Strain-promoted catalyst-free click chemistry for rapid construction of 64Cu-labeled PET imaging probes

Article abstract of DOI:10.1021/ml300236m

A rapid, efficient, and catalyst-free click chemistry method for the construction of ⁶⁴Cu-labeled PET imaging probes was reported based on the strain-promoted aza-dibenzocyclooctyne ligation. This new method was exemplified in the synthesis of ⁶⁴Cu-labeled RGD peptide for PET imaging of tumor integrin α_vβ₃ expression in vivo. The catalyst-free click chemistry reaction proceeded with a fast rate and eliminated the contamination problem of the catalyst Cu(I) ions interfering with the ⁶⁴Cu radiolabeling procedure under the conventional Cu-catalyzed 1,3-dipolar cycloaddition condition. The new strategy is simple and robust, and the resultant ⁶⁴Cu-labeled RGD probe was obtained in an excellent yield and high specific activity. PET imaging and biodistribution studies revealed significant, specific uptake of the click ⁶⁴Cu-labeled RGD probe in integrin α_vβ ₃-positive U87MG xenografts with little uptake in nontarget tissues. This new approach is versatile, which warrants a wide range of applications for highly diverse radiometalated bioconjugates for radioimaging and radiotherapy.

Full text of DOI:10.1021/ml300236m

Letter
pubs.acs.org/acsmedchemlett
Strain-Promoted Catalyst-Free Click Chemistry for Rapid
64
Construction of Cu-Labeled PET Imaging Probes
Kai Chen,*,† Xinlu Wang,†,∥,§ Wei-Yu Lin,‡,§ Clifton K.-F. Shen,‡ Li-Peng Yap,† Lindsey D. Hughes,†
and Peter S. Conti*,†
†Molecular Imaging Center, Department of Radiology, Keck School of Medicine, University of Southern California, Los Angeles,
California 90033, United States
‡Department of Molecular and Medical Pharmacology, University of California at Los Angeles, Los Angeles, California 90095, United
States
∥Department of Nuclear Medicine and PET-CT Center, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou
510010, China
S
* Supporting Information
ABSTRACT: A rapid, efficient, and catalyst-free click chemistry method for the construction of 64Cu-labeled PET imaging
probes was reported based on the strain-promoted aza-dibenzocyclooctyne ligation. This new method was exemplified in the
synthesis of 64Cu-labeled RGD peptide for PET imaging of tumor integrin αvβ3 expression in vivo. The catalyst-free click
chemistry reaction proceeded with a fast rate and eliminated the contamination problem of the catalyst Cu(I) ions interfering
with the 64Cu radiolabeling procedure under the conventional Cu-catalyzed 1,3-dipolar cycloaddition condition. The new
strategy is simple and robust, and the resultant 64Cu-labeled RGD probe was obtained in an excellent yield and high specific
activity. PET imaging and biodistribution studies revealed significant, specific uptake of the “click” 64Cu-labeled RGD probe in
integrin αvβ3-positive U87MG xenografts with little uptake in nontarget tissues. This new approach is versatile, which warrants a
wide range of applications for highly diverse radiometalated bioconjugates for radioimaging and radiotherapy.
KEYWORDS: PET imaging probe, 64Cu radiolabeling, catalyst-free click chemistry, integrin αvβ3, in vivo
same subject to be achieved by successful PET imaging over
ositron emission tomography (PET) is a nuclear imaging
Ptechnique used to map biological and physiological several hours or even days. Among these metallic radionuclides,
64Cu (t1/2 = 12.7 h; β+ 655 keV, 17.8%) has attracted
considerable interest because of its favorable decay half-life, low
β+ energy, and commercial availability.8−10 As the half-life of
64Cu is relatively short, fast, clean, and reliable chemistry which
can proceed efficiently under mild condition is required for
64Cu labeling of biomolecules. The exploration of new 64Cu-
labeling methods using a simple, versatile, and modular
approach is thus highly demanded.
