In vivo positron emission tomography (PET) imaging with an αvβ6 specific peptide radiolabeled using 18F-"click" chemistry: Evaluation and comparison with the corresponding 4-[18F]fluorobenzoyl- and 2-[18</s

Article abstract of DOI:10.1021/jm800608s

Numerous radiolabeled peptides have been utilized for in vivo imaging of a variety of cell surface receptors. For applications in PET using [ ¹⁸F]fluorine, peptides are radiolabeled via a prosthetic group approach. We previously developed solut

Full text of DOI:10.1021/jm800608s

J. Med. Chem. 2008, 51, 5901–5904
5901
a
Table 1. Radiotracers Prepared
In Vivo Positron Emission Tomography
PET) Imaging with an r ꢀ Specific Peptide
(
v
6
1
8
Radiolabeled using F-“Click” Chemistry:
Evaluation and Comparison with the
radio-
synthesis HPLC
1
8
Rxn
compd steps
Corresponding 4-[ F]Fluorobenzoyl- and
b
c
d
type
group
18
time
purity
yield
1
8
2
-[ F]Fluoropropionyl-Peptides
1
2
3
4
4
2
solid phase
solid phase
solution
[
[
[
F]FBA 137 min >99% 7.8 ( 2.2%
F]FPA 171 min >99% 4.6 ( 0.8%
1
1
8
8
F]FC5 66 min
>98% 8.7 ( 2.3%
†
†
Sven H. Hausner, Jan Marik, M. Karen J. Gagnon, and
Julie L. Sutcliffe*
a
b
A20FMDV2)NAVPNLRGDLQVLAQKVART-C(O)NH2. Preparation
of radiotracer starting from K F and the selectively deprotected peptide.
Since EOB. Decay corrected radiochemical yield based on amount of
1
8
c
d
Department of Biomedical Engineering and Center for Molecular
and Genomic Imaging, UniVersity of California DaVis,
DaVis, California 95616
1
8
K
F at EOB.
a
Scheme 1. Radiosyntheses of Imaging Tracers Evaluated
ReceiVed May 21, 2008
Abstract: Numerous radiolabeled peptides have been utilized for in
vivo imaging of a variety of cell surface receptors. For applications in
18
PET using [ F]fluorine, peptides are radiolabeled via a prosthetic group
1
8
approach. We previously developed solution-phase F-“click” radio-
1
8
labeling and solid-phase radiolabeling using 4-[ F]fluorobenzoic and
1
8
2
-[ F]fluoropropionic acids. Here we compare the three different
radiolabeling approaches and report the effects on PET imaging and
pharmacokinetics. The prosthetic groups did have an effect; metabolites
with significantly different polarities were observed.
a
Noninvasive PET imaging has become a widely used tool
1
for the detection of many diseases. Among the available
1
8
positron emitting nuclides, [ F]fluorine is widely used because
it can be produced in medical cyclotrons and combines favorable
a
Reagents and conditions: (a) K222, DMSO/acetonitrile, 100 °C. (b)
(i) NaOH, 100 °C; (ii) HCl, (iii) C18 Sep-Pak. (c) HATU, DIEA, DMF.
(d) (i) TFA/TIPS/H2O; (ii) HPLC. (e) (i) K222, acetonitrile, 100 °C; (ii)
+
decay-characteristics (T1/2 ) 110 min; mode of decay: 97% ꢀ ;
+
HPLC. (f) TEA, DMF/acetonitrile/H O, 100 °C. (g) Acetonitrile, 100 °C.
(h) (i) CuI, Na ascorbate, DIEA, DMF/acetonitrile/H2O, room temperature;
ii) HPLC.
2
maximum ꢀ energy ) 0.64 MeV) with relative chemical
2
versatility. As new disease-specific imaging targets (e.g., cell
(
surface receptors) are being identified, there is an increased
demand for targeted radiotracers. Peptides are receiving much
attention for in vivo cancer detection because excellent, tissue-
specific uptake can be achieved. Relying on well-established
synthetic chemistries, peptides are readily produced and modi-
3
peptide substrates with minimal synthetic modifications. For this,
fast, simple chemistries amendable to automation are desirable.
Many different strategies have been explored in recent years.
