Copper-free click chemistry with the short-lived positron emitter fluorine-18

Article abstract of DOI:10.1039/c1ob06034a

The copper-free strain-promoted click chemistry between ¹⁸F-labeled aza-dibenzocyclooctyne [¹⁸F]FB-DBCO and various azides is described. [¹⁸F]FB-DBCO was prepared in 85% isolated radiochemical yield (decay-corrected) throu

Full text of DOI:10.1039/c1ob06034a

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PAPER
Copper-free click chemistry with the short-lived positron emitter fluorine-18†
Vincent Bouvet, Melinda Wuest and Frank Wuest*
Received 27th June 2011, Accepted 9th August 2011
DOI: 10.1039/c1ob06034a
The copper-free strain-promoted click chemistry between 18F-labeled aza-dibenzocyclooctyne
[18F]FB-DBCO and various azides is described. [18F]FB-DBCO was prepared in 85% isolated
radiochemical yield (decay-corrected) through acylation of amino aza-dibenzocyclooctyne 1 with
N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB). [18F]FB-DBCO showed promising
radiopharmacological profil with fast blood clearance as assessed with dynamic small animal PET
studies. Metabolic stability of [18F]FB-DBCO was 60% of intact compound after 60 min post injection
in normal Balb/C mice and blood clearance half-life was determined to be 53 s based on the
time-activity-curve (TAC). Copper-free click chemistry was performed with various azides at low
concentrations (1–2 mM) which differed in their structural complexity in different solvents (methanol,
water, phosphate buffer and in bovine serum albumin (BSA) solution). Reaction proceeded best in
methanol (>95% yield after 15 min at room temperature), whereas reaction in BSA required longer
reaction times of 60 min and 40 ◦C upon completion.
An especially powerful set of bioorthogonal chemistry reactions
is summerized under the term “click chemistry”, which has
Introduction
also strongly influenced current radiopharmaceutical chemistry.4
Click chemistry describes a class of reactions using several
selective and modular building blocks to create heteroatom C–
X–C links enabling chemoselective ligation reactions to label
biologically relevant compounds. Bioorthogonal click reactions
include Staudinger ligation, Cu(I)-catalyzed [3 + 2] azide-alkyne
cycloaddition, copper-free strain-promoted [3 + 2] azide-alkyne
cycloaddition, and various reactions based on inverse-electron
demand Diels–Alder reactions. These click chemistry concepts
have been applied to the labeling of molecular probes, covalent
labeling of active enzymes, site- and residue-specific labeling
of proteins, labeling of cell surface glycans, and, more re-
cently, to in vivo chemistry for pretargeted imaging and therapy
of cancer.5
The development of radiolabeled molecular probes for molecular
imaging with positron emission tomography (PET) and single-
photon emission computed tomography (SPECT) has become a
complex chemical science. Especially PET as the most sophis-
ticated imaging methodology has evolved into a powerful non-
invasive molecular imaging technique which provides functional
information on physiological, biochemical and pharmacological
processes in living subjects.1 Among the available PET radionu-
clides, short-lived positron emitter fluorine-18 (18F, t1/2 = 109.8 min)
is particularly useful due to its favorable nuclear and chemical
properties.2 18F has found numerous applications for the labeling
of both, small molecules and compounds of high molecular weight
like peptides, proteins, and oligonucleotides. However, radiochem-
istry with 18F differs significantly from conventional chemistry.
Radiosyntheses involving 18F require fast and efficient reactions
while considering the extraordinary stoichiometry resulting from
the typically produced submicromolar amounts of radiolabeled
compounds.
Cu(I)-catalyzed [3
+ 2] azide-alkyne cycloaddition and
Staudinger ligation have successfully introduced into organic PET
chemistry with the short-lived positron emitter 18F.6,7 As a result,
numerous 18F-labeled PET radiotracers have been prepared via
Cu(I)-assisted cycloaddition between azides and terminal alkynes
and to a lesser extent through Staudinger ligation. Advances of
click chemistry for radiolabeling reactions with 18F and other
radionuclides for molecular imaging purposed have been recently
summarized in excellent reviews.4
In recent years bioorthogonal chemistry has entered into many
fields of chemical, biological, and material sciences.3 Bioorthogo-
nal chemical reporter strategy has emerged as a versatile method
for the labeling of biomolecules like nucleic acids, carbohydrates,
peptides, and proteins.
