Efficient 18F-labeling of large 37-amino-acid pHLIP peptide analogues and their biological evaluation

Article abstract of DOI:10.1021/bc3000222

Solid tumors often develop an acidic microenvironment, which plays a critical role in tumor progression and is associated with increased level of invasion and metastasis. The 37-residue pH (low) insertion peptide (pHLIP) is under study as an imaging platf

Full text of DOI:10.1021/bc3000222

Article
pubs.acs.org/bc
1
8
Efficient F‑Labeling of Large 37-Amino-Acid pHLIP Peptide
Analogues and Their Biological Evaluation
Pierre Daumar,† Cindy A. Wanger-Baumann, NagaVaraKishore Pillarsetty, Laura Fabrizio,
,§
†,§
†
†
†
∥
∥
,†,‡
Sean D. Carlin, Oleg A. Andreev, Yana K. Reshetnyak, and Jason S. Lewis*
†
‡
Department of Radiology and the Program in Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center,
New York, New York 10065, United States
∥
Physics Department, University of Rhode Island, Kingston, Rhode Island 02881, United States
ABSTRACT: Solid tumors often develop an acidic microenvironment, which plays a critical role in tumor progression and is
associated with increased level of invasion and metastasis. The 37-residue pH (low) insertion peptide (pHLIP) is under study as
an imaging platform because of its unique ability to insert into cell membranes at a low extracellular pH (pH < 7). Labeling of
e
1
8
peptides with [ F]-fluorine is usually performed via prosthetic groups using chemoselective coupling reactions. One of the most
successful procedures involves the alkyne−azide copper(I) catalyzed cycloaddition (CuAAC). However, none of the known
click” methods have been applied to peptides as large as pHLIP. We designed a novel prosthetic group and extended the use of
“
18
the CuAAC “click chemistry” for the simple and efficient F-labeling of large peptides. For the evaluation of this labeling
approach, a D-amino acid analogue of WT-pHLIP and an L-amino acid control peptide K-pHLIP, both functionalized at the
18
N-terminus with 6-azidohexanoic acid, were used. The novel 6-[ F]fluoro-2-ethynylpyridine prosthetic group, was obtained via
nucleophilic substitution on the corresponding bromo-precursor after 10 min at 130 °C with a radiochemical yield of 27.5 ±
I
6
.6% (decay corrected) with high radiochemical purity ≥98%. The subsequent Cu -catalyzed “click” reaction with the azido
functionalized pHLIP peptides was quantitative within 5 min at 70 °C in a mixture of water and ethanol using Cu-acetate and
1
8
18
sodium L-ascorbate. [ F]-D-WT-pHLIP and [ F]-L-K-pHLIP were obtained with total radiochemical yields of 5−20% after
HPLC purification. The total reaction time was 85 min including formulation. In vitro stability tests revealed high stability of the
1
8
[
F]-D-WT-pHLIP in human and mouse plasma after 120 min, with the parent tracer remaining intact at 65% and 85%,
respectively. PET imaging and biodistribution studies in LNCaP and PC-3 xenografted mice with the [ F]-D-WT-pHLIP and the
negative control [ F]-L-K-pHLIP revealed pH-dependent tumor retention. This reliable and efficient protocol promises to be
useful for the F-labeling of large peptides such as pHLIP and will accelerate the evaluation of numerous [ F]-pHLIP analogues
as potential PET tracers.
18
1
8
18
18
INTRODUCTION
such anomalies, which plays a significant role in progression and is
often associated with increased invasion and metastasis. It is
■
2
Positron emission tomography (PET) is a noninvasive func-
tional in vivo imaging modality, commonly used in tumor diagnosis.
Tremendous effort has been made toward the development of
additional efficient and widely applicable PET tracers, given the
present in 90% of tumor microenvironments and is therefore
3
,4
considered a promising environmental marker for targeting.5
A 37-residue peptide discovered in 1997 by Hunt et al. was
recently shown to display unique pH-dependent properties: the
18
limitations of [ F]-FDG, for the targeting of a large variety of
cancers. However, the development of a universal tumor targeting
PET tracer is limited due to tumor heterogeneity and differences
pH Low Insertion Peptide (WT-pHLIP, NH -ACEQNPIY-
2
WARYADWLFTTPLLLLDLALLVDADEGTG-CO H) is
2
1
within the tumor environment. Therefore, targeting a physio-
logical anomaly present in most cancers seems very promising for
the development of a widely applicable diagnostic agent in
oncology. The acidity of the tumor microenvironment is one of
Received: January 16, 2012
Revised: June 15, 2012
Published: July 12, 2012
©
2012 American Chemical Society
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Bioconjugate Chemistry
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18
Figure 1. Examples of [ F]-labeled prosthetic groups for the CuAAC “click” reaction with functionalized peptides and proteins.
18F-bearing alkyne prosthetic groups with azido-functionalized
peptides of interest was shown to result in total RCYs of 20−
soluble in aqueous solution at physiological pH and shows
transient interaction with cell membranes in tissues with neutral
extracellular pH (pH ). In tissues with an acidic pH , Asp/Glu
1
8−20
e
e
75% (d. c.).
The corresponding radiosyntheses utilizing an
18
residues of pHLIP are protonated, which increases the overall
hydrophobicity of the peptide and affinity to the cell mem-
brane. As a result, pHLIP inserts and translocates itself through
F-labeled azido prosthetic group and an alkyne-functionalized
peptide were shown to be equally efficient.
With regard to the radiochemistry, however, only a few of the
21−23
6
18
the phospholipid bilayer that is the cellular membrane. This
common F-labeled prosthetic groups like 1-(azidomethyl)-4-
18
24
18
insertion involves a change in the peptide conformation to a α-
helix, the N-terminus remaining in the extracellular space, while
the C-terminus reaches the intracellular lumen.
[
F]-fluorobenzene or 4-[ F]fluoro-N-methyl-N-(prop-2-ynyl)-
20
benzenesulfonamide are UV-detectable. A common dis-
18
advantage of [ F]-fluoroalkynes or azides is also their high
volatility, which can be technically challenging.
Finally, the preparation of [ F]-glycosylazides or azidomethyl-
F]-benzenes, for example, involves more than one step and is
therefore more laborious. Besides the inherent difficulties of a
13,18,19,21,25
The pHLIP peptide was recognized as a potentially useful
platform for tissue acidity imaging, and therefore, its potential
18
7
18
application as a universal tool in oncology was envisaged. We
[
22
successfully demonstrated that fluorescent pHLIP could be
8
used to detect tissue acidity and target tumors in mice. In an
prosthetic group radiosynthesis, to the best of our knowledge,
6
4
18
extension to the study, Cu-labeled pHLIP was evaluated for
its suitability as a PET tumor imaging agent. The novel tracer
displayed good imaging characteristics with the highest level of
radioactivity accumulation in LNCaP tumor being reached 1 h
only Hausner et al. have demonstrated an efficient F-radiolabeling
of peptides with a high molecular weight (MW > 2000 Da),
commenting on the increasing challenge to label peptides with
13
increasing complexity and size. Ramenda et al. have reported
9
18
after administration, but some demetalation and a significant
the F-radiolabeling of azide-functionalized human serum
albumin but noted that the introduction of azide residues
into HSA and subsequent radiolabeling via click chemistry
significantly altered the structural and functional integrity of
retention of activity in the GI tract were also observed.