Widely used approaches include the conjugation to free amino
groups on the peptide in solution using F-radiolabeled acids
such as 4-[ F]fluorobenzoic ([ F]FBA) acid and 2-[ F]fluo-
ropropionic ([ F]FPA) acid or their activated forms, N-succin-
13
2
3
fied. Strategies include automated syntheses with incorporation
18
of unnatural amino acids, peptidomimetics, and cyclization,
among others, to develop compounds with desirable pharma-
cokinetic properties. To make peptides amendable for PET
imaging, the [ F]fluorine-radiolabel is introduced using small
molecules (prosthetic groups). Examples of [ F]-labeled pep-
18
18
18
18
18
18
imidyl-4-[ F]fluorobenzoate ([ F]SFB) and p-nitrophenyl 2-
1
8
18
18
[
F]fluoropropionate ([ F]NFP), respectively. The successful
1
8
use of activated esters is limited to peptides bearing only one
free amino group, as otherwise a complex mixture of radiola-
beled products is obtained. Also, the low stability of [ F]SFB
4
tides for PET imaging include octreotide, vasoactive intestinal
peptide, integrin specific peptides,
5
6-8
ε
9
N -(γ-glutamyl)lysine,
18
1
0
11
12
neurotensin analogues, human C-peptide, and insulin.
The prosthetic group approach involves at least two synthetic
steps: incorporation of [ F]fluorine into the prosthetic group
and attachment to the peptide. Generally, the prosthetic group
itself should not negatively affect receptor binding, and the
synthetic approach should be applicable to many different
in solutions at a pH required for the conjugation has been found
14
as a limiting factor in this approach. The conjugation of
1
8
18
18
[
F]FBA and [ F]FPA to the selectively deprotected peptide
attached to a solid support has been developed to overcome
the above-mentioned drawbacks.
proach, the peptide is assembled on a solid support and only
15,16
In the solid-phase ap-
1
8
18
the amino group for the attachment of [ F]FBA or [ F]FPA
is selectively deprotected. The prosthetic group is then
conjugated to the peptide using in situ activation, followed
by simultaneous cleavage of the radiolabeled peptide from
the solid support and complete side chain deprotection (Table
*
To whom correspondence should be addressed. Phone: 530-754-7107.
Fax: 530-754-5739. E-mail: jlsutcliffe@ucdavis.edu.
†
Contributed equally.
a
+
Abbreviations: ꢀ , positron; DIEA, diisopropylethylamine; DMF, N,N-
dimethylformamide; DMSO, dimethylsulfoxide; EOB, end of bombardment;
K222, Kryptofix 222; PET, positron emission tomography; p.i., post
injection; Rt, retention time; T1/2, half-life; TEA, triethylamine; TFA, 2,2,2-
trifluoroacetic acid; TIPS, triisopropylsilane; % ID/g, percent of injected
radioactive dose per gram of tissue.
1
, Scheme 1).
For cases where radiolabeling on solid phase is not possible
or advantageous, such as preparations involving certain post-
1
0.1021/jm800608s CCC: $40.75
 2008 American Chemical Society
Published on Web 09/12/2008

5
902 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Letters
1
8
13
18
16,32
cleavage modifications (e.g., introduction of acid sensitive
groups, certain cyclizations) or workups requiring time-consum-
ing purification procedures, several mild, chemoselective meth-
ods have been developed to attach the radiolabeled prosthetic
labeling with [ F]FBA and [ F]FPA
followed similar
procedures, the preparation of 2 required an additional 34 min
owing to the required HPLC purification of the 9-methylan-
thranyl 2-[ F]fluoropropionate intermediate. Second, when
preparing [ F]FBA and [ F]FPA for coupling, DMF (50 µL)
was added to the final solution of the [ F]FBA or [ F]FPA to
minimize evaporative loss during removal of the acetonitrile
used as solvent during the preparation. A very gentle stream of
nitrogen (100 cm /min) and heating to not more than 100 °C
were applied, as more vigorous conditions led to substantial
loss of [ F]FBA or [ F]FPA. Addition of 5-10 µg of N,N,N,N-
tetramethylammonium hydroxide, commonly used to prevent
evaporative loss, negatively affected the yield of the subsequent
coupling reaction. For coupling to the peptide, the prosthetic
group (in 50 µL DMF) was withdrawn to a 1 mL fritted syringe
containing the preswollen resin, followed by the coupling
reagent (HATU in 30 µL DMF) and the base (DIEA in 30 µL
DMF). The order of addition of the regents has been found to
be crucial for the success of the coupling. The optimal amount
of the resin was 5 mg. Lower amounts led to lower yields, while
higher amounts did not significantly improve the yields.
1
8
1
8
18
18
group to unprotected peptides in solution (e.g., 4-[ F]fluo-
robenzaldehyde to an aminooxyacetic acid or a 6-hydrazinoni-
18 18
1
7,18 18
cotinic acid group;
[ F]fluorothiols to a chloroacetic acid
1
9
group ). All chemoselective conjugations of prosthetic groups
require modification of the peptide with a functional group that
can provide a chemistry orthogonal to all other functional groups
found in the peptide.