Click chemistry offers chemists a platform for general,
modular, and high yielding synthetic transformations for
constructing highly diverse molecules.11 The Huisgen 1,3-
dipolar cycloaddition reaction, which fuses an azide and an
processes in living subjects.1−3 Unlike morphological imaging
techniques, such as computed tomography (CT), PET requires
the injection of molecular probes in a tested subject in order to
acquire the imaging signal from molecular probes labeled with
positron-emitting radionuclides.4,5 Fluorine-18 and cabon-11
are two conventional PET radionuclides used for the
development of PET imaging probes. Due to the short half-
lives of fluorine-18 (109.8 min) and cabon-11 (20.3 min), 18F-
or 11C-labeled PET probes must be radiosynthesized with the
need for an on-site cyclotron, and the PET imaging of a subject
using 18F- or 11C-labeled PET probes must be performed
within a few hours.6 In addition to 18F and 11C, several
nonconventional metallic radionuclides, such as 64Cu, 68Ga, 86Y,
and 89Zr, have been applied to PET probes.7 These metallic
PET isotopes are usually characterized by longer half-lives,
allowing the evaluation of radiopharmaceutical kinetics in the
Received: August 10, 2012
Accepted: September 19, 2012
Published: September 19, 2012
© 2012 American Chemical Society
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alkyne together, and provides access to a variety of five-
membered heterocycles, has become of great use in labeling
studies, in the development of new therapeutics and nano-
particles, and in protein modification.12−15 However, the
Huisgen 1,3-dipolar cycloaddition reaction often requires the
presence of catalytic amounts of nonradiolabeled Cu(I) ions,
which interfere with radiometals, such as 64Cu, and make click
reaction unfavorable for the development of radiometal-labeled
PET probes. With the recent discovery of Cu(I)-free 1,3-
dipolar cycloaddition reactions,16,17 several strain-promoted
systems, such as cyclooctynes and dibenzocyclooctynes, have
been developed for 18F labeling,18,19 but few examples were
reported in radiometal labeling.20 One elegant study was to use
a Diels−Alder reaction (norbornene-tetrazine ligation) to
prepare 64Cu-labeled antibodies.21 To the best of our
knowledge, the catalyst-free aza-dibenzocyclooctyne ligation
has not yet been employed in the radiometal-labeled probes.
Herein we present a study of using aza-dibenzocyclooctyne
ligationa fast and efficient approach to synthesize 64Cu-
labeled probes. A 64Cu-labeled derivative of cyclic RGD peptide
[c(RGDfK)] (Figure 1), a well-validated integrin αvβ3
tetraamine ligands, have been developed for 64Cu label-
ing.8−10,26 Recently, a new class of BFCs, based on the cagelike
hexaazamacrobicyclic sarcophagine, has gained great attention
as potential 64Cu chelators. We and others demonstrated that
either one of the primary amines of 3,6,10,13,16,19-
hexaazabicyclo[6.6.6]eicosane-1,8-diamine (DiAmSar) or both
primary amines could be modified and coupled with bio-
logically relevant ligands.27−29 The resulting 64Cu complexes
present improved in vivo stability and radiolabeling efficiency.
On the other hand, an aza-dibenzocyclooctyne system has been
proved to be simultaneously reactive and stable.30 Therefore,
we attempted to build a DiAmSar-containing dibenzocyclooc-
tyne analog as a strained alkylne as well as a 64Cu labeling
precursor. In addition, because a poly(ethylene glycol) (PEG)
linker can fine-tune the in vivo pharmacokinetics of imaging
probes,31,32 we aimed to incorporate a PEG linker between
DiAmSar and dibenzocyclooctyne. Our synthesis started from
commercially available dibenzocyclooctyne, which was coupled
with a short PEG linker. After the activation of the carboxylic
acid group, the PEG-dibenzocyclooctyne was conjugated to the
commercially available DiAmSar in basic sodium borate buffer
to afford 3 in 45% yield (Supporting Information). Radio-
labeling of 3 with 64Cu was efficiently accomplished at 40 °C in
0.4 M NH4OAc buffer within 30 min (Figure 2a and
Figure 1. Chemical structures of relevant RGD peptides.
ligand,22−24 was exemplified by using the “click” method. We
further demonstrate that our “click” RGD probe maintains
good binding affinity to the integrin αvβ3 receptor and exhibits
excellent tumor targeting and retention properties in an
integrin αvβ3-positive mouse tumor model.
Figure 2. (a) Synthesis of [64Cu]1. Reagents and conditions: (i)
64CuCl2, 40 °C, 0.4 M NH4OAc; (ii) RGD peptide 2, 45 °C, H2O;
(iii) RGD peptide 2, room temperature, H2O; (iv) 64CuCl2, 40 °C, 0.4
M NH4OAc. (b) Radio-HPLC profiles of [64Cu]1 and [64Cu]3.