However, slow reaction kinetics as encountered for the
Staudinger ligation and the use of cytotoxic copper have led to the
search of alternative fast and copper-free click chemistry concepts
applicable to radiochemisty with the short-lived positron emitter
18F for molecular probe development and pretargeted molecular
imaging in vivo.
Department of Oncology, University of Alberta, 11560 University Ave,
Edmonton, AB T6G 1Z2, Canada. E-mail: wuest@ualberta.ca; Fax: +1
780 432 8483; Tel: +1 780 989 8150
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1ob06034a
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In this work we report the first example of radiolabeling
reactions with 18F employing copper-free click reaction based on
strain-promoted cycloaddition reactions using an 18F-labeled aza-
dibenzocyclooctyne derivative ([18F]FB-DBCO). The concept was
tested through the reaction of [18F]FB-DBCO with various azides
to evaluate versatility of the reaction for rapid incorporation of
short-lived positron emitter 18F under mild conditions (Scheme 1).
(>95%) of [18F]SFB into [18F]FB-DBCO after 30 min. After
HPLC purification, [18F]FB-DBCO was obtained in excellent
radiochemical yields of 85% (decay-corrected) within 60 min
starting from [18F]SFB. The radiochemical purity exceeded 95%.
Radiopharmacological profile of [18F]FB-DBCO was evaluated
in normal Balb/C mice using small animal PET and radiometabo-
lite analysis. [18F]FB-DBCO was completely stable for 60 min in
methanol and phosphate buffer (pH 7.4). Metabolite analysis of
arterial blood samples confirmed that compound [18F]FB-DBCO
displayed reasonable metabolic stability in vivo, reaching 60% of
intact compound after 60 min post injection in normal Balb/C
mice as determined with radio-TLC. Dynamic small animal PET
studies showed rapid clearance of [18F]FB-DBCO from the blood
and from most other tissues and organs. Most activity was found
in the bladder, gall bladder and intestines (Scheme 3).
Scheme 1 Copper-free click chemistry between ring-strained [18F]FB-
DBCO and various azides to give 18F-labeled triazoles. Only one regioiso-
mer is shown.
Moreover, we also studied metabolic stability and radiophar-
macological profile of [18F]FB-DBCO by means of small animal
PET in mice. Copper-free click chemistry reaction in buffer and
bovine serum albumin (BSA) was studied to determine potential
of the reaction for future pretargeted click chemistry in vivo.
Scheme 3 Representative small animal PET images (maximum intensity
projection) of a normal Balb/C mouse 5 s (left) and 60 min (right) after
single intravenous administration of 6 MBq of [18F]FB-DBCO.
Results and discussion
Synthesis and radiopharmacological evaluation of 18F-labeled
aza-dibenzocyclooctyne [18F]FB-DBCO
The blood clearance half-life of [18F]FB-DBCO was deter-
mined to be 53 s based on the time-activity-curve (TAC)
over the heart derived from dynamic small animal PET study
(Scheme 4).
N -(3-Aminopropionyl)-5,6-dihydro-11,12-didehydrodibenzo-
[b,f ]azocine 1 is commercially available (Click Chemistry Tools),
and a large variety of analogues can easily be synthesized through
acylation reaction using primary amine group in compound
1. For radiolabeling of compound 1 with short-lived positron
emitter 18F we decided to apply acylation reaction with prominent
bifunctional labeling agent 4-succinimidyl-[18F]fluorobenzoate
([18F]SFB).
We and others showed that [18F]SFB is a highly suitable
prosthetic group for fast and convenient radiolabeling of a broad
variety of different compounds under mild reaction conditions.
[18F]SFB can easily be prepared according to various automated
synthesis procedures recently published in literature making
prosthetic group [18F]SFB readily available for acylation reactions
with 18F.8 The preparation of [18F]FB-DBCO via acylation of
primary amine in compound 1 with [18F]SFB is depicted in
Scheme 2.
Scheme 4 Representative time activity curve (TAC) of the blood after
single administration of [18F]FB-DBCO.
The rapid blood clearance and the reasonable metabolic
stability are important requirements for using 18F-labeled aza-
dibenzo-cyclooctyne [18F]FB-DBCO as radiolabeled low molec-
ular weight compound within the proposed pretargeted strategy
in vivo based on copper-free click chemistry with corresponding
azides.
Copper-free strain-promoted click chemistry of [18F]FB-DBCO
with various azides
Scheme 2 Radiosynthesis of [18F]FB-DBCO employing acylation with
bifunctional labeling agent [18F]SFB.