3
18
18
Recently, our group successfully used the copper-catalyzed
2
0,21
Huisgen 1,3-dipolar cycloaddition (“click” chemistry)
to
1
8
conjugate ω-[ F]fluoroalkynes to peptides functionalized with
-azidopropionic acid (Table 1, Scheme 1). The formation of
,4-disubstituted 1,2,3-triazoles proceeded smoothly under mild
2
2
3
1
conditions, and the radiolabeled peptides were obtained in a
short period of time. Subsequently, this approach has been
1
8
applied for radiolabeling of different substrates with [ F]-
1
0,23-25
fluorine.
As a result of ongoing improvements in 1,3-
dipolar cycloaddition chemistry and because of its versatility
and short reaction times, “click” radiochemistry promises to
become a widely used tool for preparation of radiotracers.
When comparing the solid-phase radiolabeling of A20FMDV2
to radiolabeling of other substrates, it was found that the yields
of the coupling reactions depended mostly on the size and
complexity of the peptide. They were 22 ( 4% (n ) 5) and 13
( 3% (n ) 3) for coupling of [ F]FBA or [ F]FPA,
respectively, to the 20 amino acid peptide A20FMDV2. By
comparison, coupling yields for [ F]FBA to octapeptides
averaged over 50% but dropped to about 15% for peptides
containing over 50 amino acids. The yields for [ F]FPA
conjugation were slightly lower than those for [ F]FBA. For
both prosthetic groups, the yields of TFA-mediated cleavage
of the final product from the solid support and the simultaneous
removal of the side chain protecting groups were 71 ( 4% (n
Here we present an evaluation of the feasibility of in vivo
1
8
imaging with a [ F]-labeled “click” probe. A20FMDV2, a
1
8
18
peptide that selectively binds to the integrin Rvꢀ6, was chosen
8
,26
as model peptide (Table 1).
Expression of the epithelial-
1
8
specific integrin Rvꢀ6 is tightly regulated. It is low or undetect-
able in adult tissues but has been shown to be increased in many
different cancers, including colon, cervical, lung, and pancreatic
cancer; the integrin Rvꢀ6 has also been described as a prognostic
1
8
1
8
2
7-31
biomarker linked to poor survival.
We have shown recently
1
8
in a mouse model that [ F]FBA-A20FDMV2 (1) can be used
to selectively image Rvꢀ6-expressing tumors.
8
)
8).
The same mouse model, male athymic nude mice bearing
Rvꢀ6-positive (DX3puroꢀ6) and Rvꢀ6-negative (DX3puro, con-
trol) cell xenografts, was employed here for comparison of
“Click” radiolabeling, like any generally applicable chemose-
lective conjugation of a prosthetic group to an unprotected
peptide, requires modification of the peptide before the conjuga-
tion can be performed. Here, the 3-azidopropionyl group was
introduced at the N-terminus of A20FMDV2 for the conjugation
of 5-[ F]fluoro-1-pentyne. Again, in general, the yield of the
Cu catalyzed conjugation depended on the size and complexity
1
8
8
18
[
[
F]FBA-A20FDMV2 (1), [ F]FPA-A20FDMV2 (2), and
1
F]FC5-A20FDMV2 (3) (Supporting Information). Data pre-
sented compare tracer preparation, microPET imaging, biodis-
tribution, and an initial metabolic evaluation.
As mentioned above, compounds 1 and 2 were prepared by
1
8
I
of the peptide substrate. Short peptides provided near quantita-
solid-phase radiolabeling, while the chemoselective “click”
22
18
tive yields, but longer peptides like A20FMDV2 were obtained
in yields below 10% and required approximately 1 mg of peptide
precursor.
approach was chosen to prepare 3 from 5-[ F]fluoro-1-pentyne
and N-(3-azidopropionyl)-A20FMDV2 (Scheme 1). All pros-
thetic groups were attached to the N-terminal amino group of
the peptide chain. Their different chemical nature changed the
chemical properties of the radiotracer, that is size, lipophilicity,
and ability to form hydrogen bonds. The smallest prosthetic
In all three cases, the radiolabeled peptides were easily
separated from nonradiolabeled peptide precursors using HPLC
based on the difference in polarities caused by introduction of
the prosthetic groups (Supporting Information Figure S1).
