A Compact and Synthetically Accessible Fluorine-18 Labelled Cyclooctyne Prosthetic Group for Labelling of Biomolecules by Copper-Free Click Chemistry

Article abstract of DOI:10.1002/cmdc.201800334

A new fluorine-containing azadibenzocyclooctyne (ADIBO-F) was designed using a synthetically accessible pathway. The fluorine-18 prosthetic group was prepared from its toluenesulfonate precursor and isolated in 21–35 % radiochemical yield in 30 minutes of synthetic time. ADIBO-F has been incorporated into azide-functionalized, cancer-targeting peptides through a strain-promoted alkyne–azide cycloaddition with high radiochemical yields and purities. The final products are novel peptide-based positron emission tomography (PET) imaging agents that possess high affinities for their targets, growth hormone secretagogue receptor 1a (GHSR-1a) and gastrin-releasing peptide receptor (GRPR), with IC₅₀ values of 9.7 and 0.50 nm, respectively. This is a new and rapid labelling option for the incorporation of fluorine-18 into biomolecules for PET imaging.

Full text of DOI:10.1002/cmdc.201800334

Accepted Article
Title: A Compact and Synthetically Accessible Fluorine-18 Labelled
Cyclooctyne Prosthetic Group for Labelling of Biomolecules by
Copper-free Click Chemistry
Authors: Emily Murrell, Michael S Kovacs, and Leonard G Luyt
This manuscript has been accepted after peer review and appears as an
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To be cited as: ChemMedChem 10.1002/cmdc.201800334
Link to VoR: http://dx.doi.org/10.1002/cmdc.201800334
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ChemMedChem
10.1002/cmdc.201800334
COMMUNICATION
A Compact and Synthetically Accessible Fluorine-18 Labelled
Cyclooctyne Prosthetic Group for Labelling of Biomolecules by
Copper-free Click Chemistry
Emily Murrell, Michael S. Kovacs, and Leonard G. Luyt*[a,d,e]
[
a]
[b,c]
[
a]
E. Murrell, Dr. L. G. Luyt
Department of Chemistry
University of Western Ontario
1151 Richmond St, London, Ontario N6A 5B7, Canada
E-mail: lluyt@uwo.ca
[
[
b]
c]
Dr. M. S. Kovacs
Lawson Health Research Institute
268 Grosvenor Street, London, Ontario N6A 4V2, Canada
Dr. M. S. Kovacs
Departments of Medical Imaging and Medical Biophysics
University of Western Ontario
1151 Richmond Street, London, Ontario N6A 5B7, Canada
[
[
d]
e]
Dr. L. G. Luyt
Department of Oncology
University of Western Ontario
1151 Richmond Street, London, Ontario N6A 5B7, Canada
Dr. L. G. Luyt
London Regional Cancer Program
Lawson Health Research Institute
790 Commissioners Road East, London, Ontario N6A 4L6, Canada
Supporting information for this article is given via a link at the end of the document.
Abstract: A new fluorine-containing azadibenzocyclooctyne (ADIBO-
F) has been designed using a synthetically accessible pathway. The
fluorine-18 prosthetic group was prepared from its tosylate precursor
and isolated in 21-35% radiochemical yield in 30 minutes of synthetic
time. The ADIBO-F has been incorporated into azide-functionalized,
cancer-targeting peptides through a strain-promoted alkyne-azide
cycloaddition with high radiochemical yields and purities. The final
products are novel peptide-based PET imaging agents possessing
high affinities to their targets of GHSR-1a and GRPR with IC50 values
of 9.7 and 0.50 nM, respectively. This exhibits a new, rapid labelling
option for the incorporation of fluorine-18 into biomolecules for PET
imaging.
various imaging components under bioorthogonal conditions. The
click reaction satisfies the requirement of milder reaction
conditions for appending a prosthetic group to a biomolecule; it
also boasts the benefits of fast reaction kinetics and lack of by-
products. Because of these properties, this reaction lends itself
particularly well to synthetic radiolabelling strategies for the
production of nuclear imaging agents.[ This is especially true for
shorter-lived PET isotopes such as fluorine-18. For the use of
CuAAC in in vivo applications, the complete removal of copper
reagents remaining after reactions is necessary in order to avoid
cytotoxicity issues. These extra purification steps unfortunately
hinder the efficiency of the radiosynthesis. The strain-promoted
alkyne-azide cycloaddition (SPAAC) reaction is a convenient
copper-free alternative to click chemistry reactions which avoids
cytotoxic copper reagents.[
7]
8]
Both CuAAC and SPAAC have been extensively used to
Positron emission tomography (PET) is an important
molecular imaging technique used to non-invasively visualize and
diagnose many different biological events and pathologies.[1] The
use of fluorine-18 as a PET radionuclide is popular due to its
relatively long half life (~110 minutes) among non-metallic
isotopes, allowing ample time for radiosynthesis as well as other
favourable nuclear and chemical properties.[2] Due to its small size,
fluorine-18 has been incorporated into imaging agents of various
complexities from small molecules to larger biomolecules.[3-5]
However, an important consideration is the method of
incorporating the fluoride such that rapid and facile radiolabelling
is achieved. Direct labelling of a molecule with fluoride-18 can
require harsh nucleophilic conditions, which the biologic “targeting”
portion of an agent may not withstand. Instead, small-molecule
fluorine-18 prosthetic groups are labelled first and used to
label biomolecules such as proteins, oligonucleotides, peptides
[9-13]
and other targeting entities with fluorine-18.
