Site-selective radiolabeling of peptides by 18F- fluorobenzoylation with [18F]SFB in solution and on solid phase: Acomparative study

Article abstract of DOI:10.1007/s00726-012-1216-z

Peptides labeled with short-lived positron-emitting radionuclides are of outstanding interest as probes for molecular imaging by positron emission tomography (PET). Herein, the site-selective incorporation of fluorine-18 into lysine-containing peptides us

Full text of DOI:10.1007/s00726-012-1216-z

Amino Acids (2012) 43:1431–1443
DOI 10.1007/s00726-012-1216-z
ORIGINAL ARTICLE
18
Site-selective radiolabeling of peptides by F-fluorobenzoylation
with [18F]SFB in solution and on solid phase: a comparative study
•
Manuela Kuchar Marc Pretze Torsten Kniess
•
•
•
Jorg Steinbach Jens Pietzsch Reik Loser
•
¨
¨
Received: 7 October 2011 / Accepted: 1 January 2012 / Published online: 1 February 2012
Ó Springer-Verlag 2012
Abstract Peptides labeled with short-lived positron-emit-
ting radionuclides are of outstanding interest as probes for
molecular imaging by positron emission tomography (PET).
Herein, the site-selective incorporation of fluorine-18 into
lysine-containing peptides using the prosthetic labeling agent
N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) is descri-
bed. The reaction of [18F]SFB with four biologically relevant
resin-bound peptides was studied and optimized. For com-
parison, each peptide was 18F-fluorobenzoylated in solution
under different conditions and the product distribution was
analyzed confirming the advantages of the solid-phase
approach. The method’s feasibility for selective radiolabel-
ing either at the N-terminus or at the lysine side chain was
demonstrated. Labeling on solid phase with [18F]SFB resul-
ted in crude 18F-fluorobenzoylpeptides whose radiochemical
purities were typically greater than 90% and that could be
prepared in synthesis times from 65 to 76 min.
represent an attractive basis for the development of probes
for molecular imaging (Watt 2009). Positron emission
tomography (PET) is a present-day imaging modality used
for clinical diagnostics and research purposes routinely.
Therefore, peptides labeled with positron-emitting radio-
nuclides are of outstanding interest for the development of
radiotracers that allow to image cellular functions and
dysfunctions in vivo (Chen and Conti 2010; Tolmachev and
Stone-Elander 2010; Kuhnast and Dolle 2010). The most
widely used nonmetallic PET radionuclide is fluorine-18
(18F t = 109.8 min) due to its favorable physical and
‘
chemical properties unparalleled by any other positron
emitter. It can be conveniently produced on a small cyclo-
tron and its low-positron energy of 0.64 MeV accounts for
images of high-spatial resolution. Its half-life of 109.8 min
enables multistep radiosyntheses of tracers and imaging
studies up to 6 h after injection (Pimlott and Sutherland
2010). The conditions for the nucleophilic introduction of
radiofluorine are characterized by temperatures around
100°C, aprotic water-free solvents and precursor molecules
that preferably do not possess acidic hydrogens (Cai et al.
2008). To avoid the exposure of peptides to such harsh
reaction conditions numerous 18F-labeled prosthetic groups
that allow the indirect radiofluorination of peptides have
been developed (Wester and Schottelius 2007; Li and Conti
2010; Schottelius 2010). Among the various amine-reactive
18F-labeled prosthetic groups the acylation agent N-succ-
inimidyl 4-[18F]fluorobenzoate ([18F]SFB) is certainly the
most commonly used. This is reflected by numerous rad-
iosyntheses that have been developed for its automated
Keywords Fluorine-18 Á Solid-phase synthesis Á
Regioselectivity Á Positron emission tomography
Introduction
As peptides have the potential to address a huge number of
biomolecular targets with high affinity and selectivity they
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-012-1216-z) contains supplementary
material, which is available to authorized users.
¨
production (Mading et al. 2005; Vaidyanathan and Zalutsky
M. Kuchar Á M. Pretze Á T. Kniess Á J. Steinbach Á J. Pietzsch Á
¨
R. Loser (&)
2006; Tang et al. 2008, 2010; Glaser et al. 2009; Bejot et al.
2011). Generally, amine-reactive prosthetic groups offer the
advantage of broad applicability as most bioactive peptides
exhibit an N-terminal amino group available for acylation.
Institut fu¨r Radiopharmazie, Helmholtz-Zentrum Dresden-
Rossendorf, Bautzner Landstrasse 400, 01328 Dresden,
Germany
e-mail: r.loeser@hzdr.de
123