18F-Labeled aza-dibenzocyclooctyne [18F]FB-DBCO was further
studied in several strain-promoted click chemistry reactions
with different azides. Recently, aza-dibenzo-cyclooctynes have
been used for fast and efficient enzyme PEGylation5b and the
preparation of metabolically labeled glycoconjugates of living cells
Primary amine 1 and [18F]SFB were dissolved in acetonitrile
and incubated for 30 min at 40 ◦C. Monitoring the progress
of the reaction with radio-TLC indicated complete conversion
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through copper-free [3 + 2] cycloaddition with various azides.5f
This approach was further expanded through the use of nitrones
in strain-promoted alkyne-nitrone cycloadditions to form the
corresponding N-alkylated isoxazolines with very fast reaction
kinetics.9
We treated [18F]FB-DBCO with various azides possessing dif-
ferent structural complexities. [18F]FB-DBCO was reacted with
simple aromatic azides like 4-azidoaniline 2 and aliphatic azides
like 11-azido-3,6,9-trioxaundane-1-amine 3, carbo-hydrates like
2-azido-2-deoxyglucose 4 and 6-azido-6-deoxy-glucose 5, and
complex natural product geldanamycin 6, which has been mod-
ified with a linker containing an azide group. Geldanamycin
is an inhibitor of heat-shock protein 90 (Hsp90), which has
become an important drug target in oncology over the last
years.10
mycin derivative
solid.
6 in 78% isolated yield as a purple
All reference compounds for copper-free strain-promoted click
chemistry were obtained through treatment of azides displayed in
Scheme 5 with FB-DBCO according to Scheme 1. In contrast to
the reaction depicted in Scheme 1 employing tracer concentrations
of [18F]FB-DBCO, 1.5 to 2.0 equivalents of respective azides 2, 3,
4, 5, and 6 were reacted with one equivalent of FB-DBCO. The
reaction resulted in the formation of two distinct regioisomers
(1,4-triazole and 1,5-triazole regioisomer), which is a well known
phenomenon for this type of reaction when no copper catalyst
is used.11 However, in some cases both regioisomers were not
separable by HPLC-purification. All reactions were performed on
a mM-scale (15–30 mM), and products were isolated using HPLC
purification. Characterization of corresponding alkylated triazole
derivatives 7, 8, 9, 10 and 11 was performed with 1H NMR and/or
mass spectrometry. Cold FB-DBCO was prepared according to
Scheme 2 through reaction of amine 1 with 1.3 equivalents of SFB
in CH2Cl2 in good chemical yield of 72% after purification with
column chromatography.
Structures of all azides used in the copper-free click chemistry
reaction are given in Scheme 5.
Reaction of [18F]FB-DBCO with azides 2–6 at low concentra-
tions (1–2 mM) was performed at room temperature and 40 ◦C,
and in different solvents, including methanol, water, phosphate
buffer (pH 7.4), and bovine serum albumin (BSA) solution to
evaluate speed of the reaction under different conditions according
to Scheme 1. The results are summarized in Table 1.
In the first set of reactions we tested readily water-soluble
glucose derivatives 6-azido-6-deoxyglucose 4 and 2-azido-2-
deoxyglucose 5 (entries 1 and 2). Reaction of 6-azido-6-
deoxyglucose 4 with [18F]FB-DBCO in methanol at room tem-
perature gave desired click chemistry product [18F]7 in almost
quantitative yield after a reaction time of 15 min at room
temperature. This is consistent with the previously reported
high reactivity of aza-dibenzocyclooctynes with various azides.5b,5f
Reactivity was slightly decreased when reaction was performed in
phosphate buffer (pH 7.4) and BSA solution. In the latter case a
reaction time of 60 min was necessary to achieve a 90% conversion
of [18F]FB-DBCO into triazole [18F]7 as determined by radio-TLC
analysis.
Scheme 5 Structures of azides used for copper-free strain promoted click
chemistry.
Azides 2, 3, 4, and 5 are commercially available, whereas azido-
functionalized geldanamycin derivative 6 was prepared according
Scheme 6.
Somewhat lower conversion rates were achieved when 2-azido-
2-deoxyglucose5 was used as the azide source in the click chemistry
reaction (entry 2). This observation can be explained by the less
accessible azide group in 2-azido-2-deoxyglucose 5 in comparison
to 6-azido-6-deoxyglucose 4.