In SPAAC
examples, the most commonly used cyclooctynes are
azadibenzocyclooctynes (ADIBOs), due to their relatively fast
-1 -1
reaction kinetics (0.4-0.9 M s depending on modifications) and
[14-15]
long-term stability compared to other reported cyclooctynes.
ADIBO is commonly synthesized with an acid or amine handle,
which allows for conjugation to biomolecules through standard
coupling techniques (Figure 1, A). Fortunately, there have been a
wide variety of fluorine-18 azide-containing prosthetic groups
developed for copper-catalyzed click reactions which are also
introduce the radionuclide to
a biomolecule under milder
conditions. Careful selection of these prosthetic groups is
necessary to ensure that the addition does not elicit a change in
biological function.
The
copper-catalyzed
alkyne-azide
1,3-dipolar
cycloaddition (CuAAC) reaction as a form of “click chemistry” was
first introduced by Sharpless in 2001.[6] This concept has since
been widely explored in applications to label biomolecules with
Figure 1. Structures of A) non-labelled ADIBOs where R contains an acid or
amine for conjugation to a biomolecule, B) ADIBOS that have been indirectly
labelled with fluorine-18 and C) ADIBOS that have been further derivatized into
fluorine-18 prosthetic groups.
1
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ChemMedChem
10.1002/cmdc.201800334
COMMUNICATION
employable with cyclooctyne-modified biomolecules, allowing for
quick transition into PET imaging agents.[16]
core. Compound 5 is subsequently transformed into the alcohol 6,
which is stable to the reaction conditions used for the formation of
the cyclooctyne. Bromination across the cyclooctene and
subsequent elimination with potassium tert-butoxide produces the
cyclooctyne 7. Tosylation of the alcohol to produce a suitable
leaving group results in the precursor of the prosthetic group 8.
Conversely, the fluorine-19 standard of the prosthetic group can
be formed by halide exchange of the bromide 5 to fluoride using
tetrabutylammonium fluoride to produce 9. This can be followed
by a similar bromination/elimination of the cyclooctene to form the
cyclooctyne 10 (Scheme 1).
To approach radiolabelling via the SPAAC reaction from the
opposite
direction,
easily-accessed
azide-containing
biomolecules and targeting moieties can be employed. The
complementary piece to this azido-derivatized biomolecule
1
8
necessitates an F-labelled cyclooctyne.
Indirect labelling of ADIBOs was first shown by using the
1
8
prosthetic
group
4-succinimidyl-[ F]fluorobenzoate
to
incorporate fluorine-18 into an amine-functionalized ADIBO
17-18]
(
Figure 1, B).[
sufficiently stable for direct fluorine-18 labelling conditions.
Previous approaches in the literature make use of ADIBO-NH by
ADIBOs have also been shown to be
The tosylate precursor 8 of this novel ADIBO has been
1
8
2
labelled with dry [ F]fluoride under basic conditions in the
1
8
coupling a carboxylic acid-functionalized linker to the amine tail.
This alkyl or PEG linker connects the ADIBO to a suitable leaving
presence of Kryptofix 222. The prosthetic group [ F]10 was
purified by semi-preparative HPLC and isolated in greater than
98% radiochemical purity to give 21-35% decay-corrected
radiochemical yields in under 30 minutes of total reaction and
purification time (Scheme 2).
1
8
[19-20]
group for F-labelling of the resulting molecule (Figure 1, C).
Recently, an oxodibenzocyclooctyne was labelled with fluorine-18
showing reactivity with micromolar concentrations of azide-
modified bioconjugates.[21] While these are exciting and useful
developments for the utilization of copper-free click techniques in
fluorine-18 radiolabelling of peptides, these constructs can be
fairly large and high in molecular-weight for a prosthetic group.