Fluorine-18 is the most common PET nuclide due to its favor-
able physical properties (low positron energy, pure positron
1
0,11
20
decay, and 110 min half-life), availability, and dosimetry.
HSA. More recently, in an eloquent approach, Gill et al. have
demonstrated the utility of maleimide functionalized alkyne in
26
Therefore, the 1 F-nuclide was considered the ideal nonmetal-
8
18
based alternative for the PET-labeling of pHLIP and its
labeling antibodies with [ F]-fluorine via a click approach.
1
8
analogues. However, the F-labeling of large peptides such as
WT-pHLIP (molecular weight (MW) > 4000 Da) still remains
However, the presence of cysteine and lysine residues in the
pHLIP amino acid sequence makes this approach unrealistic in
our case, as it is necessary to limit the number of derivatization
sites to one in order to preserve the unique pHLIP properties.
1
8
challenging because nucleophilic substitution with [ F]-
fluoride requires harsh basic conditions and cannot be
12
1
8
performed directly on peptides. As a result, numerous
Therefore, it appears that further efficient F-labeling
1
8
approaches to label peptides with F were developed, including
procedures are necessary for the development of a successful
18
bioconjugation with prosthetic groups like N-succinimidyl-4-
and widely applicable [ F]-pHLIP derivative.
1
8
18
18
18
[
F]fluorobenzoate ([ F]SFB) or p-nitrophenyl-2-[ F]-
Herein, we report on the use of a novel F-labeled prosthetic
1
8
13
fluoropropionate ([ F]NFP), or more recent methodologies
involving Al F chelation or the “less conventional” use of
organosilicon-based fluoride acceptor. Among the recent
chemoselective F-labeling strategies involving indirect labeling
group for the efficient two-step “click” radiolabeling of azido-
hexanoic acid derivatized pHLIP involving a UV-detectable
prosthetic group. The ultimate aim of this study was to evaluate
18
14
15
18
18
the novel prosthetic group for its universal utilization in [ F]-
18
by means of [ F]-prosthetic groups, the popular alkyne−azide
copper(I) catalyzed cycloaddition (CuAAC) “click” reaction,
where the coupling of an azide and an alkyne leads to the
formation of a triazole ring, appeared well-suited for our
purpose. It has been shown to be widely applicable and efficient
for the labeling of peptides, mostly requiring a two-step
synthetic approach. The success of this strategy and its wide
range of application led to the development of a large variety of
peptide chemistry. Therefore, the reaction conditions were
investigated with two different peptides: D-WT-pHLIP (all
amino acids have D-configuration) and L-K-pHLIP (all amino
acids have L-configuration), which has exactly the same
sequence as WT-pHLIP, but two aspartic acid residues have
been replaced with lysines. These aspartic acid residues are
present in the transmembrane part after insertion and are
critical for pH-dependent behavior. Therefore, L-K-pHLIP
lacks pH-dependent behavior and was used as a negative
1
8
F-labeled prosthetic groups as exemplified in Figure 1 and
16,17
6,27,28
18
recently reviewed.
Thus, the one-step radiolabeling of
control in our study.
In vitro stability of the [ F]-D-WT-
suitable precursors followed by cycloaddition of the resulting
pHLIP in both human and murine plasma was also examined.
1
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Article
Scheme 1. Synthetic Route Yielding the Non-Radiolabeled Reference Compounds 3 to 5
Reagents and conditions: (a) Pd(PPh ) , CuI, Et N, DMF, RT, 24 h; (b) THF, TBAF, 1 h, RT; (c) H O, ACN, EtOH, Cu-acetate × 5 H O, sodium
3
4
3
2
2
L-ascorbate, 48-70 h, RT.
Finally, PET imaging and biodistribution studies were
250 × 10 mm, (Phenomenex, Torrance, CA); (2) analytical,
Luna 5 μm C18, 100 Å, 250 × 4.6 mm, (Phenomenex,
Torrance, CA); (3) semipreparative, Nova-Pak 6 μm C18,
60 Å, 300 × 7.8 mm, (Waters, Milford, MA). Different gradient
solvent systems were applied: (A) 20−80% MeCN in water in
the presence of 0.1% trifluoroacetic acid (TFA) in 20 min with
a flow of 5 mL/min; (B) 20−80% MeCN in water with 0.1%
TFA in 20 min with a flow of 1.5 mL/min; (C) 5−40% EtOH
in water in 5 min, 40% EtOH in water from 5 to 15 min with a
flow rate of 3 mL/min.
performed in order to determine the in vivo properties of the
18
novel [ F]-pHLIP conjugates in LNCaP and PC-3 tumor
xenografts bearing mice.
MATERIALS AND METHODS
■
General Methods. All chemicals and solvents were
purchased from Sigma-Aldrich, Anichem or Fluka and were
used without further purification unless specifically stated
otherwise. All peptide starting materials were purchased from C
S Bio Co. (Menlo Park, CA, USA). Ultrapure water was used in
Chemistry. Syntheses of cold reference compounds 2 to 5
are presented in Scheme 1.
−1
this work (>18 MΩ cm at 20 °C, Milli-Q, Millipore, Billerica,
MA, USA). All instruments were maintained and calibrated
regularly according to quality control procedures as previously
reported. Thin layer chromatography performed on precoated
silica gel 60 F245 aluminum sheets suitable for UV detection of
compounds was used for monitoring the reactions. Radio-
activity measurements were performed with a Capintec
CRC1243 Dose Calibrator (Capintec, Ramsay, NJ, USA).
Precise quantification of low radioactivity samples was achieved
with a Perkin-Elmer (Waltham, MA, USA) Automatic Wizard2
Gamma Counter. Nuclear magnetic resonance spectra were
recorded on a Bruker AVIII 500.13 MHz spectrometer with an
internal standard from solvent signals. Chemical shifts are given
in parts per million (ppm) relative to tetramethylsilane (0.00 ppm).
Values of the coupling constant, J, are given in hertz (Hz). The
2
-((tert-Butyldimethylsilyl)ethynyl)-6-fluoropyridine (2).
To a degassed solution of tert-butyl(ethynyl)dimethylsilane
190 mg, 1.30 mmol) in triethylamine (Et N, 0.6 mL) and
(
3
DMF (2 mL) was added CuI (50 mg, 0.26 mmol). To a second
degassed solution of 2-bromo-6-fluoropyridine (200 mg, 1.10 mmol)
in DMF (4 mL) was added Pd(PPh ) (15 mg, 0.013 mmol).