Our starting point was to select a suitable azide moiety
containing RGD peptide. It was found that glycosylation on the
lysine side chain of cyclic RGD peptides decreased lipophilicity
and hepatic uptake.25 This finding prompted us to consider
methods for preparing azido galacto-RGD peptide. To this end,
we synthesized 2 as shown in Figure 1. The fully protected
c[R(Pbf)GD(OtBu)fK] peptide was conjugated with Fmoc-
protected galacturonic acid derivative, followed by deprotection
of the Fmoc group, azido acetic acid coupling, and
deprotections of guanidine and acid (Supporting Information).
The synthesis was achieved in four steps with a total yield of
44%. We successfully obtained 2 in a great chemical purity
(>95%) without HPLC purification.
Supporting Information). The product [64Cu]3 was purified
by HPLC. The radioactive peak containing [64Cu]3 appeared at
17.67 min, as shown in Figure 2b. The specific activity of
[64Cu]3 was estimated to be 37 MBq·nmol−1.
To optimize the conjugation between 2 and [64Cu]3, we
systematically investigated the coupling efficiency under various
reaction conditions by changing reaction factors, including
reactant stoichiometry, solvent, reaction time, and reaction
temperature. The conjugations between 2 and [64Cu]3 were
initially carried out in deionized water and analyzed by
analytical HPLC. When [64Cu]3 (3.7 Mbq, 1 μM) was mixed
with a large excess of 2 (>100-fold) at room temperature,
[64Cu]3 was rapidly consumed within 10−15 min, and [64Cu]1
We next sought to select a suitable 64Cu-chelator complex
system, which can be readily conjugated with a strained alkyne.
Various bifunctional chelators (BFCs), including widely used
cyclam and cyclen backbones-based chelators and cross-bridged
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Table 1. Optimization of Synthesis of [64Cu]1 through the Strain-Promoted Catalyst-Free Conjugation between 2 and [64Cu]3
a
entry
2 (μM)
[64Cu]3
solvent
H2O
temp (°C)
reaction time (min)
radiochem yield (%)
1
2
3
4
5
6
7
228
114
5.7
3.7 MBq (1 μM)
3.7 MBq (1 μM)
3.7 MBq (1 μM)
3.7 MBq (1 μM)
3.7 MBq (1 μM)
1.85 MBq (0.5 μM)
3.7 MBq (1 μM)
25
25
25
45
45
45
45
15
10
10
10
15
10
15
>98
92
H2O
H2O
42
5.7
H2O
>98
>98
16
1.14
0.29
1.14
H2O
H2O
PBS buffer
>98
a
The concentration was estimated based on the specific activity of [64Cu]3 (37 MBq·nmol−1), taking into account a correction of radioactive decay.
(tR = 15.05 min, Figure 2b) formed in >92% radiochemical
yield (Table 1, entries 1 and 2). With a small excess of 2 (5.7-
fold), the radiochemical yield was decreased to 42% after
combining 2 and [64Cu]3 for 10 min at room temperature
(entry 3). However, the elevated temperature (45 °C)
significantly enhanced the [64Cu]1 yield (>98%, entry 4)
after 10-min mixing of 2 and [64Cu]3. With prolonged reaction
time (15 min), an excellent radiochemical yield (>98%) of
[64Cu]1 was still achieved by using a 1.14:1 ratio of 2 and
[64Cu]3 (entry 5). Further reducing the concentration of 2 and
[64Cu]3 resulted in a low [64Cu]1 yield (16%, entry 6). We also
investigated the efficiency of the conjugation between 2 and
[64Cu]3 in phosphate buffered saline (PBS). As shown in entry
7, [64Cu]1 was formed in a quantitative yield at 45 °C within 15
min. Among the explored reaction conditions, coupling of 2
and [64Cu]3 with a 1.14:1 ratio at 45 °C for 15 min in
deionized water or PBS buffer exhibits the highest conjugation
efficiency. In addition, after coupling of 2 and [64Cu]3, we did
not observe any major side product peaks in HPLC analysis,
indicating that the catalyst-free click reaction was rather clean
and no free 64Cu was released during radiolabeling.
determined to be 105 5 nM and 170 3 nM, respectively
(Figure S2). The slightly decreased integrin αvβ3 binding of 1 as
compared to c(RGDyK) indicates a minimum impact of a long
tail (containing galactose, triazole, and Sar moieties) on the
binding of c(RGDfK) to integrin αvβ3 receptors.