Because the copper-free click reaction is not regioselective, this
long tail also results in significantly different 1,4 and 1,5
regioisomers, which could affect receptor binding or
pharmacokinetic properties of the imaging agent. Furthermore,
Scheme 2. Radiosynthesis of [18F]ADIBO-F [18F]10.
2
the expense of ADIBO-NH and ADIBO-COOH can be prohibitive
1
8
18
The potential for [ F]ADIBO-F [ F]10 to be employed as a
prosthetic group has been tested with two peptides that are known
to be effective for targeting G protein-coupled receptors (GPCRs)
overexpressed in a variety of cancers. The first example of using
our novel prosthetic group for radiolabelling is demonstrated by
when investigating the discovery of new imaging agents. We
propose a new fluorine-containing ADIBO that seeks to: 1.
simplify synthesis from inexpensive, readily-available starting
materials; 2. minimize structural difference in regioisomers
produced by the SPAAC reaction; 3. reduce the size and
molecular weight of the prosthetic group in comparison to those
previously reported.
3
19
incorporating it into [Dpr (octanoyl),Lys ]ghrelin(1-19). It has
been shown that peptides with various imaging moieties
1
9
connected at the Lys of this peptide sequence target the GHSR-
We report
a new fluorine-containing ADIBO that is
24-26]
19
1
a receptor with nanomolar affinities.[
By modifying Lys to a
synthetically accessible in nine linear steps via modifications to
the Popik synthesis.[22-23] In brief, the inexpensive starting material
dibenzosuberenone 1 was converted to oxime 2. A subsequent
Beckmann rearrangement produces lactam 3, which is then
reduced by lithium aluminum hydride to its secondary amine 4.
The amine is acylated with bromoacetyl bromide to produce 5 with
a handle for functionalization from the azadibenzocyclooctene
lysine-azide (11), imaging moieties can be linked to the peptide
by click reactions resulting in candidate imaging agents. Through
triazole formation at the azide, our new cyclooctyne prosthetic
group was linked to this peptide in 15 minutes at 70 °C, which is
milder than typical nucleophilic fluoride addition conditions. The
SPAAC reaction allows this labelling to proceed in neutral
conditions in absence of any other reagents, and at a temperature
that allows for increased reaction kinetics while ensuring peptide
stability. Full radiochemical conversion was observed at this
temperature with no remaining prosthetic group or production of
1
8
radioactive
(
by-products.
The
product,
[ F][Dpr-
3
octanoyl),Lys (triazole-ADIBO-F)]ghrelin(1-19) [18F]12, was
19
Scheme 3. Strain-promoted alkyne-azide cycloaddition of [18F]ADIBO-F
Scheme 1. Branching synthesis of both ADIBO-OTs 8 and [19F]ADIBO-F 10
18
prosthetic group [ F]10 with an azide-modified ghrelin peptide 11. Only one
[
22-23]
standard via modifications to the Popik synthesis
of ADIBOs.
18
regioisomer of [ F]12 is shown for simplicity.
2
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ChemMedChem
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COMMUNICATION
Table 1. Characterization of the 18F-labelled ghrelin and bombesin peptides.
Peptide
Target Receptor
IC50[a]
Azido-Peptide
Concentration (mM)
Yields[b]
(%)
Purities[c] Molar Activities[d]
(nM)
(%)
(GBq/µmol)
[18
F][Dpr (octanoyl),Lys19(triazole-ADIBO-F)]ghrelin(1-19)
3
GHSR-1a
GRPR
9.7
3.7
5.6
64-66
81-88
>98
0.6-0.9
[18
F][D-Phe ,β-Ala11,Phe13,Nle14]bombesin(6-14)-
6
0.5
>98
0.6-1.1
8
PEG (triazole-ADIBO-F)
[
[
a] Determined using the 19F standard analogue. [b] Decay-corrected radiochemical yields for isolated products. [c] Radiochemical purity by analytical RP-HPLC.
d] Determined by analytical HPLC area under the curve (AUC) at 220 nm of an aliquot of the isolated fraction. (n=3 for all values)
isolated by semi-preparative HPLC resulting in good
radiochemical yields of 64-66% of the isolated product in greater
than 98% radiochemical purities (Scheme 3). The SPAAC
reaction was performed with quantities less than 200 MBq of the
prosthetic group, resulting in molar activities of 0.6-0.9 GBq/µmol
for the isolated product.
purities and molar activities of 0.6-1.1 GBq/µmol. The non-
radioactive standard of this peptide was used to validate the
identity of the labelled peptide by co-injection and for in vitro
studies (For more information refer to Supplemental Information
provided). This modified peptide-based imaging agent maintained
a high affinity to the GRPR receptor with an IC50 value of 0.50 nM,
which is comparable to that of the unmodified bombesin peptide
(0.70 nM) (Figure S8).