Both mixtures were stirred for 5 min and then combined and
allowed to react at RT for 24 h before saturated aqueous
3
4
NH Cl (7 mL) was added. The resulting solution was then
4
extracted with diethyl ether (Et O, 3 × 20 mL). The combined
organic layers were washed with water (2 × 20 mL) and brine
20 mL) before drying over Na SO . Careful evaporation
resulted in a brown oily residue. Purification by silica gel
column chromatography utilizing pentane/Et O (95:5) gave 2
(105 mg, 40%) as a colorless oil that formed white crystals at
4 °C. R = 0.8 (pentane/ethyl acetate; 9:1). H NMR (500 MHz,
CDCl ): δ 7.71 (q, J = 7.7 Hz, 1H), 7.33 (d, J = 6.5 Hz, 1H),
.88 (dd, J = 1.3, 2.5 Hz, 1H), 0.99 (s, 9H), 0.20 (s, 6H). C
NMR (500 MHz, CDCl ): 162.9 (d, J = 241.2 Hz), 141.0 (d,
J = 12.7 Hz), 125.0 (d, J = 3.9 Hz), 124.8 (d, J = 54.2 Hz),
09.6 (d, J = 36.9 Hz), 102.9, 95.0, 26.1, 16.7, 1.0. F NMR
500 MHz, CDCl ): δ −65.17. MS m/z: 236.21 (M + H) .
2
0.12 mmol) in dry tetrahydrofuran (THF, 2.5 mL) was added
tetrabutylammonium fluoride (TBAF) trihydrate (120 mg,
0.38 mmol). After stirring the reaction mixture for one hour at
RT, water (10 mL) was added. This mixture in water/THF
(4:1) was used without further purification for the synthesis of
the fluorinated reference peptides. For analytical purposes,
compound 3 was purified by semipreparative HPLC using
column 1 and the gradient solvent system A. The retention
time of compound 3 was 11.2 min. Due to the volatility of the
product and the small scale of the synthesis, concentration of
2
(
2 4
2
1
following abbreviations are used for the description of H NMR
1
and 13C NMR spectra: singlet (s), doublet (d), doublet of
f
doublets (dd), triplet (t), quartet (q). The chemical shifts of
complex multiplets are given as the range of their occurrence.
Low resolution mass spectra (LRMS) were recorded with a
Waters Acquity UPLC with electrospray ionization SQ detector
3
1
3
6
3
1
9
1
(
ESI). High resolution mass spectra (HRMS) were recorded
+
(
with a Waters LCT Premier system (ESI) and MALDI TOF
analysis was performed on a Bruker Ultraflex TOF/TOF
MALDI tandem TOF mass spectrometer. Quality control and
purification of the final products in order to confirm the purity
3
-Ethynyl-6-fluoropyridine (3). To a solution of 2 (30 mg,
≥95% of the radioactive intermediates and final products were
achieved by analytical or semipreparative high-performance
liquid chromatography (HPLC). All HPLC experiments were
performed on a Shimadzu HPLC system equipped with a Flow
Count PIN diode radiodetector from BioScan, a DGU-20A
degasser, two LC-20AB pumps, and SPD-M20A photodiode
array detector and a SPD-M20A autosampler using reverse-
phase columns: (1) semipreparative, Luna 5 μm C18, 100 Å,
1
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Bioconjugate Chemistry
Scheme 2. Radiosynthesis Yielding Radiolabeled Products [ F]-3 to [ F]-5
Article
1
8
18
18
Reagents and conditions: (a) K[ F]F-Kryptofix 222, DMSO, 130 °C, 10 min; (b) H O, EtOH, Cu-acetate × 5 H O, sodium L-ascorbate,
2
2
azidohexanoic acid derivatised peptide, 70 - 80 °C, 5 - 10 min.
the product was achieved using a C18 light cartridge (Waters)
preconditioned with 5 mL of EtOH, followed by 5 mL of water.
K222 solution (Kryptofix K222, 2.5 mg; K CO , 0.5 mg in
2 3
MeCN/water (3:1)) was slowly passed through the cartridge.
The solvents were evaporated at 110 °C under vacuum in the
presence of slight inflow of argon gas. After addition of MeCN
(1 mL), azeotropic drying was achieved under vacuum and with
a slight argon inflow. For the complete removal of water traces,
the procedure was repeated twice. A solution of the
corresponding precursor 6 (Anichem), 1.5−2 mg in 200 μL
of dry DMSO, was added to the Kryptofix complex and the
reaction mixture was heated at 130 °C for 10 min before 1 mL
of water and 0.4 mL of MeCN were added (total injection
volume 1.6 mL). Purification by semipreparative HPLC was
carried out using the reversed-phase column 3 with the gradient
solvent system C: the product was eluted with the same
The combined fractions were diluted with water and passed
1
over the cartridge. Pure product 3 was eluted with CDCl . H
3
NMR (500 MHz, CDCl ): δ 7.76 (q, J = 7.8 Hz, 1H), 7.38 (d,
3
J = 7.6 Hz, 1H), 6.94 (dd, J = 2.7, 2.9 Hz, 1H), 3.19 (s, 1H).
1
3
C NMR (500 MHz, CDCl ): δ 162.9 (d, J = 237.8 Hz), 141.2
3
(
1
d, J = 7.2 Hz), 125.4 (d, J = 96.3 Hz), 125.0 (d, J = 36.9 Hz),
10.2 (d, J = 36.8 Hz), 81.3, 78.3. 1 F NMR (500 MHz,
9
+
CDCl ): δ −65.30. MS m/z 121.89 (M + H) . HRMS calcd for
C H NF, 122.0407; found, 122.0406.
3
7
5
6-(4-(6-Fluoropyridin-2-yl)-1H-1,2,3-triazol-1-yl)hexanoyl-
D-WT-pHLIP (Fpyrtriazolo-D-WT-pHLIP, 4). 100 μL (0.01 mmol)
of a freshly prepared solution of Cu(II) acetate (18 mg/mL)
and 200 μL (0.02 mmol) of a fresh (+)-sodium L-ascorbate
solution (20 mg/mL) in water were mixed in a sealed vial
under argon. To the yellow mixture was added a solution of
azidohexanoyl-D-WTpHLIP (N (CH ) CONH-ACEQNPIY-
retention time as the cold standard 3: t = 9.2 min. Under the
R
conditions used, the bromo precursor 6 has a retention time of
18
10.8 min. Identification of [ F]-3 was achieved by coinjection
with the reference 3 on the reversed-phase column 1 and the
3
2 5
WARYADWLFTTPLLLLDLALLVDADEGTG-CO H) in
EtOH (5 mg in 300 μL, 0.0012 mmol). Further addition of
solvent system A: t = 10.2 min (Figure 2A).
2
R
1
8
Radiosynthesis of [ F]-4. Stock solutions of copper(II)
acetate (18 mg/mL) and (+)-sodium L-ascorbate (20 mg/mL),
respectively, were freshly prepared. A v-shaped 4 mL HPLC vial
(MT-IT, Prominence, Shimadzu) was equipped with a stir bar,
sealed, and set under argon before 30 μL of the Cu(II) acetate
solution and 60 μL of the (+)-sodium L-ascorbate solution were
2.7 mL of the solution of 3 (0.025 mmol) in water/THF (4:1)
was followed by stirring of the reaction mixture at RT for 70 h
before MeCN (3 mL) and water (3 mL) were added. The
product was purified using semipreparative HPLC (column 1,
solvent gradient system A): two conformational isomers (4a, 4b)
of Fpyrtriazolo-D-WTpHLIP were obtained with tR1 = 18.7 min
and tR2 = 20.1 min, respectively. HPLC analysis indicated that the
overall chemical purity of the compound 4 (4a + 4b) is >98%.