Remaining amounts of unreacted [ F]FBA and [ F]FPA were
washed away from the solid support before cleavage of the
1
8
group used was [ F]FPA; it has to be noted that this prosthetic
group is obtained as a mixture of enantiomers and therefore
the final radiolabeled peptide 2 was a mixture of diastereoiso-
18
18
18
18
mers. The medium sized [ F]FBA was expected to provide the
final product 1 with increased lipophilicity. Within the set of
prosthetic groups evaluated, the 1,4 disubstituted 1,2,3-triazole
peptide from the solid support, while unreacted 5-[ F]fluoro-
18
1-pentyne was evaporated during the drying step. All three F-
fluoropeptides were characterized by coelution with nonradio-
active standards. The specific activities of purified final products
were >37 GBq/µmol based on HPLC analysis.
Overall, the total yields of the radiolabeled products obtained
from solid-phase and chemoselective “click” approaches were
comparable but several significant differences were observed.
Although the “click” approach provided the product in less than
half of the time needed for the solid-phase syntheses and in
only two radiochemical steps, the solid-phase approaches
required smaller amounts of the starting material; “click”
18
(
[ F]FC5) in tracer 3 was the largest. However, the large dipole
moment and the ability of the nitrogen atoms in positions one
and three of the triazole ring to form hydrogen bonds decreased
the lipophilicity of the final radiolabeled peptide. The reversed-
phase HPLC retention times for the three compounds cor-
roborated these assumptions (1 16.6 min, 2 14.7 min, and 3
1
4.7 min; Supporting Information Figure S1).
Several details are worth noting when comparing the three
radiolabeling procedures summarized in Table 1. First, while

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19 5903
Chart 1. Biodistribution of Tracers 1-3 in Male Athymic Nude
a
Mice 1 h after Injection (n ) 3/tracer)
Figure 1. Representative normalized transaxial sections of microPET
scans (60-75 min p.i.) with compounds 1, 2, and 3 in mice bearing
paired human cell xenografts (R
negative parent DX3puro).
v
ꢀ
6
-expressing DX3puroꢀ6, and R
v 6
ꢀ -
conjugation required 1 mg of purified N-(3-azidopropionyl)-
A20FMDV2 versus 5 mg of resin (bearing the crude peptide)
for the solid-phase syntheses. Also, the solid-phase approach
is more easily amendable for automation of the radiochemical
procedure. In general, the chemoselective “click” method is
faster but the solid-phase approach seems to be more versatile
and cost-effective for peptides such as A20FMDV2. Thus, at
least for the moment, the chemoselective “click” approach
appears to be more suitable for short peptides (or those requiring
time-consuming postcleavage procedures) and small molecules,
while the solid-phase approach appears more advantageous for
long peptide chains.
a
Levels of radioactivity in healthy tissues, as well as Rvꢀ6-positive
(DX3puroꢀ6) and R ꢀ -negative (DX3puro) tumors are expressed as % ID/
g. Data for 1 were taken from ref 8.
v 6
a
Table 2. Uptake Ratios for Tumors and Selected Organs
b
compd DX3puroꢀ6/DX3puro DX3puroꢀ6/blood DX3puroꢀ6/kidneys
For evaluation of the effects of the prosthetic groups on
pharmacokinetics, microPET and biodistribution studies were
carried out with tracers 1-3 in athymic nude mice bearing
paired human xenografts (Rvꢀ6-positive, and Rvꢀ6-negative
c
d
e
d
e
e
1
2
3
a
3.1:1
1.9:1
3.3:1
2.5:1
1.8:1
2.3:1
b
1:5.4
1:4.1
1:9.4
c
Based on biodistribution (1 h p.i.). For P-values, see Chart 1. P <
8
d
e
control). MicroPET imaging data were acquired as dynamic 4
0.01. P < 0.05. P < 0.001.
×
15 min scans (n ) 3/tracer), beginning 15 min after injection.
As depicted in Figure 1, all three tracers were able to target the
Rvꢀ6-positive DX3puroꢀ6 tumor. Tracers 1 and 3 showed better
DX3puroꢀ6/DX3puro and DX3puroꢀ6/background ratios than
unmetabolized radiotracer (Supporting Information Figure S3).
Instead, three radioactive metabolites were observed for 1 (Rt
)
9.0, 10.4, 10.8 min vs Rt(1) ) 16.6 min), while 2 yielded
2
. Overall, the PET data paralleled the biodistribution data,
two metabolites with very short retention times (i.e., high
hydrophilicities; Rt ) 2.1, 3.2 min vs Rt(2) ) 14.7 min).