The in vivo tumor-targeting efficacy of [64Cu]1 was evaluated
in nude mice bearing U87MG human glioblastoma xenograft
tumors (n = 5) by static microPET scans at 2, 4, and 20 h after
tail-vain injection of [64Cu]1. Representative coronal slices that
contained the tumor are shown in Figure 3. U87MG tumors
Figure 3. Decay-corrected whole-body microPET images of U87MG
tumor bearing mice (n = 5) at 2, 4, and 20 h after intravenous injection
of [64Cu]1. The image obtained with coinjection of c(RGDyK) (10
mg/kg body weight) is shown for a 2 h blockade (right). Tumors are
indicated by arrows.
To compare with “click” labeling of [64Cu]1, direct labeling
of 1 with 64Cu was also conducted (Figure 2a). The
conjugation of 2 and 3 was initially carried out at room
temperature. As anticipated, RGD peptide 1 was formed rapidly
in an excellent yield (95%) after HPLC purification
(Supporting Information). Radiolabeling of 1 with 64Cu could
be achieved at 40 °C in 0.4 M NH4OAc buffer within 30 min.
The HPLC retention time (15.05 min) of product from direct
64Cu labeling of 1 was consistent with that of “click” 64Cu
labeling product (Figure 2b), suggesting the products from two
labeling methods were identical. It is also noteworthy that the
specific activity of [64Cu]1 from the direct labeling method
(30−37 MBq·nmol−1) was close to that from the “click”
labeling approach (30 MBq·nmol−1).
were clearly visualized at all time points examined. Region-of-
interest (ROI) analysis on microPET images showed the tumor
uptake values were 4.96 0.73, 4.11 0.54, and 2.41 0.31%
ID/g at 2, 4, and 20 h postinjection (pi), respectively (Table 2).
At 2 h pi, the tumor/muscle, tumor/liver, and tumor/kidneys
ratios reached 11.88
1.31, 2.85
0.28, and 2.22
0.26,
respectively. Consequently, the high tumor-to-normal tissue
Table 2. Decay-Corrected Biodistribution of [64Cu]1 in
The in vitro stability of [64Cu]1 was evaluated after 1, 6, and
24 h of incubation in PBS or mouse serum by radio-HPLC
(Figure S1). Chromatographic results demonstrated no release
of 64Cu from the conjugate over a period of 24 h. This high
stability is attributed to a Sar cage in the conjugate. The
octanol/water partition coefficient (log P) for [64Cu]1 was
U87MG Tumor-Bearing Mice Quantified by micoPET
a
Imaging (n = 5)
b
tissue
2 h
2 h (blockade)
4 h
20 h
Percent Injected Dose/gram (% ID/g)
T
M
L
4.96 0.73
0.42 0.05
1.76 0.35
2.23 0.21
0.71 0.30
0.27 0.04
1.16 0.15
2.09 0.28
4.11 0.54
0.36 0.07
1.64 0.23
1.79 0.20
2.41 0.31
0.35 0.05
1.25 0.23
0.69 0.26
determined to be −1.94
0.10 (Supporting Information),
suggesting that [64Cu]1 is rather hydrophilic. In addition, it is
known that the U87MG human glioblastoma cell line
overexpresses integrin αvβ3 receptor.33 Therefore, we used
the U87MG cells to measure the integrin αvβ3 binding affinity
of 1 by a competitive cell-binding assay,33 where 125I-echistatin
was employed as integrin αvβ3-specific radioligand for
competitive displacement. The IC50 values of c(RGDyK) and
1, which represent the concentrations required to displace 50%
of the 125I-echistatin bound to the U87MG cells, were
K
Tumor-to-Normal Tissue Uptake Ratio
T/M
T/L
T/K
11.88 1.31
2.85 0.28
2.22 0.26
2.52 0.81
0.60 0.22
0.33 0.11
11.65 1.83
2.51 0.16
2.32 0.35
7.03 1.22
1.96 0.26
3.87 1.26
a
b
The results are presented as mean
muscle; L, liver; K, kidneys.
