Ortho-stabilized 18F-azido click agents and their application in PET imaging with single-stranded DNA aptamers

Article abstract of DOI:10.1002/anie.201505927

Azido ¹⁸F-arenes are important and versatile building blocks for the radiolabeling of biomolecules via Huisgen cycloaddition ("click chemistry") for positron emission tomography (PET). However, routine access to such clickable agents is challenged by inefficient and/or poorly defined multistep radiochemical approaches. A high-yielding direct radiofluorination for azido ¹⁸F-arenes was achieved through the development of an ortho-oxygen-stabilized iodonium derivative (OID). This OID strategy addresses an unmet need for a reliable azido ¹⁸F-arene clickable agent for bioconjugation reactions. A ssDNA aptamer was radiolabeled with this agent and visualized in a xenograft mouse model of human colon cancer by PET, which demonstrates that this OID approach is a convenient and highly efficient way of labeling and tracking biomolecules. Markedly apt: A high-yielding direct radiofluorination strategy via ortho-oxygen-substituted iodonium derivatives is described. A ssDNA aptamer (sgc8), labelled with the resulting ¹⁸F azido agent through click chemistry, was used for PET imaging and provides the first example for the noninvasive in vivo PET imaging of protein tyrosine kinase 7 (PTK-7).

Full text of DOI:10.1002/anie.201505927

Angewandte
Chemie
International Edition: DOI: 10.1002/anie.201505927
German Edition: DOI: 10.1002/ange.201505927
Imaging Agents
Ortho-Stabilized 18F-Azido Click Agents and their Application in PET
Imaging with Single-Stranded DNA Aptamers
Lu Wang, Orit Jacobson, Din Avdic, Benjamin H. Rotstein, Ido D. Weiss, Lee Collier,
Xiaoyuan Chen,* Neil Vasdev,* and Steven H. Liang*
Abstract: Azido 18F-arenes are important and versatile build-
ing blocks for the radiolabeling of biomolecules via Huisgen
cycloaddition (“click chemistry”) for positron emission tomo-
graphy (PET). However, routine access to such clickable
agents is challenged by inefficient and/or poorly defined
multistep radiochemical approaches. A high-yielding direct
radiofluorination for azido 18F-arenes was achieved through
the development of an ortho-oxygen-stabilized iodonium
derivative (OID). This OID strategy addresses an unmet
need for a reliable azido 18F-arene clickable agent for
bioconjugation reactions. A ssDNA aptamer was radiolabeled
with this agent and visualized in a xenograft mouse model of
human colon cancer by PET, which demonstrates that this
OID approach is a convenient and highly efficient way of
labeling and tracking biomolecules.
which only limited examples are attractive for routine
production for PET imaging. This can be attributed to lengthy
multistep syntheses with short half-life 18F and/or the require-
ment for special handling owing to the strong ionization
energy. For instance, [18F]fluoroethyl azide was the first
aliphatic 18F-azide and was prepared in high yield (55%,
decay corrected) through a nucleophilic displacement reac-
tion with [18F]fluoride.[6] A disadvantage of this type of low-
molecular-weight agent is its high volatility and the need for
intermediate purification by distillation, which is challenging
to carry out in radiochemical laboratories.[6,7] To the best of
our knowledge, 4-[18F]fluorobenzyl azide is the only azido 18F-
arene used in PET imaging studies.[8] However, the prepara-
tion of 4-[18F]fluorobenzyl azide involves laborious multistep
processes[8b,d] or the utilization of a specialized flow device.[8e]
We have recently reported a manual one-step synthesis of
[18F]fluorobenzyl azide with high radiochemical yield and
specific activity,[9] but the subsequent problems associated
with high volatility made this agent unsuitable for widespread
use. Since 18F-arenes are generally stable in vivo (high
resistance to deradiofluorination)[2a,8e, 10] and possess
a strong chromophore (UV-detectable), there is an unmet
need for a convenient and highly efficient radiofluorination
method to provide azido 18F-arene click agents for bioconju-
gation through robust click chemistry.