Calculated mono-isotopic mass for Fpyrtriazolo-D-WTpHLIP
18
mixed. The prosthetic group [ F]-3 (150−500 MBq) was
directly collected from the HPLC into the prepared 4 mL
HPLC vial (V = 1.2−1.6 mL). A solution of azidohexanoyl-D-
WT-pHLIP (N (CH ) CONH-ACEQNPIYWARYADWLF-
3
2 5
(
4
C H FN O S): 4426.18. 4a: found, MS (MALDI) m/z =
426.65, 4b: found, MS (MALDI) m/z = 4426.56.
TTPLLLLDLALLVDADEGTG-CO H, 1 mg, 0.2 μmol) in EtOH
206 301
48 58
2
(150 μL) was added and the mixture was heated at 70 °C for
5−10 min. After addition of MeCN (100 μL), purification of
the product was achieved using semipreparative HPLC on
reversed-phase column 1 with solvent system A (total injection
volume 1.5−2.0 mL). Two main radioactive peaks were eluted
with the same retention time as the cold reference compounds
4a and 4b that are conformational isomers. HPLC analysis
indicated that the overall radiochemical purity of the compound
4 (4a + 4b) was >98%. The radiolabeled peptide could not be
isolated from the peptide starting material, resulting in lowered
6-(4-(6-Fluoropyridin-2-yl)-1H-1,2,3-triazol-1-yl)hexanoyl-
L-K-pHLIP (Fpyrtriazolo-L-K-pHLIP, 5). This compound was
prepared in an analogous way to compound 4. The mixture
containing the azidohexanoyl-L-K-pHLIP (N (CH ) CONH-
ACEQNPIYWARYAKWLFTTPLLLLKLALLVDADEGTG-
CO H, 7.7 mg, 1.18 μmol) was stirred at RT for 48 h. Purification
of the product was achieved by semipreparative HPLC on column
1
Calculated monoisotopic mass for peptide 5 (C H FN O S):
4
3
2 5
2
using the gradient solvent system A: t = 15.7 min; 99% purity.
R
210 315
50 54
18
452.31; found, MS (MALDI) m/z = 4452.724.
Radiochemistry. Radiosyntheses of [ F]-3 to [ F]-5 are
apparent specific activity. Only [ F]-4a, obtained in MeCN/
water (3:1, 0.1% TFA), was used for in vivo evaluation. After
dilution with 7 mL of water, the solution was passed over a C18
light Sep Pak cartridge (Waters), preconditioned with 5 mL of
EtOH followed by 5 mL of water. The cartridge was washed
with further 3 mL of water in order to remove traces of MeCN
1
8
18
presented in Scheme 2.
1
8
18
Radiosynthesis of [ F]-3. [ F]-fluoride (1480−2220 MBq)
1
8
18
was obtained via the O(p,n) F reaction of 11-MeV protons
18
in an EBCO TR-19/9 cyclotron using enriched O-water.
QMA light cartridges (Waters) preconditioned with 0.5 M
K CO (5 mL) and water (5 mL) were used for trapping of
1
8
18
and TFA, and the trapped [ F]-D-WT-pHLIP ([ F]-4a) was
1
8
2
3
eluted with 0.5−1 mL of EtOH. Identification of [ F]-4a was
achieved by comparison of the retention time with the cold
reference compound 4 eluted with identical retention time
1
8
−
18 −
F from the aqueous solution. In order to elute F from the
cartridge into a sealed 5 mL reaction vial, 1 mL of Kryptofix
1
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Bioconjugate Chemistry
Article
then shaken for 15 min. For phase separation, the samples were
centrifuged at 2348 rcf for 5 min. 50 μL aliquots of each layer
were withdrawn carefully from each phase and pipetted into
microcentrifuge tubes (Eppendorf) for measurement of the2
distribution of the activity with a gamma-counter (Wizard,
Perkin-Elmer). The experiment was performed in quintuplicate.
In Vitro Stability test. For the determination of the in vitro
stability of 18F-labeled pHLIP analogues, 20 μL aliquots of the
18
18
model peptide [ F]-D-WT-pHLIP ([ F]-4) in EtOH were
added to both human and murine plasma (380 μL) and
incubated at 37 °C. After 60 and 120 min, aliquots of 180 μL
were removed and added to an equal amount of MeCN for
precipitation of the present proteins. After 5 min of
centrifugation (21 130 rcf), 4 °C), the supernatant was analyzed
under semipreparative HPLC conditions as described for the
purification of the radiolabeled peptides.
Tumor Cell Culture. Human prostate cancer cell lines
LNCaP and PC-3 from the American Tissue Culture Collection
(
ATCC, Manassas, VA, USA) were cultured in a 5% CO2
atmosphere at 37 °C. LNCaP cells were cultured in RPMI 1640
medium containing 10% fetal calf serum (FCS), 2 mM
L-glutamine, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L
sodium bicarbonate, and 100 U/mL of penicillin and
streptomycin. PC-3 cell were cultured in F-12 Kaighn's medium
containing 10% FCS, 2 mM L-glutamine, 1.5 g/L sodium
bicarbonate, and 100 U/mL of penicillin and streptomycin.
Cells were trypsinized in the absence of magnesium or calcium
ions using a cocktail of 0.25% trypsin and 0.53 mM EDTA in
Hank's buffered salt solution.
LNCaP and PC-3 Tumor Xenografts. Male athymic mice
NCRNU-M, 20−25 g) were obtained from Taconic Farms,
(
Figure 2. HPLC chromatograms with radio and UV-traces (UV traces
were recorded at 254 nm and retention times are given with respect to
Inc. (Hudson, NY, USA) and were kept in the MSKCC
vivarium for one week before any experimental handling was
performed. The animals were allowed free access to water and
food, and all animal care and experimental procedures were
approved by the Institutional Animal Care and Use Committee
1
8
the radiotrace): (A) coinjection of compound 3 and [ F]-3 with t =
1
R
18
0.4 min on column 1; (B) purification of [ F]-4a and -4b after 5 min
at 70 °C with t = 18.7 min and tR = 20.2 min, respectively; (C)
R
18
purification of [ F]-5 after 5 min reaction at 70 °C (t = 15.8 min).
R
HPLC chromatograms were recorded utilizing column 1 applying
elution conditions A.
(
IACUC). LNCaP and PC-3 tumor xenografts were induced on
the right and the left shoulder of each mouse, respectively, by
subcutaneous injection of the corresponding cell suspension in
Matrigel (BD, Collaborative Biomedical Products, Inc.,
under the same HPLC conditions but injecting only 50 μL of
the collected solution for analysis. After evaporation of the
solvent under vacuum and a slight argon inflow, which was
completed within 6−8 min, the product was formulated in
saline with 3% ethanol for injection.