1432
M. Kuchar et al.
Furthermore, lysine residues bearing an amino group in the
side chain are very abundant in peptide sequences. On the
other hand, this implies the problem of site-selective 18F-
labeling in the presence of a free N-terminus and one or
multiple lysine residues. This problem has been tackled by
taking advantage of the slight difference between the pKb
values of the N-terminus and the e-amino group of lysine
(Wester et al. 1995; Hermanson 2008; Hoppmann et al.
2008) but the success of this strategy is limited and depends
on the particular peptide or protein to be radiolabeled.
As an alternative to labeling by 18F-fluoroacylation,
methods based on chemoselective reactions such as Huis-
gen’s [3 ? 2] azide-alkyne cycloaddition were developed
(Mamat et al. 2009; Glaser and Robins 2009; Ross 2010).
However, this approach requires the introduction of artifi-
cial moieties into the peptide that can potentially impair its
bioactivity and very often involves volatile 18F-containing
reagents. Furthermore, this method is not necessarily more
efficient concerning the radiochemical yields of the labeled
peptides compared to 18F-fluoroacylation (Hausner et al.
2008; Schottelius 2010).
(Becaud et al. 2009; Jacobson et al. 2011). However, this
technique depends on the presence of electron-withdrawing
groups in ortho position to the leaving group and thus
requires a higher degree of modification of the parent
peptide. Furthermore, by applying this methodology the
separation of the radiolabeled product from the precursor
can be sometimes difficult (Jacobson et al. 2011).
Due to the mentioned drawbacks of the different devel-
oped approaches the selective labeling of peptides with
[18F]SFB remains still a challenge. A site-selective modi-
fication with this reagent can be only achieved by reacting
peptides at one reactive amino group while all others are
protected. As fully protected peptides are often difficult to
handle in solution, radiolabeling should be preferably car-
ried out with peptides attached to a polymeric support. This
is easy to realize because peptides bound to solid support by
their C-terminus can be generated via Fmoc-based solid-
phase synthesis. The 18F-labeling of peptides by fluoroa-
cylation on solid supports has been sporadically reported
(Sutcliffe-Goulden et al. 2000, 2002; Marik et al. 2006) but
not by employing [18F]SFB and never by a direct compar-
ison to fluoroacylation in solution. Therefore, the aim of this
study was to evaluate the potential to label various lysine-
containing peptides with [18F]SFB at defined sites by solid-
phase synthesis. The obtained results were systematically
In a different prosthetic group-based approach the ra-
diofluorine was directly introduced into peptides contain-
ing benzoyl moieties bearing a trimethylammonium or
nitro leaving group by nucleophilic aromatic substitution
NH2
NH2
OH
18F
OH
O
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H
N
H
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
H
H
N
N
N
N
N
N
NH2
N
N
N
H
N
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
H
H
H
O
O
O
O
O
O
O
O
O
O
O
O
HO
O
18F
NH2
O
HO
HO
18F]4
[
18F]2
HO
O
[
O
HO
HO
NH2
NH2
OH
OH
O
H
N
O
OH
O
O
O
O
O
O
O
O
O
O
O
O
H
H
N
H
H
N
H
N
H
H
N
H
N
H
N
H
N
H2
N
N
N
N
N
N
H
N
N
H
N
H
N
H
NH2
N
H
H2
N
N
H
N
N
H
N
H
N
H
N
H
H
O
O
O
O
O
O
O
O
O
O
O
O
NH2
O
HO
HO
HO
3
HO
O
1
O
O
HO
HO
O
O N
O
18F
N-Succinimidyl-4-
[
18F]fluorobenzoate ([18F]SFB)
HN
NH2
H2
N
NH
H2
N
NH
H2
N
O
NH2
NH2
O
O
NH2
HN
O
HO
O
O
HN
O
HN
O
HO
O
O
O
O
O
O
O
H
H
H
N
H
N
H
H
N
H
N
H
N
H
N
H
H
N
N
N
NH2
O
N
N
N
N
N
N
H2
N
N
H
N
H
N
H
NH2
N
H
N
H
N
H2
N
N
N
N
H
H
H
H
H
O
O
N
O
N
O
O
O
O
O
O
O
O
O
H2
7
HN
5
O
HN
NH2
NH
NH2
NH2
NH
NH2
NH2
18F
HN
NH2
H2
N
H2
N
HN
O
NH2
O
NH2
O
O
O
NH2
HO
O
O
HN
O
HN
O
HN
O
HO
H2
O
O
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
NH2
O
N
N
N
N
H
N
N
N
H
N
H
N
H
NH2
N
N
H
N
N
H
N
H
N
H
N
H
H
H
O
N
O
O
O
O
O
O
O
O
O
O
O
18F
H2
N
18F]8
[
18F]6
HN
[
O
HN
NH2
NH2
NH2
NH2
NH2
Fig. 1 Structures and desired sites of 18F-fluorobenzoylation of the investigated peptides
123