As an aromatic azide, 4-azidoaniline 2 showed comparable
reactivity in methanol as the solvent as found for both azido-
substituted glucose derivatives 4 and 5 (entry 3). However, solubil-
ity problems prevented performance of the click reaction in phos-
phate buffer and BSA solution. Complex azido-functionalized
geldanamycin derivative 6 also posed solubility problems, and re-
action with [18F]FB-DBCO proceeded in a DMSO/water mixture
to afford a 69% conversion of [18F]FB-DBCO into click chemistry
product [18F]10 after a reaction time of 60 min at 40 ◦C (entry 4).
Aliphatic azide 11-azido-3,6,9-trioxaundane-1-amine 3 represents
an interesting azide-containing linker for potential conjugation
and functionalization of specific targeting vectors like antibodies
and proteins for subsequent pretargeting. Compound 3 is readily
soluble in water, and the reaction with [18F]FB-DBCO proceeded
in good radiochemical yields of 75% based upon conversion of
Scheme 6 Reaction conditions: (i) 11-azido-3,6,9-trooxaundane-1-amine
3, DMF, room temperature, 30 min.
As a methoxy quinone, geldanamycin rapidly undergoes
Michael addition and b-elimination with primary amines to
furnish the corresponding vinylogous amine product.10c,d Treat-
ment of geldanamycin with a five-fold excess of 11-azido-3,6,9-
trioxaundane-1-amine 3 afforded azido-functionalized geldana-
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Table 1 Copper-free click chemistry with [18F]FB-DBCO
[18F]FB-DBCO into triazole [18F]11 after a reaction time of 30 min
at 40 ◦C (entry 5).
Reaction
Entry
1
conditionsa
RCY (%)b
98
Productc
Summary and conclusions
A
We have developed a convenient and simple method for copper-
free click chemistry with the short-lived positron emitter fluorine-
18. To the best of our knowledge, this is the first report on
the application of copper-free strain-promoted click chemistry
involving aza-dibenzocyclooctynes in 18F chemistry. The commer-
cial availability of amine-functionalized aza-dibenzocyclooctyne
1 and the readily availability of prosthetic group [18F]SFB make
the proposed 18F-labeled building block [18F]FB-DBCO an ideal
starting material for subsequent copper-free strain-promoted click
chemistry reactions.
B
C
97
90
2
A
85
The copper-free strain-promoted click chemistry with [18F]FB-
DBCO was tested with various azides, which differed in their
structural complexity ranging from simple aliphatic and aromatic
azides, azido-functionalized carbohydrates to complex natural
products like azido-functionalized geldanamycin. The presented
experiments clearly demonstrate the feasibility to use copper-free
strain-promoted click chemistry between aza-dibenzocyclooctyne
[18F]FB-DBCO and various azides at low concentrations as little as
1–2 mM for the convenient radiolabeling of small molecules under
mild conditions. Moreover, this approach has the potential to be
extended to other classes of compounds like peptides, proteins
and oligonucleotides. The opportunity to perform the reaction in
the absence of copper under physiological conditions makes this
approach especially attractive for the mild radiolabeling of pro-
teins and antibodies with the short-lived positron emitter fluorine-
18. However, compared to alternative bioorthogonal reactions
like tetrazine/trans-cyclooctene cycloaddition, our approach pro-
ceeded with much slower reaction rate which seems not to be
feasible for in vivo chemistry-based pretargeting.4h Alternative
strategies which include more reactive aza-dibenzocyclooctynes
and the application of nitrones as more reactive coupling partners
are currently under investigation.
B
C
69
75
3
A
82
4
D
69
Experimental
General methods
1H NMR and 13C NMR spectra were recorded on a 500 MHz
or 600 MHz (Varian Unity) spectrometers. Chemical shifts are
given in ppm referenced to internal standards (s = singlet, bs =
broad singlet, d = doublet, dd = doublet of doublet, ddd = doublet
of doublet of doublet, t = triplet, m = multiplet). Mass spectra
were recorded using a Micromass ZabSpec Hybrid Sector-TOF by
positive mode electrospray ionization. Thin layer chromatography
(TLC) was monitored using HF254 silica gel. All solvents were dried
and/or distilled prior to utilization. Crude reaction mixtures were
analyzed by TLC and HPLC. HPLC analysis was performed on a
5
E
75
˚
semi-preparative Luna C18 column (100 A, 10 mm, 250 ¥ 10 mm).
a All reactions were carried out with 1–2 mM of the respective azide. The
following reaction conditions were used: A) methanol, room temperature,
15 min; B) phosphate buffer, 40 ◦C, 30 min; C) 3.50% bovine serum albumin
(BSA) solution in water, room temperature, 60 min; D) DMSO/water
(1/1), 40 ◦C, 60 min.; E) water, 40 ◦C, 30 min. b Radiochemical yield
determined by radio-TLC representing the percentage of the F-labeled
triazole product present in the reaction mixture. c Only one regioisomer
formed during the reaction is shown.
