Strain-promoted copper-free "click" chemistry for 18F radiolabeling of bombesin

Article abstract of DOI:10.1002/anie.201105547

Click for PET: The GRP-receptor-specific peptide bombesin, which is often used for nuclear imaging of tumors, can be labeled with ¹⁸F in a mild and rapid manner by using a copper-free azide-alkyne "click" reaction. A range of azides can be used to provide peptides with different hydrophobicities. The resulting ¹⁸F radiopharmaceutical tracers (see scheme) maintain their high affinity for the targeted receptor in vitro in human prostate cancer cells. Copyright

Full text of DOI:10.1002/anie.201105547

DOI: 10.1002/anie.201105547
Isotopic Labeling
Strain-Promoted Copper-Free “Click” Chemistry for 18F Radiolabeling
of Bombesin
Lachlan S. Campbell-Verduyn, Leila Mirfeizi, Anne K. Schoonen, Rudi A. Dierckx,
Philip H. Elsinga,* and Ben L. Feringa*
Bombesin is a 14 amino acid (Pyr-Gln-Arg-Leu-Gly-Asn-
Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) neuropeptide,
11C (t1/2 ꢀ 20 min) and 13N (t1/2 ꢀ 10 min), 18F has the distinct
advantage of allowing for off-site production and transporta-
tion of the radionuclide as well as allowing for scans to be
carried out over several hours. Furthermore, the use of 18F
results in images with higher resolution than other radio-
nuclides because of its low positron energy.[5] Very few
instances of 18F-labeled bombesin have been reported to date,
which is predominantly due to the synthetic challenges
associated with the introduction of 18F when compared with
simple chelation techniques used for metallic radionuclides.[6]
Notably, the synthetic time frame is also much reduced when
compared with metallic radionuclides such as 64Cu (t1/2
ꢀ 12 h). A major disadvantage is the need for the multistep
synthetic procedures required to synthesize current prosthetic
groups such as [18F]succinimidyl 4-fluorobenzoate ([18F]SFB)
or [18F]4-fluorobenzaldehyde, which are commonly used to
introduce 18F in the presence of a free amine.[7] Ideally, a
prosthetic group should be easily synthesized, introduce the
radionuclide in the last step of the synthesis, and should
require only the mildest of conditions to attach it to the
biomolecule of interest.
which binds with high affinity to the gastrin-releasing peptide
receptor (GRPR). Bombesin has received much attention in
the field of nuclear imaging because the GRPR is massively
overexpressed on a variety of tumor cells, including breast
and prostate tumor cells, thus making bombesin a promising
radioligand for the diagnosis and imaging of cancer.[1] Much
effort has been invested in the development of labeled
bombesin analogues.[2] Bombesin is often modified in the
form of Lys[3]-bombesin, which allows for site-selective
introduction of the radionuclide at the terminal amino
The azide–alkyne cycloaddition has been popularized
under the general term “click” chemistry since the discovery
that it proceeds regioselectively at room temperature in the
presence of catalytic amounts of CuI ions.[8] The bioorthogo-
nality of the azide and the alkyne has proven unparalleled.
The robustness and versatility of this reaction along with its
mild conditions makes it attractive for labeling target
molecules with radionuclide-containing prosthetic groups.
Many groups have exploited the bioorthogonality of this
reaction to allow for fast and straightforward labeling of sugar
and peptide targets with 18F and other radionuclides.[9] The
obvious limitation of this methodology for biological systems
is the cytotoxicity of copper. Potential contamination of
labeled compounds with traces of copper is a major drawback
and this reaction is thus not suitable for development of
in vivo pretargeting methodologies. In recent years, great
progress has been made in developing copper-free method-
ologies through, for example, the use of strained cyclo-
octynes;[10] in one instance this reaction has been used for the
introduction of 111In to a target peptide for single photon
emission computed tomography (SPECT) imaging.[11] We
envisioned the use of Lys-[3]bombesin modified with a
strained alkyne to allow for rapid and facile labeling with
18F in the absence of possible copper contamination. A further
advantage of this methodology would be the possibility to
fine-tune the properties of the resulting labeled peptide. The
azide group can be designed to tune the hydrophilicity, bulk,
or charge of the peptide in question. Furthermore, though we
group of lysine. Amino acids 7–14 are known to be essential
for receptor binding, thus modification in the third amino acid
reduces potential for interference.[3] A variety of bombesin
analogues for nuclear imaging have been synthesized and are
predominantly labeled with large metal-based radionuclides
(64Cu, 111In, 68Ga) through the commonly introduced chelating
groups
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid (DOTA) and 1,4,7-triazacyclononane-1,4,7-triacetic
acid (NOTA).[4]
Positron emission tomography (PET) is a nuclear imaging
technique used extensively in diagnostic medicine and drug
development. In the last decade, 18F (t1/2 ꢀ 110 min) has been
popularized as a nonmetallic PETradioisotope. With a longer
half-life than other nonmetallic radioisotopes for PET, such as
[*] L. S. Campbell-Verduyn,[+] A. K. Schoonen, Prof. Dr. B. L. Feringa
Stratingh Institute for Chemistry, University of Groningen
Nijenborgh 4, Groningen 9747AG (The Netherlands)
E-mail: b.l.feringa@rug.nl
L. Mirfeizi,[+] Prof. Dr. R. A. Dierckx, Prof. Dr. P. H. Elsinga
Department of Nuclear Medicine and Molecular Imaging
University Medical Center Groningen, University of Groningen
Hanzeplein 1, Groningen, 9713GZ (The Netherlands)
E-mail: p.h.elsinga@umcg.nl
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201105547.
Angew. Chem. Int. Ed. 2011, 50, 11117 –11120
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
11117