Site-selective radiolabeling of peptides
1433
Table 1 Sequences of
investigated peptides
Peptide
Original sequence
Sequence modified for this study
Derivative of a1(I)-N-telopeptide
1(I)-N-telopeptide
k7-fragment
GGGDPKGGGGG
GGGDPKGGGGG-NH2
LSYGYDEKSTGISVP-NH2
KKRKAKKKRKFA-NH2
SNEWILPRLPQHK-NH2
(1)
(3)
(5)
(7)
pQLSYGYDEKSTGISVP
KKRKAKKKRKFA
SNEWILPRLPQH
SNEW peptide
compared to those of 18F-fluorobenzoylation in solution
regarding radiochemical yield and radiochemical purity of
the labeled peptides. The selection of peptides subjected to
the radiolabeling experiments and the corresponding
desired labeled products are shown in Fig. 1 and their
sequences can be found in Table 1.
nonradioactive peptides by semi-preparative HPLC was
done using a Hewlett Packard 1050 system with automatic
injector and UV detector at a volumetric flow rate of 4 mL/
min and a Varian Prepstar system equipped with UV
detector (Prostar, Varian) and automatic fraction collector
Foxy 200 at a flow rate of 20 mL/min. Chromatographic
purification of radiolabeled peptides was performed with a
Jasco HPLC system equipped with UV detector (UV-2075)
and radioactivity detector (type Gabi, Raytest). A Nucleosil
100-5 C18 column (VP 250/10, Macherey–Nagel) served
as stationary phase using gradient systems B, D, E, or G.
[18F]SFB was produced in an automated radiosynthesis
Materials and methods
General remarks
All chemical reagents and solvents were obtained from
commercial suppliers and used without further purification.
Fmoc-protected amino acids and coupling reagents were
purchased from MultiSynTech (Witten, Germany) and Iris
Biotech (Marktredwitz, Germany). Fmoc-Rink-Amide
MBHA polystyrene resin was obtained from Multisyntech.
Mass spectra were obtained on a Quattro/LC mass spec-
trometer (Micromass) equipped with an electrospray ioni-
zation source. Generally, mixtures of water and acetonitrile
each containing 0.1% TFA were used for HPLC with the
wavelength for UV detection at 220 nm. Gradient systems
and flow rates are specified in Table 2 and Supplementary
Material. Analytical HPLC and radio-HPLC were per-
formed on an Agilent Technologies 1200 system equipped
with automatic injector, UV detector and radioactivity
detector (type Gabi, Raytest). Analyses were carried out
with a Nucleosil 100-5 C18 Nautilus column (VP 250/10,
Macherey–Nagel) operated by system A, Nucleosil 100-5
C18 PPN column (VP 250/4, Macherey–Nagel) operated
by system C and Nucleosil 100-7 C18 EC (VP 250/4,6;
Macherey–Nagel) operated by system F. Purification of
using a remotely controlled synthesis module (GE
¨
TracerLab) in a hot cell as described (Mading et al. 2005)
with implemented refinements as reported recently (Kapty
et al. 2011). The stability of [18F]SFB was assessed by
radio-TLC performed on Merck silica gel F-254 aluminum
plates using a mixture of ethyl acetate and petroleum ether
(1:1) as mobile phase. Detection was performed by radio-
luminography in a Fuji BAS 2000 scanner. The results
were confirmed by radio-HPLC using system C. Nonra-
dioactive SFB as standard for chromatography was syn-
thesized according to Wester et al. (1996).
Peptide synthesis
Peptides were synthesized by microwave-assisted, fully
automated solid-phase peptide synthesis based on the Fmoc-
protecting group strategy with appropriately side chain-
protected L-amino acids as building blocks using a CEM
Liberty Microwave Peptide Synthesizer combined with a
CEM Discover microwave reactor. The Rink-amide MBHA-
functionalized resin was Fmoc-protected and had a loading
capacity of 0.6 mmol/g. A coupling cycle took 45 min and
started with the deprotection using 0.1 M N-hydroxyben-
zotriazole (HOBt) in 20% piperidine/DMF (2 9 7 mL)
followed by washing with DMF. Coupling of the amino acids
was performed by addition of a 0.2 M solution of the cor-
responding Fmoc-protected amino acid in DMF (5 equiv.,
2.5 mL), 0.45 M O-benzotriazolyl-N,N,N’,N’-tetramethyl-
uronium hexafluorophosphate (HBTU) in DMF (1.0 mL),
and 2 M diisopropylethylamine (DIPEA) in N-methylpyrr-
olidone (0.5 mL). The automated synthesis ended with a
final deprotection of the N-terminal Fmoc group. Resins
loaded with N-terminally deprotected peptides 1, 3, and 5
Table 2 HPLC systems
Peptide
Analytical HPLC
system
Preparative HPLC
system
Derivative of
a1(I)-N-telopeptide
System A
System B
System D
a1(I)-N-telopeptide
k7-fragment
System C
System E
System F
SNEW peptide
System G
Details can be found in Supplementary Material
123

1434
M. Kuchar et al.
were used as precursors for acylation with fluorobenzoyl
chloride as well as for solid-phase radiolabeling with
[18F]SFB. In addition, peptides 1, 3, and 7 were synthe-
sized with an Alloc-protected lysine side chain. In these
cases, N-terminal Fmoc deprotection was followed by tert-
butoxycarbonylation of the free N-terminus. As fully
deprotected peptides were required for 18F-labeling in
solution as well as standards for HPLC, portions of the
resins loaded with N-terminally deprotected peptides 1, 3,
Alloc-3, 5, and 7 were cleaved before further modification.
Generally, TFA-mediated cleavage and deprotection was
carried out in a syringe equipped with a frit filter at the
bottom using 10 mL of 95% TFA/H2O. Exceptionally,
peptides 7, 8, and iso-8 were released from the resin with
10 mL of reagent K (85% TFA, 5% H2O, 5% thioanisole,
2.5% ethanedithiol) (King et al. 1990). The solutions
containing the crude peptides were concentrated in a
stream of nitrogen, precipitated by addition of cold diethyl
ether, and the solids were collected by filtration. The
peptides were purified by HPLC and characterized by mass
spectrometry.
Analytical data for Na-4-fluorobenzoyl peptides:
2. HPLC retention times: 19.7 min (system A), 28.0 min
(system B); MS (ESI) m/z 937.2 ([M ? H]?), 469.5
([M ? 2H]2?
)
4. HPLC retention times 17.0 min (system C), 42.5 min
(system D); MS (ESI) m/z 1740.1 ([M ? H]?), 881.5
([M ? 2H]2?
)
Alloc-4. HPLC retention time 21.7 min (modified-sys-
tem C); MS (ESI) m/z 909.0 ([M ? 2H]2?
6. HPLC retention time 28.2 min (system E); MS (ESI)
m/z 869.6 ([M ? 2H]2?), 580.4 ([M ? 3H]3?
)
)
iso-8. HPLC retention times 11.0 min (system F),
30.5 min (system G); MS (ESI) m/z 1740.8
([M ? H]?), 870.8 ([M ? 2H]2?).
Synthesis of Ne-4-fluorobenzoyl peptides
The fluorobenzoylation of the side chain for the synthesis
of peptides iso-2, iso-4, and 8 was carried out for peptidyl
resins containing 1, 3, and 7 with Alloc-protected lysine
side chains. The preswollen resins (amount corresponding
to 0.1 mmol of resin-bound peptide) were incubated with
di-tert-butyl dicarbonate (44 mg, 0.2 mmol) and triethyl-
amine (28 lL, 0.2 mmol) in DMF (5 mL) for 3 h followed
by washing as described above and deprotection of the
lysine residue with diethylamine (21 lL, 0.2 mmol)
Pd(PPh3)4 (58 mg, 0.05 mmol) in dichloromethane (5 mL)
for 2 h. The Pd catalyst was removed by washing with
DMF, 0.5% sodium N,N-diethyldithiocarbamate in DMF
(m/v), dichloromethane (3 mL each, washing procedure
was repeated three times) (Shepherd et al. 2004). A portion
of peptidyl resin containing 7 treated in this manner was
used as precursor for labeling with [18F]SFB on resin. The
nonradioactive fluorobenzoylations of the lysine side
chains were performed in the same fashion as the N-ter-
minal fluorobenzoylations described above. Cleavage of
the peptides from the resin was done as described.
Analytical data for unmodified peptides:
1. HPLC retention times 15.4 min (system A), 18.0 min
(system B); MS (ESI) m/z 815.1 ([M ? H]?), 408.3
([M ? 2H]2?
)
3. HPLC retention times 11.4 min (system C), 29.5 min
(system D); MS (ESI) m/z 1617.1 ([M ? H]?) 809.2
([M ? 2H]2?
)
Alloc-3. HPLC retention time 37.6 min (modified-sys-
tem C); MS (ESI) m/z 1698.5 ([M-H]-), 848.5 ([M-
2H]2-
)
5. HPLC retention time 21.5 min (system E), MS (ESI)
m/z 808.3 ([M ? 2H]2?), 539.6 ([M ? 3H]3?
)
7. HPLC retention times 3.3 min (system F); 22.8 min
(system G); MS (ESI) m/z 1619.4 ([M ? H]?), 810.4
([M ? 2H]2?).
Synthesis of Na-4-fluorobenzoyl peptides
Analytical data for Ne-4-fluorobenzoyl peptides:
iso-2. HPLC retention times 22.5 min (system A),
40.8 min (system B); MS (ESI) m/z 937.1 ([M ? H]?),
4-Fluorobenzoyl chloride (FBzCl) was reacted with the
deprotected N-termini of peptides 1, 3, 5, and 7 for the
synthesis of peptides 2, 4, 6, and iso-8. The preswollen
resins (amount corresponding to 0.1 mmol of resin-bound
peptide) loaded with peptides 1, 3, Alloc-3, 5, and 7 were
incubated with a solution of 4-fluorobenzoyl chloride
(60 lL, 0.5 mmol) and triethylamine (71 lL, 0.5 mmol) in
DMF (5 mL) for 1 h followed by washing the resin with
dichloromethane (10 mL), DMF (10 mL) and ethanol
(10 mL). The completion of the reaction was proven by the
Kaiser test (Kaiser et al. 1970) before the fluorobenzoy-
lated peptides were cleaved from the resin as described
above.
480.3 ([M ? 2H]2?
)
iso-4. HPLC retention times 13.8 min (system C),
41.0 min (system D); MS (ESI) m/z 867.9 ([M-2H]2?
)
8. HPLC retention times 9.8 min (system F), 29.1 min
(system G); MS (ESI) m/z 1741.3 ([M ? H]?), 871.4
([M ? 2H]2?).
Synthesis of N-succinimidyl 4-[18F]fluorobenzoate
[18F]SFB was synthesized according to the procedure
18
described in (Mading et al. 2005). [ F]Fluoride was
¨
123