Similarly, two main metabolites with intermediate retention
times were detected for 3 (Rt ) 5.9, 8.6 min vs Rt(3) ) 14.7
min). This initial analysis indicated that, despite identical peptide
sequence and comparable overall biodistribution, significant
pharmacokinetic differences do exist and that they have to be
attributed to the prosthetic groups. It can be assumed that the
showing highest levels of activity in kidneys and the urinary
bladder (Supporting Information Figure S2). Thus, the ra-
diotracer 3 prepared by “click” chemistry can be considered
comparable to other radiotracers bearing established pros-
thetic groups, yielding images similar to those of the
1
8
[
F]FBA-tracer 1.
Biodistribution studies revealed generally similar uptake-
patterns of 1-3 for most organs and the tumors (n ) 3/tracer,
h p.i., Chart 1, Supporting Information Table S1). Renal
(
1
comparatively minor) differences in % ID/g-values between
, 2, and 3 seen for individual organs can be linked at least
1
clearance was the dominant route of elimination, with 1 resulting
in highest levels of activity in the urine (1 1082 ( 279% ID/g,
partially to different excretion characteristics of the metabolites
as well. The differences would likely have been more pro-
nounced were it not for the fact that all the metabolites examined
here were more hydrophilic (i.e., had shorter HPLC retention
times) than the intact tracers, resulting in rapid renal excretion.
However, such a favorable clearance behavior can not neces-
sarily be expected for metabolites of other tracers.
2
311 ( 133% ID/g, 3 501 ( 332% ID/g), while 3 appeared to
result in moderately increased levels of radioactivity in the
kidneys and the liver.
Uptake levels in the Rvꢀ6-positive tumor 1 h after injection
were 0.66 ( 0.09% ID/g, 1.18 ( 0.28% ID/g, and 1.01 ( 0.09%
ID/g for 1, 2, and 3, respectively. While 2 showed the highest
uptake, the Rvꢀ6-positive/Rvꢀ6-negative tumor uptake ratio was
comparatively low (1.9:1 vs > 3:1 for 1 and 3, Table 2). A
similar trend was found for the Rvꢀ6-positive tumor/blood ratio.
These differences are noteworthy, as they demonstrate the effect
of the prosthetic groups on tumor-targeting and pharmacoki-
netics. The results are even more surprising in light of the
identical HPLC-retention times of 2 and 3. Furthermore, 1 had
the longest HPLC-retention time (highest lipophilicity), which
would be expected to favor liver uptake, yet it resulted in highest
levels of radioactivity in the urine.
1
8
In conclusion, we compared three F-prosthetic groups,
F]FBA, [ F]FPA, and [ F]FC5, the latter introduced by
click” chemistry, for peptide radiolabeling and in vivo studies.
1
8
18
18
[
“
All three prosthetic groups were readily introduced at the
N-terminus of a tumor targeting model peptide with similar
overall radiolabeling yields. The “click” radiolabeling approach
was fastest but required a relatively large amount of purified
peptide precursor. During in vivo animal studies, we observed
that the prosthetic groups had a noticeable effect on pharma-
cokinetics, notably tumor uptake and metabolic fate, thus
underscoring the necessity for the investigation of different
prosthetic groups that allow combination of convenient chem-
istries with favorable pharmacokinetics for each individual tracer
under development.
A possible answer to this may lie in the metabolic fate of
the compounds. As stated above, renal clearance was the main
route of excretion for all three tracers. When urine samples taken
1
h p.i. were analyzed by radio-HPLC, none of them contained

5
904 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 19
Acknowledgment. We thank David L. Kukis, Salma Jivan,
Letters
1
8
biodistribution of F-labelled linear peptides. Eur. J. Nucl. Med. Mol.
Imaging 2002, 29, 754–759.
16) Marik, J.; Hausner, S. H.; Fix, L. A.; Gagnon, M. K. J.; Sutcliffe,
Craig Abbey, John F. Marshall, Catherine E. Stanecki, and Julia
Choi. This work was supported by NIH grant no. R21
CA107792.
(
18
J. L. Solid-phase synthesis of 2-[ F]fluoropropionyl peptides. Bio-
conjugate Chem. 2006, 17, 1017–1021.
(
17) Poethko, T.; Schottelius, M.; Thumshirn, G.; Hersel, U.; Herz, M.;
Henriksen, G.; Kessler, H.; Schwaiger, M.; Wester, H. J. Two-step
Supporting Information Available: Experimental details and
analytical data for precursors, nonradioactive analogues and ra-
diotracers, and information about the imaging studies. This material
is available free of charge via the Internet at http://pubs.acs.org.
18
methodology for high-yield routine radiohalogenation of peptides: F-
labeled RGD and octreotide analogs. J. Nucl. Med. 2004, 45, 892–
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