The eluting solvent started with an acetonitrile/water gradient
from (15/85 to 50/50, v/v) for 8 min at a flow rate of 3 mL min-1,
followed by a 8 min gradient from (50/50 to 70/30, v/v) and
finally a 14 min at 70/30. UV detection was performed at 220 nm
and 254 nm. Radioactivity detection was performed using a well-
scintillation NaI (Tl) detector.
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Chemical syntheses
FB-DBCO-coupled triazole-10. Compound 6 (5.1 mg, 6.8
mmol) and FB-DBCO (2 mg,
5 mmol) were stirred in
5,6-Dihydro-11,12-didehydrodibenzo-[b,f ]azocino-3-oxoprop-
yl-4-fluorobenzamide (FB-DBCO). N-(3-Aminopropionyl)-5,6-
dihydro-11,12-didehydrodibenzo-[b,f ]azocine 1 (34 mg, 123 mmol)
was dissolved in 1 mL of dichloromethane and added drop-wise to
a solution of SFB (39 mg, 164 mmol) in dichloromethane (4 mL).
The reaction mixture was stirred for 20 min at 25 oC and progress
of the reaction was monitored by TLC (hexane/ethyl acetate 1/1).
Upon completion, the reaction mixture was concentrated under
reduced pressure and the residue was then purified by column
chromatography (hexane/ethyl acetate 1/1) to afford 35 mg (72%)
of the desired product as a pale-yellow oil. TLC analysis, Rf 0.5;
1H NMR (500 MHz, CDCl3): d 2.08 (ddd, 1H, J = 3.9 Hz, J = 7.7
Hz, J = 16.6 Hz), 2.53(ddd, 1H, J = 3.8 Hz, J = 7.2 Hz, J = 16.7
Hz), 3.42–3.56 (m, 2H), 3.67 (d, 1H, J = 13.9 Hz), 5.16 (d, 1H, J =
13.9 Hz), 6.66 (t, 1H, J = 12.0 Hz), 6.98–7.04 (M, 2H), 7.18 (dd,
2H, J = 0.8 Hz, J = 7.5 Hz), 7.29–7.43 (M, 6H), 7.47–7.52 (M,
2H), 7.71 (d, 1H, 7.5 Hz). 13C NMR (150.8 MHz, CDCl3): 34.86,
35.63, 55.52, 107.75, 114.7, 115.3, 115.4, 122.5, 122.9, 125.6, 127.3,
128.2, 128.5, 128.6, 128.9, 129.1, 129.2, 130.7, 132.2, 147.9, 150.9,
166.0, 172.3. m/z (ESI) C25H19FN2O2 ([M+Na+]) calcd. 421.1322,
found 421.1322.
MeOH/CH2Cl2 (1 : 1, 1.5 mL) for 10 h at 40 oC. The two formed
regioisomers could not be separated by HPLC. Regioisomers
1
eluted between 18.6–18.9 min. Yield: 51%. H NMR (500 MHz,
CDCl3): 0.98 (d, 3H, J = 6.5 Hz), 1.11 (d, 3H, J = 6.5 Hz), 1.64–
1.82 (M, 9H), 2.02 (s, 3H), 2.08 (ddd, 1H, J = 3.9 Hz, J = 7.7 Hz,
J = 16.6 Hz), 2.40 (dd, 1H, J = 10.7 Hz, J = 10.3 Hz), 2.53(ddd,
1H, J = 3.8 Hz, J = 7.2 Hz, J = 16.7 Hz), 2.67 (d, 1H, J = 13.0 Hz),
2.74 (m, 1H), 3.26 (s, 3H), 3.35 (s, 3H) 3.42–3.62 (m, 20H), 3.67
(d, 1H, J = 13.9 Hz), 4.30 (d, 1H, J = 10 Hz), 5.16 (d, 1H, J = 13.9
Hz), 5.19 (s, 1H), 5.85 (d, 1H, J = 15.5 Hz), 5.87 (d, 1H, J = 15.7
Hz), 6.09 (bs, 1H), 6.25 (bs, 1H), 6.58 (t, 1H, J = 11.5 Hz), 6.66
(t, 1H, J = 12.0 Hz), 6.95–7.04 (M, 3H), 7.18 (dd, 2H, J = 0.8 Hz,
J = 7.5 Hz), 7.22–7.26 (M, 3H), 7.29–7.43 (M, 5H), 7.47–7.52 (M,
2H), 7.71 (d, 1H, 7.5 Hz). m/z (ESI) C61H74FN8O13 ([M+Na+]) 12
calcd. 1167.5, found 1167.5.