Site-selective radiolabeling of peptides
1435
produced on a PET cyclotron Cyclone 18/9 (IBA, Bel-
gium); [18O]H2O was irradiated with protons exploiting the
18O(p,n)18F nuclear reaction. After bombardment, the
[18O]H2O containing [18F]F- was transferred from the
cyclotron to an anion exchange cartridge (Sep-PakÒ Light
Waters AccellTM Plus QMA cartridge) placed in a com-
mercially available synthesizer TRACERlabFDG for the
nucleophilic fluorination (GE Medical Systems, Mu¨nster,
Germany). The automated radiosynthesis of [18F]SFB was
performed as described in modified form in (Kapty et al.
2011). In a typical experiment starting from 16 GBq
[18F]fluoride 4 GBq [18F]SFB could be obtained after final
solid-phase extraction in radiochemical purity [95%. For
determination of the radiochemical and chemical purity of
the final product an HPLC System (Hewlett Packard series
1050) was used, including pump, injector and UV-detector
(254 nm) coupled in series with a radioactivity detector
GABI (raytest, Straubenhardt, Germany) and Nucleosil
Nautilus C-10 column (5 lm, 250 9 4.6 mm ? precol-
umn 4 9 4 mm). HPLC analyses were carried out at a flow
rate of 0.5 mL/min with the following gradient of eluents
(A = MeCN with 0.1% TFA; B = H2O with 0.1% TFA):
0 min, 10% A/90% B; 10 min, 60% A/40% B; 14 min,
100% A: 25 min, 100% A.
by semi-preparative HPLC was tried exemplarily for [18F]4
formed at pH 9 and 25°C.
18F-Fluorobenzoylation of Alloc-3 in solution
Radiolabeling was done as described above under the fol-
lowing conditions: pH 7, 50°C; pH 9, 25°C; pH 9, 50°C.
After 30 min of incubation a mixture of 0.05 eq Pd(PPh3)4
(3.5 mg, 0.003 lmol) and morpholine (12 lL of a 0.1 M
solution in THF) in THF (100 lL) was added and incu-
bated for 20 min at the corresponding reaction tempera-
tures. The reaction mixture was subjected to solid-phase
extraction using a Chromafix C-18 cartridge (200 mg,
Macherey–Nagel) preconditioned with 5 mL of THF fol-
lowed by 5 mL of water). The product was eluted from the
cartridge with THF (200 lL) and analyzed by radio-HPLC.
18F-Fluorobenzoylation of resin-bound peptides
Reactions were carried out in a polypropylene syringe
equipped with a frit at the bottom and a cartridge adapter
on top. The peptidyl resin (preswollen in DMF) was sus-
pended in a solution of [18F]SFB (500–2500 MBq) in water
(100 lL), DMF (200 lL) and 0.15 M sodium phosphate
buffer pH 7 (100 lL). The reaction mixture was gently
agitated in a heating bath at 50°C for 30–50 min ([18F]2
and [18F]4: 30 min, [18F]6: 40 min, [18F]8: 50 min). The
liquid phase was removed by filtration and the resin was
washed with DMF and water (2 9 1 mL each) to remove
unreacted [18F]SFB. The resin-bound radioactivity was
measured in an activity meter. The cleavage reagent
([18F]2, [18F]4, and [18F]6: 400 lL 95% TFA/water; [18F]8:
400 lL reagent K) was added to the resin and the resulting
mixture incubated at 50°C for 20 min in a thermomixer.
The product-containing solution was collected by filtration
and concentrated in a stream of argon. The radioactivity of
the filtrate and of the resin was determined. Aliquots of the
crude products were analyzed by radio-HPLC. In the
meantime, the products were purified by semi-preparative
radio-HPLC followed by the evaporation of the eluent.
Data are shown in Table 4.
Stability of N-succinimidyl 4-[18F]fluorobenzoate
The stability of [18F]SFB was evaluated in 0.15 M sodium
phosphate at pH values of 7.0, 9.0, 9.2, 9.4, 9.6, 9.8, and
10.0 at 25°C as well as at 50°C. The composition of the
[18F]SFB solutions were analyzed by radio-TLC after 3,
10, 20, 30, and 60 min and for pH 7.0, 25°C in addition by
radio-HPLC after 1 h using system C. Additionally, the
aqueous [18F]SFB obtained after radiosynthesis was ana-
lyzed immediately and after 5 h by radio-TLC as well as by
radio-HPLC using system C. The results are shown in
Table S1 of Supplementary Material.
18F-Fluorobenzoylation of peptides 1, 3, 5, and 7
in solution
A solution of [18F]SFB (50–100 MBq) in water (25 lL) as
obtained by the radiosynthesis was added to a solution of
the corresponding peptide (0.5–1.0 mg) in 0.15 M sodium
phosphate (200 lL). The pH values of the phosphate-buf-
fered peptide solutions were 7 and 9 (compounds 1, 3, and
7) and 6, 7, and 9 (compound 5). The reaction mixtures
were incubated at 25°C and 50°C in a thermomixer for
30 min. The radiochemical yields of the formed 18F-flu-
orobenzoylated peptides were determined by analyzing the
reaction mixtures by radio-HPLC. The results are shown in
Table 3 and all radio-chromatograms are included in
Supplementary Material. Isolation of a radiolabeled peptide
Results and discussion
All peptide sequences considered in this study were
assembled by microwave-assisted solid-phase peptide
synthesis using Rink-amide linker-functionalized polysty-
rene-based resins to obtain C-terminal amides in quantita-
tive yields. The purities of the crude peptides ranged
between 72 and 94% and their identities were confirmed by
ESI–MS. To enable an accurate analysis of labeling with
[18F]SFB in solution, the nonradioactive standards of both
123