6
Bedford, MA, USA) and media (1:1) containing (5.0 × 10 )−
7
10 cells (viability >90%) per injection (200 μL). All procedures
were carried out under anesthesia (1.5% isoflurane). Because of
the slow rate of growth of the LNCaP tumors, these tumors
were implanted 14 days prior to the PC-3 tumor implantation
to have similarly sized tumors for the imaging and
biodistribution studies. Palpable tumors of similar size
developed after 4 to 5 weeks after the initial tumor xenograft
induction.
1
8
Radiosynthesis of [ F]-5. The radiosynthesis and purifica-
1
8
tion were achieved in an analogous way to [ F]-4. For the
1
8
formulation of an injectable solution of [ F]-5, a cartridge
purification step was performed as described above. The
product was eluted with 1.5 mL of acidified EtOH containing
0
.02% 2 N HCl and the solvent was removed under vacuum
Small-Animal PET Imaging Studies. PET imaging studies
were conducted on a microPET Focus 120 rodent scanner
(Concorde Microsystems). Dual tumor bearing mice (n = 4)
1
8
and a slight argon inflow. [ F]-5 was formulated in phosphate
buffer for injection after addition of 50 μL of 0.1 N NaOH for
neutralization. Identification of [ F]-5 was achieved by
comparison of the retention time with the cold reference
compound 5 eluted with identical retention time under the
same HPLC conditions.
1
8
3
with LNCaP tumor (right shoulder, V = 100−200 mm ) and
3
PC-3 (left shoulder, V = 100−200 mm ) were administered
18
[ F]-4 [160−170 μCi (5.9−6.3 MBq)] in 200 μL formulated
1
8
solution (3% EtOH in saline) and control peptide [ F]-5
[260 μCi (9.8 MBq)] in 200 μL formulated solution (phosphate
buffered saline) via tail vein injection. Approximately 3 min prior
to the PET scan, mice were anesthetized by inhalation of 2%
isoflurane (Baxter Healthcare, Deerfield, IL, USA)/ oxygen gas
mixture and placed on the bed of the scanner, while anesthesia
was maintained using 1.5% isoflurane/gas mixture. PET data for
each mouse were recorded via a 15 min static acquisition 2 and
1
8
18
Determination of the Lipophilicity of [ F]-4 and [ F]-
2
9
5
(logDpH7.4). As described by Wilson et al. for the shake
flask method, phosphate buffer (pH 7.4) saturated octanol
500 μL) and octanol saturated phosphate buffer (500 μL)
(
were added to a microcentrifuge tube (1.5 mL, Eppendorf).
Following the addition of 10 μL of the radiotracer solution
(
approximately 37 kBq), the samples were first vortexed and
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4
2
h post injection (p.i.). For the static images, a minimum of
0 million coincident events were detected.
which demonstrates the challenge of labeling large peptides
efficiently without affecting their integrity.
An energy window of 350−700 keV and a coincidence
timing window of 6 ns were used. Data were sorted into two-
dimensional histograms by Fourier rebinning, and transverse
We experienced numerous difficulties during our first
attempts to synthesize 18F-labeled WT-pHLIP via CuAAC
18
using a previously described [ F]fluoro-PEG-alkyne. Indeed,
images were reconstructed by filtered back-projection into a
even though such a prosthetic group has been used successfully
for the radiolabeling of RGD constructs, no “click coupling”
3
18
128 × 128 × 63 (0.72 × 0.72 × 1.3 mm ) matrix. The images
were normalized to correct for nonuniformity of response of
the PET, dead-time count losses, positron branching ratio, and
physical decay to the time of injection, but no attenuation,
scatter, or partial-volume averaging correction was applied. The
measured reconstructed spatial resolution for the Focus 120 is
approximately 1.6 mm in full width at half-maximum at the
center of the field of view. The counting rates in the re-
constructed images were converted to percent of injected dose
per weight (%ID/g) by use of a system calibration factor
derived from the imaging of a mouse-sized water-equivalent
with azido-derivatized pHLIP analogues was observed in the
standard CuAAC conditions that we used (Cu(II) acetate/
(+)-sodium L-ascorbate in H O/CH CN 1:1). We were
2
3
therefore interested in the development of a novel fluorinated
prosthetic group and a corresponding synthetic procedure
(Scheme 1), suitable for the radiolabeling of large peptides. We
chose fluoropyridinealkyne 3 because of the commercial
availability of the corresponding bromo-precursor 6, its UV-
18
detectability, and the fact that 2-[ F]fluoropyridines were
31
shown to display high in vivo stability. Moreover, the small
size of 3 was expected to have a very limited impact on the
pharmacokinetic of peptides as large as pHLIP.
1
8
phantom containing F. Images were evaluated by region-of-
interest (ROI) analysis using ASIPro VM software (Concorde
Microsystems).
18
Radiochemistry. [ F]-3 was prepared in one step by
nucleophilic fluorination of the corresponding bromo precursor
6 (Scheme 2) using Kryptofix and K CO . Optimized
Combined Histological/Autoradiographic Analysis.
After the completion of the last imaging session, vascular
perfusion marker Hoechst 33342 (15 mg/kg in 0.2 mL of
sterile saline) was administered via the tail vein catheter. Series
of contiguous fresh-frozen tumor sections of 10 μm thickness
were cut and exposed to a phosphor-imaging plate (Fujifilm
BAS-MS2325, Fuji Photo Film, Japan) for an appropriate
length of time at −20 °C. Digital images of radioactivity
distribution at 50 μm resolution were obtained. The same
sections were subsequently used for immunofluorescence
imaging of Hoechst 33342, and finally stained with H&E.
Autoradiographic and histological images were registered using
Adobe Photoshop CS3 software.
2
3
1
8
conditions (DMSO, 10 min, 130 °C) led to [ F]-3 in RCYs
of 27.5 ± 6.6% (n = 11) decay-corrected (d.c.), with conversion
rates between 70% and 90%. This high labeling efficacy was not
translated into higher RCYs because a loss of radioactivity was
systematically observed during the transfer to the HPLC,
18
presumably due to the volatility of [ F]-3. Although a
distillation-based purification might allow for higher yields,
18
like those obtained with volatile [ F]fluoroalkynes prosthetic
19
groups, it would also introduce MeCN, which in our case was
not favorable for the subsequent “click” reaction. Instead,
technical improvements or pH adjustments could potentially
minimize the effect of the pyridine alkyne volatility issue. Our
Post-Mortem Biodistribution Studies. Dual tumor-
bearing mice (LNCaP and PC-3 tumors) were randomized
1
8
HPLC purification has a principal advantage to afford [ F]-3 in
high radiochemical purity (RCP > 98%, Figure 2A) in a mixture
of ethanol and water, which appeared to be a perfectly suitable
solvent system for the CuAAC with pHLIP. Indeed, full
before the experiment. Only mice with palpable tumors (V =
3
0
.5−1.5 cm ) were utilized. In order to investigate the
1
8
biodistribution of radioactivity upon [ F]-D-WT-pHLIP
18
18
(
1
4
[ F]-4) injection, the tracer [∼39 μCi (1.4 MBq), 15 nmol,
50−250 μL] was administered intravenously to the mice (n =
per time point, 16−24 g). Four mice were euthanized at 2
incorporation of [ F]-3 was achieved within 10 min at 70 °C as
demonstrated by the HPLC chromatograms in Figure 2B,C,
18
where no unreacted material [ F]-3 was detected.
and 4 h post injection (p.i.), respectively. The blood was
collected immediately followed by quick removal of the tumors.