Communications
focus in this work on the use of bombesin, we expect that the
potassium tert-butoxide in THF to form 7. Basic hydrolysis
afforded carboxylic acid 8, and the N-hydroxysuccinimide
ester 9 was formed to allow coupling of the strained alkyne to
the peptide.
Of key importance to the field of radiochemistry is high
reactivity, which allows for the rapid introduction of the short-
lived radiolabel under biocompatible conditions. Before we
proceeded with the modification of bombesin, we tested the
reactivity of the aza-benzocylooctyne with several fluorine-
containing azides in pseudo in vivo conditions (Scheme 2).
technique would be amenable to the use of other biomole-
cules as well. We present herein the first instance of 18F
radiolabeling using copper-free “click” chemistry, and its
application to the synthesis of a 18F-labeled analogue of
bombesin, a potent ligand for tumor imaging. We further
demonstrate that the “click” radiolabeling does not compro-
mise the GRP receptor in vitro binding affinity in human
prostate cancer cells.
Our starting point was to find a suitable strained alkyne
with the optimal balance of reactivity and stability. Van Hest,
van Delft, and co-workers reported an aza-dibenzocyclooc-
tyne (7), which proved to be simultaneously reactive and
stable.[12] For our purposes, which involve rapid “clicking” of a
short-lived radioisotope and eventual in vitro and in vivo
studies, 7 appeared an ideal choice. However, we opted for an
alternate and shorter synthetic route to our target molecule 9
(Scheme 1). Our synthesis started from commercially avail-
able dibenzosuberenone (1), which was heated to reflux in the
presence of hydroxylamine hydrochloride to form oxime 2 in
95% yield. The crucial step of this synthesis is the subsequent
Beckmann rearrangement, which gave amide 3 in 67%
yield.[13] The amide was reduced with Dibal-H to amine 4,
which was thus reached in three steps, rather than five. The
synthesis starts with the very inexpensive precursor 1 (1 g
ꢀ 1 E) rather than the reported 2-ethynylaniline (1 g
ꢀ 55 E). A short linker can be introduced by reaction of 4
with methyl succinyl chloride in the presence of triethylamine.
The bromination proceeded smoothly in 88% yield and was
followed by subsequent debromination with a solution of
Scheme 2. Strain-promoted “click” chemistry for 18F labeling.
Strained alkyne 7 could be reacted with a 18F-labeled azide by
simply stirring the two reactants together in a mixture of
human plasma and DMSO. We found that alkyne 7 could be
fully converted to the corresponding triazoles within
15 min.[14] With the hydrophilic [18F]PEGylated azide 10,
triazole 11 was isolated with a radiochemical yield (RCY) of
42%. With the more lipophilic [18F]fluorobenzyl azide 12, the
product was isolated with an RCY of 31%. Furthermore, it
should be noted that the reaction is performed in human
plasma. Considering the eventual application of radiolabeling
in vivo, it was important to confirm that the selected strained
alkyne was not too fragile to withstand any exposure to a
biological environment. Given that alkyne 7 could be fully
converted to triazoles in less than 15 min, we concluded that
the reaction was fast enough for the desired time scale for
labeling with 18F.
Succinimidyl ester 9 was therefore conjugated to Lys[3]-
bombesin under basic conditions (Scheme 3). Full conversion
to the product Aza-DBCO-BN was achieved after 24 h as
determined by reverse-phase (RP) HPLC, and subsequently
Aza-DBCO-BN was purified by RP-HPLC and characterized
by mass spectrometry (ESI-MS). Now that the target bom-
besin analogue was modified with a strained alkyne, we tested
the efficiency of the copper-free [3+2] cycloaddition with
various 18F-containing azides.
Scheme 1. Reagents and conditions: a) NH2OH·HCl (3.0 equiv), pyri-