1436
M. Kuchar et al.
NH2
NH2
OH
F
OH
O
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H
H
N
H
H
H
N
H
H
N
H
N
H
H
H
N
H
N
N
N
N
N
N
N
N
N
NH2
N
N
N
N
N
N
N
H
N
N
H
N
H
N
H
N
H
H
H
H
H
H
H
H
O
O
O
O
O
O
O
O
O
O
O
O
O
NH2
F
O
HO
HO
HO
2
HO
O
F
O
4
HO
HO
F
HN
O
O
OH
HN
O
OH
O
H
N
OH
O
O
O
O
O
O
H
H
N
H
H
H
N
H2
N
N
N
N
N
O
N
N
N
H
N
H
N
H
N
H
N
H
O
O
O
O
O
H
H
H
H
H
N
H
H
O
O
O
O
O
O
O
N
N
N
N
NH2
N
NH2
H2
N
N
N
N
O
N
HO
HO
H
H
H
H
O
O
HO
O
O
O
HO
O
iso-4
HO
iso-2
HO
O
F
HN
HN
NH2
HN
O
O
O
NH2
HO
O
O
HO
H2
O
O
O
O
O
H
N
H
H
N
H
N
H
N
N
NH2
N
N
N
H
N
N
H
N
H
N
N
H
H
O
N
O
O
N
O
O
O
H2
HN
O
8
HN
HN
NH2
H2
N
O
O
NH2
HN
O
HO
O
O
HO
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
NH2
N
N
H
N
H
N
N
H
N
H
N
N
H
H
O
O
N
O
O
N
O
O
F
H2
HN
O
HN
iso-8
Fig. 2 Isomers of fluorobenzoylated peptides 2, 4, and 8
the desired as well as the alternative 18F-fluorobenzoylation
products of the peptides 1, 3, and 7 were prepared. The
pairs of isomeric fluorobenzoylated products are depicted
in Fig. 2. The synthesis of the nonradioactive reference
compounds 2, 4, 6, and iso-8 was accomplished by reacting
the corresponding resin-attached peptides with 4-fluor-
obenzoyl chloride on their N-terminus. The side-chain
fluorobenzoylated peptides iso-2, iso-4, and 8 were syn-
thesized by incorporation of Fmoc-Lys(Alloc)-OH to allow
a selective deprotection of the lysine e-amino group. Pro-
tection of the N-terminus and deprotection of the e-amino
group was done as shown for [18F]8 in Scheme 3, fluor-
obenzoylation and cleavage/deprotection were carried out
congruently to the N-terminally modified peptides.
be advantageous for 18F-fluorobenzylation of lysine e-
amino groups, however this pH is the upper limit beyond
which substantial hydrolysis is taking place (Wester et al.
1995; Hermanson 2008). Indeed, at pH values above 9.5 a
rapid degradation of the radiolabeling agent could be
observed even at 25°C (see Supplementary Material).
Initial investigations were focused on the radiolabeling
of peptides 1 and 3. Compound 1 is based on a model
sequence derived from the N-terminal telopeptide of the
a1-chain of type I-collagen (Nagan and Kagan 1994)
whereas 3 represents the authentic telopeptide (Helseth
et al. 1979) whose N-terminal pyroglutamate residue was
removed to allow replacement by a 4-fluorobenzoyl group.
The central lysine residues of both derivatives undergo
modification in vivo and, therefore, a radiolabeling that is
selective for their N-terminus is desirable. These biologi-
cally relevant peptides containing only one lysine residue
seemed to be suitable for systematic investigations con-
cerning the outcome of 18F-fluorobenzoylation in solution.
Peptides 1 and 3 were subjected to reaction with [18F]SFB
in solution for 30 min at pH 7 and pH 9 each at 25 and
50°C. Labeling in solution of 1 resulted in the formation of
[18F]2 as the main component independent of the employed
conditions (Table 3). The highest yield of 70% for [18F]2
The separation of the isomeric fluorobenzoylated pep-
tides by analytical HPLC required careful individual opti-
mization regarding both the mobile and stationary phase.
Details can be found in Supplementary Material.
Prior to the labeling experiments the stability of
[18F]SFB in aqueous solution at different pH and temper-
ature was investigated. The 18F-fluorobenzoylation agent
revealed more stable at pH 7/50°C than at pH 9/25°C.
These pH values were selected as pH 7 is known to favor
radiolabeling at the N-terminus and pH 9 is considered to
123