The lung, liver, heart, kidneys, spleen, bone, muscle, stomach,
small intestines (SI), large intestines (LI), and testes were taken
as well. The individual tumors and further organs were weighed
and the radioactivity in each sample was measured in the
gamma counter. Activity concentrations were calculated as
percentage injected dose per tissue weight (%ID/g wet tissue).
The two peaks observed in Figure 2B are due to acidic
HPLC conditions, which promotes the formation of two pH-
dependent conformational isomers (4a and 4b). When either of
the peaks was collected separately and reinjected, we observed
two peaks in HPLC indicating that these are same chemical
species. This fact has also been confirmed by HPLC-MS. No
dimerization of peptide (through cysteine-cysteine disulfide
linkage) was observed. It is also interesting to note that
conformational isomers were only observed in the case of WT-
pHLIP (Figure 2B), which reflects the non-acidity-dependent
behavior of K-pHLIP (Figure 2C). Figure 2 also reveals that
1
8
As a negative control, a formulated solution of [ F]-5 [55 μCi
2 MBq), 3.5 nmol)] was administered to a further group of
(
dual tumor bearing mice (n = 4, 19−22 g). These mice were
euthanized at 2 h p. i. and biodistribution performed as above.
18
18
the radiolabeled peptides [ F]-4 and [ F]-5 could not be
separated from the azido-derivatized starting materials. Con-
sequently, the apparent specific activities were low (0.1−1
RESULTS AND DISCUSSION
■
1
8
Recently, the CuAAC “click reaction” has attracted increasing
GBq/μmol), which should not question the use of [ F]-pHLIP
as an efficient PET tracer anyway. Indeed, given the mechanism
of pHLIP insertion across the cell membrane, the number of
insertion sites is theoretically unlimited wherever an acidic
18
interest for the F-labeling of peptides, mainly because of the
1
8
recent development of novel [ F]-prosthetic groups and
1
6,17,30
efficient protocols for this type of chemistry.
However,
18
7
the feasibility of such methods for the synthesis of large [ F]-
peptides (MW > 2000 Da) has yet to be proven. Indeed, the
microenvironment is present, as opposed to receptor-based
32
targeting systems.
1
8
18
preparation of an F-labeled α β specific 20 amino acids
Considering the size and the complexity of the [ F]-pHLIP
v
6
13
peptide by Hausner et al. in 2008 is the only successful report,
analogues, the detailed radiochemical data obtained with
1
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Bioconjugate Chemistry
Article
1
8
18
18
Table 1. Analytical Data Determined for Radiolabeled Peptides [ F]-4a, [ F]-4b (n = 4), and [ F]-5 (n = 3) after
Semipreparative HPLC Purification
RCP [%]
total RCY [%]
specific activity (EOS) [GBq/μmol]
total preparation time [min]
logDpH7.4
[18
[18
[18
F]-D-WT-pHLIP ([ F]-4a)
18
≥95
−
≥98
13.6 ± 6.0
13.6 ± 6.0
5−13
0.1−1.0
−
0.1−0.8
85
85
85
−0.70 ± 0.04
−0.70 ± 0.20
−1.30 ± 0.03
18
F]-D-WT-pHLIP ([ F]-4b)
18
F]-L-K-pHLIP ([ F]-5)
[18
F]-4 and [ F]-5 (Table 1) show the high efficacy of our
18
PET Imaging Studies. In order to prove the ability of
1
8
18
novel strategy using [ F]-3. Total RCYs of up to 20% could be
[ F]-4a to target acidity, we chose two prostate carcinoma
tumor models, LNCaP and PC-3. Indeed, as determined by
magnetic resonance, Vavere et al. reported a significantly
1
8
achieved with respect to the starting amount of [ F]-fluoride
activity in a short synthesis time (≤85 min) and in one step,
whereas several of the UV-detectable prosthetic groups that
were used for peptide labeling were produced after multistep
more acidic average pH for LNCaP tumors when compared
e
9
with the PC-3 tumors. Therefore, we were hopeful that this
2
2,23,33
18
radiosyntheses.
The total radiochemical yields also reflect
difference could be observed with PET following [ F]-4a
that some radioactivity was lost during the transfers and on the
HPLC, which leads to reduced overall yields. In our preparative
HPLC conditions, we never observed any unreacted [ F]-3 or
administration.
As a matter of fact, the LNCaP tumor could be clearly
visualized at both time points (Figure 3) and exhibited a
18
other byproducts in the reaction. Although better yields were
16
reported with small-sized peptides, the CuAAC for was never
achieved in such high yields with large peptides. By
comparison, Hausner et al. radiolabeled a 20-amino-acid
1
3
peptide via “click” reaction in yields below 10%. Finally, our
novel 18F-labeling protocol was performed with low amounts
of copper, and a minimum of 1 mg peptide starting material
1
8
was necessary to achieve complete incorporation of [ F]-3,
1
8
which is in line with previously reported F-“click” reactions
1
3
on larger peptides.
For the characterization of the novel 18F-labeled pHLIP
analogues, a logD determination was carried out (Table 1).
pH7.4
18
18
[
F]-4a and [ F]-4b eluted from the HPLC system with
different retention times and relative lipophilicity, exhibited the
same logDpH7.4 of −0.7 presumably due to isomerization and
18
formation of the same equilibrium state at pH 7.4. [ F]-5 was
eluted from the HPLC system with shorter retention time, and
it accordingly displayed a logD
of −1.30 ± 0.03. It is well
pH7.4
1
8
accepted that most of the [ F]-prosthetic groups (Figure 1)
1
8
lead to more lipophilic F-labeled peptides, especially as the
“
click” approach involves the formation of a lipophilic triazole
1
6,34
ring, often considered a surrogate for the amide bond.
Figure 3. Representative coronal PET images of mice bearing LNCaP
18
18
18
(right) and PC-3 (left) tumors injected with [ F]-4 (A) and [ F]-5 (B).
Although a similar effect is expected with the use of [ F]-3, the
small size of the prosthetic group and the triazole ring should
only have a limited impact on the pharmacological properties of
future investigated peptides.
The stability of the [ F]-prosthetic group in the [ F]-
peptide construct is important in terms of clearance and
metabolism. In particular, in vivo radio-defluorination is easily
radioactivity accumulation of 8.3 ± 1.5 and 8.5 ± 1.3%ID/g at
and 4 h p. i., respectively. The PC-3 tumor showed lower
radioactivity accumulation of 4.2 ± 0.4 to 5.4 ± 0.5%ID/g at
and 4 h p.i, respectively, and was poorly visualized. The lower
2
1
8
18
2
uptake is a good indication for the successful application of the
pHLIP principle for PET imaging of acidic tumors. Another
indication for the specificity of the pHLIP was the lack of any
tumor accumulation observed with the negative control peptide
32
detectable because of the related bone uptake of radioactivity.
18
Therefore, [ F]-4a, as a prominent pHLIP analogue, was first
subjected to an in vitro stability test in human and murine
plasma at 37 °C. The tracer displayed good stability: after
1
8
[ F]-K-pHLIP (Figure 3).