There is a new emerging class of targeting vector for
molecular imaging, namely aptamers or single stranded
oligonucleotides, which show highly specific binding to
protein targets with efficient tissue penetration and rapid
blood clearance.[11] Aptamers are chemically assembled and
can be easily modified to improve stability and pharmacoki-
netic profiles without notable immunogenicity.[11b,c] There are
ongoing efforts to explore the feasibility of labeled aptamers
for in vivo imaging.[12] The single-stranded DNA aptamer sgc8
was identified as specifically targeting protein tyrosine kinase
7 (PTK-7),[12b] which is over-expressed in several human
malignancies.[13] However, the mapping of PTK-7 in various
tumors has not been realized in clinical use, mainly owing to
the lack of a method to noninvasively probe this target
in vivo. A reliable and efficient method to radiolabel an
aptamer targeting PTK-7 would enable us to utilize PET
imaging to further our understanding of this kinase in cancer
biology and could lead to the development targeted therapies
through precision medicine.
18
+
1
B
iologics labeled with fluorine-18 ( F, b , t = = 109.7 min),
2
including nucleic acids, peptides, and proteins, are extensively
used for molecular imaging in positron emission tomography
(PET).[1] 18F incorporation in these sensitive biomolecules
requires fast, high yielding, and site-specific bioconjugation
methods that can be carried out under mild conditions.[2]
Huisgen reactions (chemoselective and regiospecific 1,3-
dipolar cycloaddition between an azide and an alkyne) are
the most commonly used bioconjugation strategy.[3] The
resulting 1,2,3-triazole linker is stable in vivo[4] and is
considered to be an isosteric surrogate for peptide bonds.[5]
Compared to the many applications of azides in non-radio-
active bioconjugation, only a small number of 18F-labeled
azides have been developed for PETradiochemistry,[3] among
[*] L. Wang,[+] D. Avdic, Dr. B. H. Rotstein, Dr. L. Collier,
Prof. Dr. N. Vasdev, Prof. Dr. S. H. Liang
Division of Nuclear Medicine and Molecular Imaging
Massachusetts General Hospital & Department of Radiology
Harvard Medical School, Boston, MA 02114 (USA)
E-mail: vasdev.neil@mgh.harvard.edu
liang.steven@mgh.harvard.edu
Dr. O. Jacobson,[+] Prof. Dr. X. Chen
Laboratory of Molecular Imaging and Nanomedicine
National Institute of Biomedical Imaging and Bioengineering
National Institutes of Health, Bethesda, MD 20892 (USA)
E-mail: shawn.chen@nih.gov
Dr. I. D. Weiss
Laboratory of Molecular Immunology
National Institute of Allergy and Infectious Diseases
National Institutes of Health, Bethesda, MD 20892 (USA)
With the goal to develop a highly efficient radiolabeling
method for azido 18F-arene click agents and a robust platform
for aptamer radiolabeling with application to PTK-7, we
designed an unusual precursor scaffold based on iodine(III)
chemistry (Scheme 1). On the basis of early discoveries of an
[+] 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.201505927.
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Scheme 1. A new 18F-azido agent for PET imaging of a ssDNA aptamer
in a xenograft tumor model.
ortho effect on iodonium species[9,14] and a secondary bonding
interaction observed in hypervalent iodine compounds,[15] we
speculated that ortho-oxygen-coordinated iodonium deriva-
tive (OID) would provide stabilization for iodine(III) to yield
thermally stable and highly reactive labeling precursors. We
synthesized a series of labeling precursors featuring different
lengths and positions of the aliphatic linkers (1–5) and ortho
coordinating substituents (6 and 7; Scheme 2). The synthesis
was highly efficient from commercially available starting
materials (Scheme S1–S4 in the Supporting Information).
