.
2002, 114, 2708–2711; Angew. Chem. Int. Ed. 2002, 41, 2596–2599.
[35] T. Kniess, M. Kuchar, J. Pietzsch, Appl. Radiat. Isot. 2011, 69, 1226–1230.
[36] B. Shi, M. F. Greaney, Chem. Commun. 2005, 886–888.
[37] P. Grosse-Gehling, F. R. Wuest, T. Peppel, M. Kçckerling, C. Mamat, Radio-
chim. Acta 2011, 99, 365–373.
[38] U. Sirion, H. J. Kim, J. H. Lee, J. W. Seo, B. S. Lee, S. J. Lee, S. J. Oh, D. Y.
Chia, Tetrahedron Lett. 2007, 48, 3953–3957.
[39] M. Chikira, J. Inorg. Biochem. 2008, 102, 1016–1024.
[40] S. Juran, M. Walther, H. Stephan, R. Bergmann, J. Steinbach, W. Kraus, F.
Emmerling, P. Comba, Bioconjugate Chem. 2009, 20, 347–359.
[41] J. M. Collins, N. E. Leadbeater, Org. Biomol. Chem. 2007, 5, 1141–1150.
[42] C. Glaser, Ber. Dtsch. Chem. Ges. 1869, 2, 422–424.
[43] HPLC conditions: Phenomenex Aqua C18 column (250ꢁ4.6 mm, 5 mm,
100 ꢂ; eluent: CH3CN/H2O (60:40 isocratic); flow rate: 1.0 mLminꢀ1
.
[44] M. Galibert, P. Dumy, D. Boturyn, Angew. Chem. 2009, 121, 2614–2617;
Angew. Chem. Int. Ed. 2009, 48, 2576–2579.
[45] C. W. Tornøe, C. Christensen, M. Meldal, J. Org. Chem. 2002, 67, 3057–
3064.
[46] R. K. Jain, J. Controlled Release 1998, 53, 49–67.
[47] D. S. King, C. G. Fields, G. B. Fields, Int. J. Peptide Protein Res. 1990, 36,
255–266.
[48] T. Kniess, M. Laube, R. Bergmann, F. Sehn, F. Graf, J. Steinbach, F. Wuest,
J. Pietzsch, Bioorg. Med. Chem. 2012, 20, 3410–3421.
[16] C. Mamat, B. Mosch, C. Neuber, M. Kçckerling, R. Bergmann, J. Pietzsch,
ChemMedChem 2012, 7, 1991–2003.
[17] C. Giorgio, I. H. Mohamed, L. Flammini, E. Barocelli, M. Incerti, A. Lodola,
M. Tognolini, PLoS One 2011, 6, e18128.
[18] M. Tognolini, M. Incerti, I. Hassan-Mohamed, C. Giorgio, S. Russo, R.
Bruni, B. Lelli, L. Bracci, R. Noberini, E. B. Pasquale, E. Barocelli, P. Vicini,
M. Mor, A. Lodola, ChemMedChem 2012, 7, 1071–1083.
Received: February 4, 2013
Published online on April 4, 2013
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2013, 8, 935 – 945 945