6
0 min, 80% and 100% of the parent tracer remained intact in
The MR studies reported by Vavere et al. determined the
human and murine plasma, respectively; 65% and 85% were
still present after 120 min. The degradation products were
more hydrophilic and displayed retention times (t ) suggesting
potential peptide fragments. [ F]-Fluoride is very hydrophilic
and an elution from the HPLC column within the first 3 min is
expected. However, no radioactive peaks were detected during
the first 10 min. The fact that peptides derived from D-amino
acids were shown to display higher in vivo stability,
the good in vitro stability of the D-WT-pHLIP analogue,
[
total volume average pH in the tumors, but the heterogeneity
e
of the tumor microenvironment suggests the formation of
acidic and nonacidic areas in tumors. Accordingly, the PET
R
1
8
18
images acquired after [ F]-4a administration displayed a
heterogeneous distribution of the tracer in the tumor since
hot spots within the LNCaP tumor could be visualized,
indicating the specific enrichment of pHLIP analogues in acidic
regions. Therefore, following PET imaging at 4 h p. i., tumors
were excised, sectioned, and evaluated by digital auto-
3
5,36
and
1
8
18
F]-4a, encouraged the in vivo evaluation of the novel pep-
radiography for F-distribution. This distribution was sub-
tide tracers.
sequently compared to the distribution of the vascular
1
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Bioconjugate Chemistry
Article
18
Figure 4. Combined histological/autoradiographic analysis obtained after [ F]-4 injection (4 h p. i.). Data from a single 10 μm frozen section
obtained from a LNCaP tumor. H&E staining (panel A), the vascular perfusion marker Hoechst 33342 (blue, panel B) and autoradiograph of
radioactivity distribution (panel C) are shown. Panel D shows co-registration of autoradiograph (pseudocolor green) overlaid with Hoechst 33342
(
blue). Panel E contains a high-magnification region from the image shown in Panel D. Scale bar = 1 mm.
perfusion marker Hoechst 33342 using the same section. A
1
8
clear pattern of higher F uptake in tumor regions lacking
vascular perfusion was observed (Figure 4). Previous studies
have shown that tumor regions with low vascular perfusion are
associated with low pO and anaerobic glycolysis, resulting in
2
37
lactate accumulation and reduced pH. The accumulation of
18
[
F]-4a in tumor regions lacking vascular perfusion is con-
sistent with the proposed mechanism of pHLIP accumulation,
and corroborates the specificity underlying the heterogeneous
tumor distribution observed in the PET imaging.
For both tracers, the PET images exhibited the highest
18
radioactivity concentrations in the liver indicating that [ F]-4a
18
and [ F]-5 are mainly excreted via the hepatobiliary system.
Little radioactivity accumulation was detected in the bone in all
1
8
PET imaging studies performed with the [ F]-pHLIP
radiotracer that were radiolabeled using our novel [ F]-
18
18
18
Figure 5. Biodistribution of [ F]-4 and [ F]-5 at 2 and 4 h p.i. with
labeling protocol, pointing to a high in vivo stability of the
uptake values expressed as %ID/g.
18
[
F]-prosthetic groups. The high activity observed in the liver
Table 2. LNCaP Tumor-to-Tissue Ratios Determined upon
Biodistribution Studies with the Tracers Administered to
Tumor Bearing Mice
might preclude the use of the tracer for imaging tumors near
the liver.
Biodistribution. Biodistribution studies were performed
18
18
with [ F]-4a and [ F]-5 in mice with dual PC-3 (left) and
LNCaP (right) tumors on the shoulders. On the basis of our
preliminary imaging studies, we chose the 2 and 4 h time points
for the biodistribution study. Figure 5 shows the distribution of
tumor-to-tissue ratio [18F]-4 2 h p. i. [18F]-4 4 h p. i. [18F]-5 2 h p. i.
tumor/blood
tumor/muscle
tumor/liver
0.2
4.5
0.2
0.3
0.4
5.7
0.2
0.3
0.1
0.4
0.2
0.4
1
8
18
radioactivity in mice of [ F]-4a at 2 and 4 h p.i. and of [ F]-5
tumor/kidney
18
at 2 h. For [ F]-4a, the highest accumulation of radioactivity in
LNCaP tumors was observed at 4 h p.i.. The uptake in PC-3
tumors was much lower at both time points. At 4 h p.i., LNCaP
tumors had uptake of 8.0 ± 0.7%ID/g as compared to PC-3
tumors which had uptake 5.3 ± 0.7%ID/g. These results are in
(
2
LNCaP)-to-kidney ratios are 0.2 and 0.3, respectively, at both
and 4 h. From our limited biodistribution studies, it is difficult
to differentiate between excretory and metabolic factors
contributing to these high values. As shown in Table 2, the
9
64
line with our earlier publication with [ Cu]-DOTA-pHLIP
18
negative control peptide [ F]-5 exhibits similar ratios in liver
(
all L-amino acids) and with PET imaging studies.
and kidney. It can be hypothesized that the activity in these
organs is due to excretion rather than metabolism. The tumor-
The highest accumulation of radioactivity at both time points
1
8
was detected in the liver (32.5 ± 4.3%ID/g and 34.9 ± 5.8%
ID/g at 2 and 4 h p. i., respectively), followed by the kidneys.
As expected, with large hydrophobic peptides the blood
to-muscle ratio of 4.5 measured at 2 h p.i. with [ F]-4a in
1
8
comparison to 0.4 with [ F]-5 demonstrates that we are
18
specifically targeting the tumor with [ F]-4a and is not a result
of nonspecific accumulation. The tumor-to-muscle ratio is even
higher at 4 h p.i. with a value of 5.7. These studies clearly
demonstrate that this click approach with prosthetic group 3
has no negative impact on the biological properties of pHLIP.
Additionally, higher %ID/g values in LNCaP tumors as
6
4
clearance was slow. [ Cu]-pHLIP shows highest accumulation
in LNCaP tumors at 1 h p.i. (4.5 ± 1.7%ID/g) and highest
9
tumor-to-blood ratio at 24 h p.i.
1
8
18
The tumor-to-tissue ratios for [ F]-4a and [ F]-5 are
shown in Table 2. The tumor (LNCaP)-to-liver and the tumor
1
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Bioconjugate Chemistry
Article
compared to the PC-3 tumor indicate that the accumulation of
the tracer reflects acidity of the tissue.
(2) Gillies, R. J. (2001) Causes and consequences of hypoxia and
acidity in tumors. Novartis Foundation symposium. Trends Mol. Med.
1
8
7
(2), 47−49.
[
F]-pyridines labeled at ortho-position to the nitrogen have
31
(3) Cairns, R., Papandreou, I., and Denko, N. (2006) Overcoming
been reported to be stable against in vivo defluorination.
However, we observe some bone uptake with [ F]-5 (1.31 ±
.63%ID/g) and [ F]-4a (4.14 ± 1.12%ID/g) 2 h p.i. In
comparison, [ F]-fluoropentyne-based prosthetic group shows
very minimal bone uptake (0.39%ID/g at 1 h p.i.). In our case,
it is unclear whether the uptake is the result of defluorination or
specific accumulation, because the two peptides radiolabeled
with the same prosthetic group show different bone uptake
values.