Site-selective radiolabeling of peptides
1437
could be obtained at pH 9/25°C and the ratio of [18F]2 to
[18F]iso-2 did not change much with the different
conditions.
Compared to 1, the conversion of [18F]SFB with 3 was
poor at pH 7 and 25°C with 50% of unconsumed acylation
agent remaining. This emphasizes the dependence of the
radiolabeling efficiency on the peptide sequence. The ratio
of the isomeric 18F-fluorobenzoylated peptides was at pH
7/25°C with 65:35 less favorable than in the case of 1.
Almost equal amounts of radiolabeled isomers were
formed at pH 7/50°C and pH 9/25°C (Fig. 3) whereas the
ratio reversed by increasing both pH and temperature.
Attempts to isolate [18F]4 by semi-preparative HPLC failed
due to poor separation of the fluorobenzoylated isomers. In
conclusion, an efficient and selective radiolabeling of 3 in
solution revealed as not feasible.
Labeling with [18F]SFB on solid phase was optimized
for peptides 1 and 3 regarding the volume of solvent and
temperature. Concerning the amount of peptide-loaded
resin, the precursor masses and solvent volumes that cor-
respond to those that are typically employed for reaction of
peptides with [18F]SFB in solution were chosen (Richter
et al. 2009), lower amounts led to less favorable results.
The optimization of the reaction conditions was done on
the basis of determining the ratio of the resin-bound
radioactivity related to the applied activity of [18F]SFB
(see Table S2 in Supplementary Material). As acylations
with N-hydroxysuccinimide esters are very often per-
formed in aqueous media (Sewald and Jakubke 2002;
Hermanson 2008) 0.15 M sodium phosphate at pH 7 was
tested as reaction medium beside different organic solvents
common for solid-phase organic synthesis on polystyrene-
based resins. A pH of 7 was also preferable on basis of the
results obtained in the [18F]SFB-stability assays. Interest-
ingly, performing the reaction with the preswollen peptide-
loaded resin suspended in a mixture of aqueous phosphate-
buffered solution and DMF revealed as superior to pure
organic solvents. An elevated temperature of 50°C was
clearly advantageous over lower temperatures. In the
optimized procedure peptidyl resins (amounts correspond-
ing to 2–3 lmol of resin-bound peptide) preswollen in
DMF were incubated with a solution of [18F]SFB in DMF/
aqueous buffer pH 7 (1:1) at 50°C for 30 min (Scheme 1).
After washing to remove unreacted acylation agent and
TFA-mediated deprotection and cleavage from the resin
[18F]2 and [18F]4 could be obtained in a radiochemical
purity of 98–99 and 83–97%, respectively (Fig. 4).
Exemplary protocols of activity distribution for radio-
labeling of 1 and 3 can be found in Table S2 of the Sup-
plementary Material. Noteworthy, the resin-bound activity
could be quantitatively released by TFA-mediated cleavage
with no unconverted [18F]SFB being detectable in the
crude peptides indicating that unspecific binding of the 18F-
123

1438
M. Kuchar et al.
(A)
3.0×1006
(B)
300000
18F]4
[
18F]iso-4
[
18F]4
[
18F]iso-4
[
2.0×1006
1000000
0
200000
100000
0
0
5
10
15
20
25
0
5
10
15
20
25
time (min)
time (min)
Fig. 3 Radiolabeling of 3 with [18F]SFB in solution (0.15 M sodium phosphate buffer). Radio-HPLC-chromatograms of reaction mixtures after
30 min for a pH 7, 50°C; b pH 9, 25°C
Scheme 1 N-terminal 18F-
fluorobenzoylation of 1 on solid
phase. Reagents and conditions:
a [18F]SFB, DMF/0.15 M
sodium phosphate pH 7 (1:1),
50°C, 30 min; b TFA, 50°C,
20 min. Solid-phase
O
HN
O
O
O
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
N
H
H2N
N
H
N
H
N
N
H
N
H
O
O
O
O
O
O
O
radiolabeling of 3 and 5 was
done accordingly
O
O
a
O
HN
O
O
O
O
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
N
H
N
H
N
H
N
H
N
N
H
N
H
O
O
O
O
O
O
18F
O
O
O
b
NH2
O
O
O
O
O
O
O
H
N
H
N
H
N
H
N
H
N
NH2
N
H
N
H
N
H
N
N
H
N
H
O
O
O
O
O
18F
HO
O
Fig. 4 Radiolabeling of 1 and 3
with [18F]SFB on solid phase.
Shown are the preparative
(A)
150
(B)
30
[
18F]4
radiochromatograms during
purification of [18F]2 (a) and
18F]4 (b) cleaved from the resin
[
18F]2
[
20
10
0
100
50
0
0
10
20
30
40
50
60
0
10
20
30
40
50
60
time (min)
time (min)
123