SD (n = 5). T, tumor; M,
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ACS Medicinal Chemistry Letters
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ratios provided excellent contrast for PET imaging. A blocking
experiment was conducted to confirm the integrin αvβ3
specificity of [64Cu]1. In the presence of a blocking dose (10
mg/kg) of c(RGDyK), the U87MG tumor uptake was reduced
to the background level (0.71 0.30%ID/g) at 2 h pi (Figure 3
and Table 2). The uptake values of normal tissues (e.g., muscle,
liver, and kidneys) were also lower than those without
coinjection of c(RGDyK) (Table 2). The ex vivo biodistribution
of [64Cu]1 was examined in U87MG tumor-bearing mice at 20
h pi after a microPET scan with and without coinjection of
c(RGDyK) (10 mg/kg of mouse body weight). The percentage
injected dose per gram of tissue (%ID/g) was shown in Figure
S3. The biodistribution results were consistent with the
quantitative analysis of microPET imaging. At 20 h pi, the
U87MG tumor uptake of [64Cu]1 reached 2.26 0.17%ID/g,
whereas the presence of c(RGDyK) peptide significantly
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures, cell-based integrin αvβ3 receptor
binding, and biodistribution data. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*Tel: 323-442-3858. Fax: 323-442-3253. E-mail: K.C.,
chenkai@usc.edu.
*E-mail: P.S.C., pconti@usc.edu
Present Address
2250 Alcazar Street, CSC103, Molecular Imaging Center,
Department of Radiology, Keck School of Medicine, University
of Southern California, Los Angeles, CA 90033, USA.
Author Contributions
§These authors contributed equally.
reduced the tumor uptake to 0.45
0.12%ID/g (P < 0.01)
in the blocking group. In addition, [64Cu]1 displayed little
accumulation and retention in liver and kidneys at 20 h pi. For
the nonblocking group, 1.21 0.17%ID/g and 0.65 0.11%
ID/g remained in the liver and kidneys, respectively.
Furthermore, similar to microPET imaging analyses, the
presence of c(RGDyK) peptide decreased the overall uptake
of [64Cu]1 in most tissues and organs. Based on the
biodistribution results, the contrast ratios of tumor to normal
organs for the nonblocking and blocking groups were
calculated. For the nonblocking group, the ratio of tumor
uptake to muscle, liver, and kidneys uptake at 20 h pi was
Funding Sources
This work was supported by the research fund (#IRG-58-007-
51) from the American Cancer Society and by the USC
Department of Radiology.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
PET, positron emission tomography; CT, computed tomog-
raphy; HPLC, high performance liquid chromatography;
DiAmSar, 3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane-1,8-di-
amine; BFCs, bifunctional chelators; PEG, poly(ethylene
glycol); pi, postinjection; PBS, phosphate buffered saline
calculated to be 5.65
respectively, while the corresponding values for the blocking
group were 1.41 0.10, 0.64 0.11, and 0.83 0.13,
0.11, 1.87
0.17, and 3.48
0.14,
respectively. Overall, the biodistribution pattern of the 64Cu-
labeled “click” RGD probe is quite similar to what we
previously obtained for 64Cu-labeled non-“click” RGD probes.27
In conclusion, a new catalyst-free click chemistry approach
based on strain-promoted aza-dibenzocyclooctyne ligation has
been developed for 64Cu-labeling of biomolecules. In our new
approach, we first prepared a 64Cu-labeled alkyne-containing
component (prosthetic group), followed by conjugation of
biomolecule via click chemistry. Successful employment of
catalyst-free click chemistry in the preparation of 64Cu-labeled
probes, which was demonstrated in our work, can eliminate the
contamination problem of catalyst Cu(I) ions under the
conventional Cu-catalyst 1,3-dipolar cycloaddition condition.
The strain-promoted click reaction proceeds with a fast rate at
low concentration, making it superior to other types of
conjugation reactions for radiolabeling with short-lived
isotopes, such as 64Cu. Although we focused on the use of
integrin αvβ3-specific RGD peptide for proof of principle, the
technique is versatile and can be applied to other 64Cu-labeled
or other radiometal-labeled probes. More importantly, this new
catalyst-free click approach creates a modular platform in which
a biomolecule can be modified with a wide variety of chelators
and radiometals. Given the fact that different radiometals often
require different chelators, this methodology could no doubt
assist in the rapid and robust construction of highly diverse
radiometalated bioconjugates for in vitro and in vivo screenings,
and radioimaging and radiotherapy applications.
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