1
8
physiologic barriers to cancer treatment by molecularly targeting the tumor
microenvironment. Molecular Cancer Research: MCR 4 (2), 61−70.
(4) Sakamoto, S., Ryan, A. J., and Kyprianou, N. (2008) Targeting
vasculature in urologic tumors: mechanistic and therapeutic
significance. J. Cell. Biochem. 103 (3), 691−708.
1
8
0
18
(
5) Hunt, J. F., Rath, P., Rothschild, K. J., and Engelman, D. M.
1997) Spontaneous, pH-dependent membrane insertion of a
transbilayer alpha-helix. Biochemistry 36 (49), 15177−15192.
6) Reshetnyak, Y. K., Andreev, O. A., Lehnert, U., and Engelman, D.
(
(
M. (2006) Translocation of molecules into cells by pH-dependent
CONCLUSION
insertion of a transmembrane helix. Proc. Natl. Acad. Sci. U.S.A. 103 (17),
■
6
(
460−6465.
We have extended the use of the CuAAC “click chemistry” to
the development of 2-ethynyl-6-[ F]fluoropyridine as a new
prosthetic group suitable for the F-labeling of large peptides.
This approach should be widely applicable and was shown to be
efficient for the radiolabeling of pHLIP analogues with a
7) Andreev, O. A., Dupuy, A. D., Segala, M., Sandugu, S., Serra, D.
1
8
A., Chichester, C. O., Engelman, D. M., and Reshetnyak, Y. K. (2007)
Mechanism and uses of a membrane peptide that targets tumors and
other acidic tissues in vivo. Proc. Natl. Acad. Sci. U.S.A. 104 (19),
1
8
7
(
893−7898.
1
8
molecular weight over 4000 Da. Two F-labeled analogues
8) Reshetnyak, Y. K., Yao, L., Zheng, S., Kuznetsov, S., Engelman, D.
1
8
were injected in mice in order to acquire the first in vivo [ F]-
pHLIP data. The WT-pHLIP construct showed good in vitro
stability, and only mild in vivo defluorination was observed in
both cases. A milestone in the development of an 18F-labeled
pHLIP tumor imaging agent was achieved, as the use of [ F]-3
will allow for fast production and evaluation of second-
generation pHLIP analogues, designed for higher accumulation
in the tumors and faster clearance from nontarget tissues.
M., and Andreev, O. A. (2011) Measuring tumor aggressiveness and
targeting metastatic lesions with fluorescent pHLIP. Mol. Imaging Biol.
13 (6), 1146−1156.
(9) Vavere, A. L., Biddlecombe, G. B., Spees, W. M., Garbow, J. R.,
Wijesinghe, D., Andreev, O. A., Engelman, D. M., Reshetnyak, Y. K.,
and Lewis, J. S. (2009) A novel technology for the imaging of acidic
prostate tumors by positron emission tomography. Cancer Res.
1
8
6
(
9 (10), 4510−4516.
10) Ametamey, S. M., Honer, M., and Schubiger, P. A. (2008)
Molecular imaging with PET. Chem. Rev. 108 (5), 1501−1516.
(11) Okarvi, S. M. (2001) Recent progress in fluorine-18 labelled
peptide radiopharmaceuticals. European J. Nucl. Med. 28 (7), 929−938.
AUTHOR INFORMATION
■
*
Corresponding Author
Tel: 1-646-888-3038. Fax: 1-646-422-0408. E-mail: lewisj2@
mskcc.org.
(
12) Olberg, D. E., and Hjelstuen, O. K. (2010) Labeling Strategies
18
of Peptides with F for Positron Emission Tomography. Curr. Top.
Med. Chem. 10 (16), 1669−1679.
Author Contributions
These authors contributed equally to this work.
(13) Hausner, S. H., Marik, J., Gagnon, M. K. J., and Sutcliffe, J. L.
§
(
2008) In Vivo Positron Emission Tomography (PET) Imaging with
18
Grant Support
an αvβ6 Specific Peptide Radiolabeled using F-“Click” Chemistry:
Evaluation and Comparison with the Corresponding 4-[ F]-
1
8
Funded in part by the NIH/NCI R01CA138468 (JSL) and the
Office of Science (BER) - U.S. Department of Energy (Award
DE-SC0002456; JSL). Technical services provided by the
MSKCC Small-Animal Imaging Core Facility were supported
in part by NIH grants R24-CA83084 and P30-CA08748.
18
Fluorobenzoyl- and 2-[ F]Fluoropropionyl-Peptides. J. Med. Chem.
1 (19), 5901−5904.
14) McBride, W. J., D’Souza, C. A., Sharkey, R. M., Karacay, H.,
5
(
Rossi, E. A., Chang, C. H., and Goldenberg, D. M. (2010) Improved
18
F Labeling of Peptides with a Fluoride-Aluminum-Chelate Complex.
Notes
Bioconjugate Chem. 21 (7), 1331−1340.
(15) Schirrmacher, R., Bradtmoller, G., Schirrmacher, E., Thews, O.,
The authors declare no competing financial interest.
Tillmanns, J., Siessmeier, T., Buchholz, H. G., Bartenstein, P.,
Waengler, B., Niemeyer, C. M., and Jurkschat, K. (2006) F-18-labeling
of peptides by means of an organosilicon-based fluoride acceptor.
Angew. Chem., Int. Ed. 45 (36), 6047−6050.
ACKNOWLEDGMENTS
■
Funded in part by the NIH/NCI R01CA138468 (JSL) and the
Office of Science (BER) - U.S. Department of Energy (Award
DE-SC0002456; JSL). Technical services provided by the
MSKCC Small-Animal Imaging Core Facility were supported
in part by NIH grants R24-CA83084 and P30-CA08748.
Special thanks to Valerie Longo, Vadim Divilov, Kuntalkumar
Sevak, and Nicholas Ramos for technical support, Calvin Lom
and Howard Sheh from the MSKCC Radiochemistry-Cyclo-
tron Core, and George Sukenick from the MSKCC Nuclear
Magnetic Resonance (Analytical).
(16) Glaser, M., and Robins, E. G. (2009) ‘Click labelling’ in PET
radiochemistry. J. Labelled Compd. Radiopharm. 52 (9−10), 407−414.
(17) Mamat, C., Ramenda, T., and Wuest, F. R. (2009) Recent
Applications of Click Chemistry for the Synthesis of Radiotracers for
Molecular Imaging. Mini-Rev. Org. Chem. 6 (1), 21−34.
(
18) Li, Z.-B., Wu, Z., Chen, K., Chin, F. T., and Chen, X. (2007)
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Click Chemistry for F-Labeling of RGD Peptides and microPET
Imaging of Tumor Integrin αvβ3 Expression. Bioconjugate Chem.
1
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8 (6), 1987−1994.
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preparation of [ F]fluoropeptides using CuI catalyzed 1,3-dipolar
cycloaddition. Tetrahedron Lett. 47 (37), 6681−6684.
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