A non-radioactive fluorine-19 containing analogue of 12 was
synthesized to serve as a reference standard. Co-injection of the
1
8
nonradioactive standard 12 with the labelled peptide [ F]12, in
addition to an overlay of the UV chromatogram for the standard
and the radio-chromatogram of the labelled peptide confirms the
success of the radiolabelling SPAAC reaction (Figure 2).
Interestingly, the two regioisomers produced had no
distinguishable difference in retention times, even with various
HPLC gradients, reflecting the minimal structural difference
between these regioisomers. Using the nonradioactive 12, an IC50
of 9.7 nM was obtained from a competitive binding assay for the
receptor GHSR-1a. This value is comparable to that of similar
known ghrelin-targeting imaging probes (9.5-25.8 nM)[24-25]
We have reported a novel ADIBO-F and its tosylate
precursor which can be synthesized on the side chain of the
ADIBO core structure with minimal additional sterics or molecular
weight. The tosylate precursor is accessible in a scalable
synthetic procedure over eight steps in 15% overall yield from
inexpensive starting materials via modifications to the Popik
1
8
synthesis. The radiolabelling of the [ F]ADIBO-F precursor was
carried out efficiently in yields of 21-35% and isolated for use as
a prosthetic group in approximately an hour from fluoride-18
trapping. The availability of a minimally-sized ADIBO-based
fluorine-18 prosthetic group in the radiolabelling toolbox allows for
copper-free click labelling of a wide range of biomolecules and
targeting moieties for PET imaging agents.
(
Figure S7) and shows that this peptide maintains a strong affinity
for the receptor of interest with the addition of our new prosthetic
group, making it potentially suitable as a cancer-targeting PET
imaging probe. The stability of the fluorine towards hydrolysis was
assessed using a 1 mg/mL solution of 12 in water at 37 °C over
As proof-of-principle this prosthetic group’s utility was
shown by incorporating it into two different cancer-targeting
peptides. These reactions were carried out in high yields with
simple and quick purifications. The entire two-step labelling
process can be performed manually within two hours including
purifications resulting in good molar activities. The excellent
radiochemical yields of this click reaction in mild, aqueous
reaction conditions lends itself favourably to the labelling of
sensitive biomolecules.
24 h. No degradation of the peptide or fluoride lability was
observed as indicated by HPLC-MS analysis.
This novel cyclooctyne prosthetic group demonstrates a
new, rapid labelling option for the incorporation of fluorine-18 into
azide-containing biomolecules to make imaging agents.
Experimental Section
Electronic Supplementary Information (ESI) available: Full synthetic
details and characterization; assays and traces.
Figure 2. Overlaid analytical C18 RP-HPLC chromatograms at 25 to 80%
1
8
(
2
ACN/H O, 0.1% TFA) over 15 minutes of purified peptide [ F]12 (solid trace,
Acknowledgements
radio-chromatogram) and its non-radioactive [19F]12 analogue (dashed trace,
UV chromatogram, λ = 220 nm).
This work was funded by the Natural Sciences and Engineering
Research Council of Canada (NSERC). We thank the Lawson
Cyclotron & PET Radiochemistry Facility at St. Joseph’s Hospital
in London, Ontario, Canada, for donating [ F]F- for these
procedures. We also thank Dr. Lihai Yu for assistance in
radiochemistry and the Molecular Imaging Collaborative Program
at the University of Western Ontario.
6
In
a
similar
manner,
[D-Phe ,β-
1
1
13
14
Ala ,Phe ,Nle ]bombesin(6-14) is known to target all four of the
gastrin-releasing peptide receptor (GRPR) subtypes with sub-
nanomolar affinities.[27] An N-terminal modification of bombesin-
therefore [D-
Phe ,β-Ala ,Phe ,Nle ]bombesin(6-14) with an azido-PEG
linker on the N-terminus was synthesized and linked to
18
[28-31]
targeted peptides does not disrupt affinity,
6
11
13
14
8
1
8
[
F]ADIBO-F in the same process as described above resulting
in full radiochemical conversion and excellent isolated
radiochemical yields of 81-88%. The labelled peptide was purified
by semi-preparative RP-HPLC in greater than 98% radiochemical
3
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Keywords: Click Chemistry • Fluorine • Imaging Agents •
Peptides • Radiopharmaceuticals
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4
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Entry for the Table of Contents
An efficient synthesis and peptide conjugation of a new fluorine-18 azadibenzocyclooctyne prosthetic group for the SPAAC
radiolabelling toolbox.
5
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