Site-selective radiolabeling of peptides
1439
Table 4 Results of radiolabeling with [18F]SFB on solid phase
Protected and resin- Radiochemical purity (%)
bound peptide
precursor
Isolated ? decay-corrected Activity range of Synthesis time Total
radiochemical yield (%)
n
isolated product
(MBq)
for crude synthesis
product (min) time (min)
After cleavage After purification
1
3
5
7
98
90
99
1
7
1
[99
98
–
14
14
11
5
2
4
1
2
80–125
70–172
30–35
65
64
73
76
5
5
3
6
135
137
143
153
5
3
3
7
28
11
4
2
1
54 25
95
15–36
2
fluorobenzoylation agent to the polystyrene matrix does not
occur.
peptide 3 containing an Alloc-protected e-amino group
(Alloc-3; Scheme 2). The 18F-fluorobenzoylation was
performed under conditions identical to liquid-phase
labeling of 1 and 3 except that reaction at pH 7 and
25°C was omitted. Subsequently to reaction with
[18F]SFB the reaction mixtures were treated with
Pd(PPh3)4 and morpholine for side chain deprotection.
Substantial formation of [18F]fluorobenzoic acid could be
observed in the radio-HPLC-chromatograms with ana-
lytical radiochemical yields of [18F]4 not exceeding 10%.
Therefore, the potential of this approach for site-selective
labeling with [18F]SFB seems to be limited and clearly
disadvantageous compared to 18F-fluorobenzoylation on
solid phase.
The following purification by semi-preparative HPLC to
separate labeled and unlabeled peptide provided [18F]2 in a
radiochemical purity of 98–99%, a high chemical purity
and a decay-corrected radiochemical yield of 12–15%.
Purification of [18F]4 led to a radiochemical purity of
96–99%, a high chemical purity and a decay-corrected
radiochemical yield of 8–14% (Table 4, Fig. S7B of Sup-
plementary Material).
To further evaluate the advantages of the solid-phase
approach, it should be compared to the reaction of
peptides containing a protected lysine side chain with
[18F]SFB in solution. This was done exemplarily for
Scheme 2 Fluorobenzoylation
of Alloc-protected 3 in solution.
Reagents and conditions:
O
HN
O
O
OH
a [18F]SFB, 0.15 M sodium
O
OH
OH
phosphate buffer pH 7 and 9,
30 min; b Pds(PPh3)4,
morpholine, 20 min
O
O
O
O
O
O
H
H
N
H
H
N
H
H
N
H2N
N
N
N
N
N
N
N
H
N
H
N
H
N
H
N
H
H
H
O
O
O
O
O
O
O
NH2
O
HO
HO
HO
O
HO
HO
a
O
HN
O
O
OH
18F
OH
O
OH
O
O
O
O
O
O
H
N
H
H
N
H
H
N
H
H
N
N
N
N
N
N
N
N
H
N
H
N
H
N
H
N
H
H
H
O
O
O
O
O
O
O
O
NH2
O
HO
HO
HO
O
HO
HO
b
NH2
O
OH
18F
OH
O
OH
O
O
O
O
O
O
H
N
H
H
N
H
H
N
H
H
N
N
N
N
N
N
N
N
H
N
H
N
H
N
H
N
H
H
H
O
O
O
O
O
O
O
O
NH2
O
HO
HO
HO
O
HO
HO
123

1440
M. Kuchar et al.
Fig. 5 Radiolabeling of 5 with
18F]SFB in solution (0.15 M
(A)
20
(B)
30
[
sodium phosphate buffer).
Radio-HPLC-chromatograms of
reaction mixtures after 30 min
at 25°C for a pH 7; b pH 9; c pH
6
[
18F]6
15
10
5
20
10
0
[
18F]6
[
18F]SFB
[
18F]SFB
0
0
10
20
30
40
50
60
0
10
20
30
40
50
60
time (min)
time (min)
(C)
10
8
[
18F]6
6
[
18F]SFB
4
2
0
0
10
20
30
40
50
60
time (min)
Very often biologically active peptides comprise mul-
tiple lysine residues as exemplified with the k7-fragment 5,
a cell-penetrating peptide (Rennert et al. 2008). This seven
lysine residues containing peptide was used for investiga-
tion towards labeling with [18F]SFB. The acylation in
solution was tried at 25°C, pH 7 and 9. As expected, the
18F-fluorobenzoylation in solution resulted in a complex
mixture of products (Fig. 5a, b) whose minor component
was the desired product accompanied by side-chain labeled
peptides represented by broad peaks in the chromatograms
indicating the random distribution of the 18F-label among
the different lysine residues. To shift the product spectrum
to the favor of the N-terminally labeled peptide, the reac-
tion of 5 with [18F]SFB was tried at pH 6. As obvious from
Fig. 5c, this did not result in a more selective radiolabeling.
Considering these results it was refrained from labeling of
5 at 50°C.
essentially binding to the extracellular domain of EphB2, a
member of the Eph/ephrin tyrosine kinase receptor family
(Koolpe et al. 2005; Chrencik et al. 2007). One peptide of
the latter class was selected to demonstrate the feasibility
of the solid-phase approach for selective radiolabeling at
lysine side chains. For this purpose the parent sequence
SNEWILPRLPQH was C-terminally extended by a lysine
residue. To allow selective deprotection, lysine was
incorporated with an Alloc-protected side chain. After
protecting the N-terminus of the peptide with Boc, the side-
chain amino group of the C-terminal lysine was deprotec-
ted by Pd(0)-catalyzed allyl transfer to diethyl amine. This
40
[
18F]6
30
20
10
0
In contrast, 18F-fluorobenzoylation of resin-bound pro-
tected 5 provided [18F]6 in an excellent radiochemical
purity (Fig. 6) of 99% and an overall decay-corrected yield
of 11 1% (n = 4) (Table 4).
The examples of radiolabeled peptides discussed so far
all contained a functional lysine residue. However, there
are several peptides whose biological functionality is crit-
ically dependent on a free N-terminus. Examples are
ligands of protease-activated receptors (Barry et al. 2006)
or peptides containing the N-terminal sequence SNEW
0
10
20
30
40
50
60
time (min)
Fig. 6 Radiolabeling of 5 with [18F]SFB on solid phase. Shown is the
radio-HPLC-chromatogram of the crude product
123