While most of the precursor molecules were crystalline,
precursors with long alkyl chains (4 and 5) led to the
formation of semisolids or oils (Figure S1 in Supporting
Information), which are not ideal for handling in PET
radiochemistry and were thus not further pursued. With the
aim to decrease 18F-product volatility (see below) and
generate a readily reactive crystalline precursor, we studied
the combined effect of a para bromine atom and an ortho
oxygen-containing group in precursors 6 and 7, and we
discovered that this combination increases not only the
radiochemical yield but also the thermostability of the
precursors. In the 18F-labeling experiments, benzyl azides
1A–3A and 9A provided 24–70% 18F-incorporation yields,
while OIDs 6A and 7A afforded 69% and 90% conversions
with excellent product yields of 51% and 82%, respectively.
This is the first time that the ortho effect in hypervalent
iodine(III) chemistry has been utilized in the design of 18F-
labeled agents. The OIDs 6A and 7A also proved to be highly
efficient in 18F-lableling compared to the traditional nucleo-
philic aromatic substitution (SNAr) approach. A control
experiment was carried out to test labeling efficiency from
the nitro precursor (Scheme 2, compound 8) via the SNAr
pathway, but this approach did not afford any desired product
and gave 35% conversion to other unidentified byproducts.
This was also supported by a literature report of limited
conversion (< 0.5%) from 1-bromo-4-nitrobenzene.[16]
Scheme 2. Synthesis and radiolabeling of different iodonium(III) pre-
cursors. a) Conditions: precursor (4 mg), Et4NHCO3 (2 mg), DMF
(0.4 mL), [18F]FÀ(1–3 mCi), 1208C. b) RCC=radiochemical conversion.
c) RCY=radiochemical yield. d) No desired product was found.
Figure 1. Characterization and comparison of labeling precursors and
18F-azido clickable agents. A) Precursor (2 mg), Et4NHCO3 (1 mg),
DMF (0.4 mL), [18F]fluoride (1–3 mCi). B) Precursor (4 mg), DMF
(0.4 mL),[18F]fluoride (1–3 mCi), 1208C. C) Precursors (2 mg) were
stored at ambient temperature. D) [18F]1B or [18F]7B (3–5 mCi) in
CH3CN (1 mL) was concentrated under N2. DMF=N,N-dimethylfor-
mamide.
time and amount of precursor loading (Figure S2 in the
Supporting Information). For bioorthogonal reactions that
are routinely carried out in aqueous media at pH 4 or 10, the
resulting product [18F]7B showed superior stability in alkaline
or acidic solutions (Figure S3 in the Supporting Information).
The in vitro stability of [18F]7B in mouse serum was assessed
by HPLC and no degradation was observed after incubation
In comparison to the labeling precursor 1A for
[18F]fluorobenzyl azide, OID 7A showed superior radio-
chemical conversions in terms of reaction temperature (Fig-
ure 1A) and base loading (Figure 1B), as well as reaction
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at 378C for 2 h. OID 7A also demonstrated extraordinary
thermostability, a critical and practical consideration for the
routine production of radiotracers for PET, while compound
1A only showed a shelf half-life of 10 days (Figure 1C; and
Figure S4 in the Supporting Information), which was partially
attributed to secondary bonding at the iodine(III) center.[15]
We also synthesized an ortho-substituted carbon analogue 9A
(see Scheme 2) and this compound showed inferior radio-
1
chemical yield (37%) and thermostability (shelf t = = 15 days)
2
compared to its oxygenated counterpart 7A, thus suggesting
that there may be a stabilizing interaction between the ortho
oxygen and the iodine. Since the biological agents to be
labeled (e.g., aptamers and nucleic acids) are usually avail-
able in small quantities (low mg), 18F-bioconjugations of these
materials are usually performed in a small volume (mL scale)
to provide fast reaction kinetics. One of major limitation for
4-[18F]fluorobenzyl azide is associated with its high volatility,
which means that it is difficult to recover in microliter level
after concentration, whereas the new azide [18F]7B exhibited
up to 92% recovery and is suitable for further coupling
reactions (Figure 1D). The radiosynthesis of [18F]7B was also
automated on a production scale by using a commercial
radiosynthesis module, which produced approximately
400 mCi of [18F]7B in greater than 50% non-decay-corrected
radiochemical yield, with more than 95% (radio)chemical
purity and greater than 2 Ci mmolÀ1 specific activity (Fig-
ure S5 in the Supporting Information). Among all the
parameters evaluated, the non-volatile clickable agent
[18F]7B, which was prepared from thermally stable OID 7A
in high yield and with outstanding stability under either basic
or acidic conditions, revealed 7 to be a promising scaffold for
bioorthogonal reactions and this scaffold was thus transi-
tioned for further evaluation in click chemistry and in vivo
PET imaging studies.