dine/ethanol (1:1), reflux; b) TCT (1.0 equiv), DMF, RT; c) Dibal-H
(5.0 equiv), CH2Cl2, RT; d) Et3N (2.0 equiv), methyl succinyl chloride
(1.5 equiv), CH2Cl2, RT; e) Br2 (1.0 equiv), CH2Cl2, 08C; f) 1.0m tBuOK
in THF, THF, À408C, Ar atmosphere; g) LiOH (2.0 equiv), H2O, RT;
h) EDC (1.1 equiv), NHS (1.1 equiv), CH2Cl2, RT. TCT=trichlorotria-
zine, DMF=dimethylformamide, Dibal-H=diisobutyl aluminum hy-
dride, THF=tetrahydrofuran, EDC=N-(3-dimethylaminopropyl)-N’-
ethylcarbodiimide, NHS=N-hydroxysuccinimide.
Three 18F-containing azides were selected to react with
Aza-DBCO-BN (Scheme 4). One advantage of this method-
ology is the ease with which the properties of the resulting
peptidic tracer can be modified by simply changing the azides.
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tively. The logarithmic partition coefficients were deter-
mined for all three tracers and were found to be À0.43,
1.27, and 0.26, respectively. This methodology provides us
with tracers that range from the quite hydrophilic
[18F]PEGTOxBN
[18F]BnTOxBN.
to
the
more
hydrophobic
The binding affinity of all three tracers for gastrin-
releasing peptide receptors was tested using PC3 human
prostate cancer cells. The in vitro binding was determined
by performing a competitive receptor binding assay with
the receptor specific radioligand [125I]Tyr[4]-BN (a dis-
placement assay). The 50% inhibitory concentrations
(IC50) were determined to be 40 nm, 29 nm, and 30 nm for
15, 16, and 17, respectively (see Figures 1–3 in the
Supporting Information). All three tracers maintain high
affinity for the GRPRs even after modification.
We have achieved rapid radiolabeling of bombesin
with [18F] by using a very straightforward protocol. Simple
stirring of the radionuclide-
Scheme 3. Modification of bombesin with 9. DIPEA=N,N-diisopropylethyl-
amine.
containing azide with the
modified bombesin ana-
logue for 10–15 min at
room temperature suffices
to reach the target peptides
in modest to good yields.
Furthermore, the azide can
be readily varied from a
more lipophilic aromatic
azide to
a
hydrophilic
PEGylated azide. As
a
result, peptides with differ-
ent properties are readily
accessible from the same
modified peptide, thus
allowing for rapid modifica-
tion and fine-tuning. In this
way, the optimal lipophilic-
ity for cellular uptake and
metabolic clearance can be
achieved. To the best of our
knowledge, this is the first
example of 18F radiolabeling
using copper-free click
chemistry. We have also
developed a simplified and
relatively inexpensive route
to the target aza-dibenzocy-
clooctyne, and have hope-
fully rendered it more acces-
sible for future use in a
clinical setting. Although
we describe herein the
modification and labeling
Scheme 4. Reagents and conditions: a) K[18F], MeCN, 1108C; b) DMF, RT. R=Pyr-Gln, R1 =Leu-Gly-Asn-Gln-
Trp-Ala-Val-Gly-His-Leu-Met-NH2, Pyr=pyroglutamic acid.
[18F]PEGylated azide 10, [18F]fluorobenzyl azide 12, and
[18F]fluoroazidobutane 14 were reacted with Aza-DBCO-BN
at room temperature in DMF for 15 min, during which time
complete conversion of the starting material could be
detected by radio-TLC to give triazoles [18F]PEGTOxBN
(15), [18F]BnTOxBN (16) and [18F]BuTOxBN (17) respec-
of bombesin and demonstrate that it maintains high affinity
for the targeted receptors, ideally, this methodology would be
applied to imaging by pretargeting. For molecules such as
antibodies that have slower pharmacokinetics and are thus
not amenable to the use of the short-lived 18F, it would be
highly advantageous to be able to administer the 18F radio-
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Communications
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Keywords: click chemistry · imaging agents · isotopic labeling ·
peptides · radiopharmaceuticals
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