Site-selective radiolabeling of peptides
1441
O
O
S
O
O
P
Ph
Ph
Ph
NH
HN
HN
O
NH
O
O
O
NH
O
O
O
O
O
O
O
O
O
H
N
H
H
N
H
N
H
H
N
N
N
N
N
N
H
N
H
N
H2N
N
N
H
H
H
O
O
O
O
O
O
O
H
O
Ph
Ph
N
N
O
N
Ph
O
O
N
Ph
Ph
Ph
O
O
a
O
S
O
P
Ph
Ph
Ph
NH
HN
HN
O
NH
O
O
O
NH
O
O
O
O
O
O
O
O
O
O
H
N
H
H
N
H
H
N
H
N
N
N
N
N
N
H
N
H
N
O
N
N
N
H
H
H
H
O
O
O
O
O
O
O
H
O
Ph
Ph
N
N
O
N
Ph
O
O
N
Ph
Ph
Ph
O
b
O
O
S
P
Ph
Ph
Ph
NH
HN
HN
NH2
O
O
O
NH
O
O
O
O
O
O
O
O
O
O
H
N
H
N
H
N
H
H
N
H
N
N
N
H
N
N
H
N
H
N
O
N
H
N
N
H
H
O
O
O
O
O
O
O
H
O
Ph
Ph
N
N
O
N
Ph
O
O
N
Ph
Ph
18F
Ph
O
O
c
O
S
P
Ph
Ph
Ph
NH
HN
HN
O
NH
O
O
O
NH
O
O
O
O
O
O
O
O
O
O
H
H
N
H
N
H
H
N
H
N
N
N
N
N
N
H
N
H
N
O
N
H
N
N
H
H
H
O
O
O
O
O
O
O
H
O
Ph
Ph
N
N
O
N
18F
Ph
O
O
N
Ph
Ph
Ph
d
NH2
HN
O
NH
O
O
O
NH2
HN
O
HO
O
O
HO
O
O
O
H
H
H
N
H
H
N
N
NH2
O
N
N
N
N
N
N
H
N
H2N
N
N
H
H
H
H
O
O
O
O
O
H2N
N
HN
O
HN
[
18F]8
Scheme 3 Side-chain 18F-fluorobenzoylation of the e-amino group of
7 on solid phase. Reagents and conditions: a (tBuOCO)2O, DMF,
TEA, rt, 2 h; b Pd(PPh3)4, diethylamine, DCM, rt, 2 h; c [18F]SFB,
DMF/0.15 M sodium phosphate buffer pH 7 (1:1), 50°C, 30 min;
d reagent K, 50°C, 20 min
provided 7 attached to the solid phase ready for function-
alization with [18F]SFB and for preparation of the nonra-
dioactive reference compound 8 (Scheme 3). It should be
mentioned that the chromatographical separation of the
isomeric fluorobenzoylated reference peptides 8 and iso-8
revealed as cumbersome and required tedious optimization.
This is indicated by the close retention times in the chro-
matograms (Supplementary Material, Fig. S11). Reaction
of the SNEW peptide 7 with [18F]SFB in solution had a
slight tendency to the formation of [18F]8 over [18F]iso-8,
especially at pH 9, while there was a high variation
between the single labeling experiments (Table 3). Reac-
tion of resin-bound 7 with [18F]SFB led to [18F]8 in varying
radiochemical purities that were less satisfying compared
to the other peptides investigated (Table 4) while the iso-
lated radiochemical yield of the purified labeling products
was similar to those of the other tracers.
To summarize the results of labeling in solution, the 18F-
fluorobenzoylation of 1 resulted in [18F]2 as main product
nearly independent of the pH value. In contrast to this,
123

1442
M. Kuchar et al.
one-step 18F-labeling of peptides via nucleophilic aromatic
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Bejot R, Elizarov AM, Ball E, Zhang J, Miraghaie R, Kolb HC,
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radiolabeling of 7 produced predominantly [18F]8 even at
pH 7.
The influence of the chosen conditions on selectivity was
most pronounced for peptide 3 while there was a tendency
for an equal distribution of the radiolabeling products at pH
7/50°C and pH 9/25°C. Theses results show that the out-
come of the radiolabeling reaction is unpredictable for any
peptide and difficult to control by the pH value.
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In this study a method for the site-selective labeling of
peptides with fluorine-18 on solid support using [18F]SFB,
the most common prosthetic reagent for introduction of
radiofluorine into biomolecules, was developed. Its
advantages over the conventional radiolabeling in solution
could be exemplarily demonstrated by systematic com-
parison of radiolabeling on solid phase and in solution for
four different biologically relevant peptides. The elabo-
rated methodology revealed especially advantageous for
the radiolabeling of peptides containing multiple lysines
such as k7-fragment 5 and can be also applied for labeling
at sites distinct from the N-terminus as exemplarily shown
for SNEW peptide 7. Labeling on solid phase was in each
case more reliable and efficient as a site-selective reaction
with [18F]SFB in aqueous solution revealed as impossible
for every considered peptide despite systematic variation of
pH and temperature. The benefits of the solid-phase
approach become especially obvious if one considers the
time-consuming optimizations that were necessary to sep-
arate the isomeric fluorobenzoylated peptides chromato-
graphically. Labeling with [18F]SFB on solid support has
certainly the potential for straight-forward introduction of
fluorine-18 into almost every peptide that is interesting as
imaging probe for PET.
Acknowledgments The dedicated assistance of Uta Lenkeit and
Peggy Wecke in the radiosynthesis of [18F]SFB is gratefully
acknowledged.
King DS, Fields CG, Fields GB (1990) A cleavage method which
minimizes side reactions following Fmoc solid-phase peptide
synthesis. Int J Peptide Protein Res 36:255–266
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binding peptides identified by phage display enable design of an
Conflict of interest The authors declare that they have no conflict
of interest.
antagonist with ephrin-like affinity.
280:17301–17311
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Biol Chem
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with fluorine-18: Three decades of research. Curr Radiopharm
3:174–201
Li Z, Conti PS (2010) Radiopharmaceutical chemistry for positron
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