A variety of terminal alkyne-containing small or biolog-
ical molecules was evaluated in the coupling reaction with
[18F]7B under biocompatible conditions (Scheme 3). The new
azide showed excellent radiochemical conversions in click
Scheme 3. Application of the 18F-azide agent [18F]7B in copper-medi-
reactions with
a phenylalanine derivative (94%) and
strained-induced DBCO cycloaddition (98%). We have
previously labeled a ssDNATsC aptamer with N-succinimidyl
4-[18F]fluorobenzoate ([18F]SFB) through peptide bond for-
mation but obtained the corresponding 18F-labeled TsC
aptamer in only 1.5% yield.[12g] Significantly increased
bioconjugation yield (> 95%, radiochemical conversion;
48% yield of isolated product) was obtained with [18F]7B
and the alkyne-linked TsC aptamer, thereby demonstrating
an up to 30-fold improvement in site-specific coupling
efficiency. Encouraged by these results, we also radiolabeled
sgc8 with [18F]7B, which proceeded with 49% radiochemical
yield, for further in vivo evaluation.
PTK-7 is an excellent template protein to illustrate the use
of the novel 18F-click agent, especially since this would be the
first in vivo PET imaging study of this important target in
a disease model. The affinity of the labeled aptamer [18F]7B-
sgc8 towards PTK-7 was determined by receptor saturation
binding assay, which showed a single digital nanomolar Kd
value of 1.1 nm. The uptake of [18F]7B-sgc8 was rapid and was
blocked by the addition of excess unlabeled aptamer, thus
ated (A) and strain-induced (B) click reactions, as well as copper-
mediated bioconjugation reactions with the ssDNA aptamers TsC (C)
and sgc8 (D).
demonstrating high specificity for PTK-7 (Figure 2A).
[18F]7B-sgc8 binding to PTK-7 was not due to internalization
and washed away over time, thus suggesting that PET imaging
should be completed within few hours following injection of
the radiotracer. To map tumoral PTK-7 expression, we
selected a colon cell line because this protein is expressed in
colon carcinomas but not in normal colon tissue and thus may
be utilized as a biomarker for tumor progression.[13a,b] Flow
cytometry analysis of the colorectal carcinoma cell line
HCT116 showed positive and high PTK-7 expression (Fig-
ure 2B). In vivo studies were performed by administering
[18F]7B-sgc8 to female nude mice with HCT116 tumors
followed by PET imaging (Figure 2C). [18F]7B-sgc8 clearly
visualized PTK-7-positive tumors at all time points, with
tumoral uptake of 0.71 Æ 0.07% ID/g and a reasonable tumor-
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provide 18F-azido click agents in a high-yield one-step
[18F]fluorination. The click agent [18F]7B demonstrated low
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award.
Keywords: aptamers · bioconjugation · fluorine · PET imaging ·
radiopharmaceuticals
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Received: June 29, 2015
Published online: August 20, 2015
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