FB-DBCO-coupled triazole-11. Yield: 49%. Formation of
inseparable regioisomers. tR
= 13.5–13.8 min. m/z (ESI)
C33H37FN6O5 ([M+Na+]) 13 calcd. 639.3, found 639.2.
Radiosyntheses
No-carrier added aqueous [18F]fluoride was produced using the
TR19/9 (Advanced Cyclotron systems Inc.) cyclotron at the
Edmonton PET Center (Ep = 17.8 MeV) by irradiation of enriched
[18O]water (3.0 mL, Rotem Germany, >98% enrichment) via the
18O(p,n)18F nuclear reaction. [18F]SFB was synthesized following
the procedure reported by Ma¨ding et al.8a Overall synthesis was
usually carried out in less than 45 min providing [18F]SFB in decay-
corrected yield of 60% and in a radiochemical purity greater 95%.
17-[11-Azido-3,6,9-trioxaundane-1-amino]-17-desmethoxy gel-
danamycin 6. Geldanamycin (100 mg, 178.4 mmol) and 11-azido-
3,6,9-trioxaundane-1-amine 3 (177 mL, 892 mmol) were dissolved
in DMF (5 mL). The mixture was stirred at room temperature for
30 min. HCl (1 N, 50 mL) was added and the mixture was extracted
with dichloromethane. After drying with MgSO4 and evaporation
of the solvent, the residue was purified by column chromatography
(methanol/CH2Cl2 1/9) to give 104 mg (78%) of the desired
product as a purple solid. TLC analysis (methanol/CH2Cl2 1/9),
Rf 0.7. HPLC analysis, tR = 18.3 min. 1H NMR (500 MHz, CDCl3):
d 0.97 (d, J = 6.5 Hz, 3H), 0.99 (d, J = 6.5 Hz, 3H), 1.80 (s, 3H,
CH3), 2.02 (s, 3H, CH3), 3.27 (s, 3H, OCH3), 3.36 (s, 3H, OCH3),
3.39 (t, J = 5.2 Hz, 2H), 3.45 (m, 1H), 3.48–3.57 (m, 2H), 3.69 (m,
6H), 4.30–4.32 (m, 2H), 5.19 (s, 1H), 5.86 (t, J = 10.9 Hz, 1H),
5.90 (d, J = 9.4 Hz, 1H), 6.58 (t, J = 10.9 Hz, 1H), 6.66 (m, 1H),
6.95 (bd, J = 10.9 Hz, 1H), 7.27 (s, 1H), 9.17 (s, 1H). m/z (ESI)
C36H54N6O11 ([M+H]+) calcd. 747.3929, found 747.3929.
5,6-Dihydro-11,12-didehydrodibenzo-[b,f ]azocino-3-oxoprop-yl-
4-[18F]fluorobenzamide ([18F]FB-DBCO). N-(3-Aminopropion-
yl)-5,6-dihydro-11,12-didehydrodibenzo-[b,f ]azocine 1 (1.5 mg,
5.4 mmol) was dissolved in acetonitrile (500 mL) of containing
100 MBq of [18F]SFB. The reaction was stirred for 30 min
at 40 oC. Progress of the reaction was monitored by radio-
TLC (hexane/ethyl acetate 1/1). Upon completion, the reaction
mixture was concentrated and the residue was purified using
semi-preparative HPLC. [18F]FB-DBCO was isolated in 95%
radiochemical yield (decay-corrected). The radiochemical purity
exceeded 95%. Radio-TLC (hexane/ethyl acetate 1/1) Rf 0.5, tR =
18.6 min.
General procedure for copper-free click chemistry of FB-DBCO
with azides 2–6. 25 mmol of respective azide compound and
5 mg (12.5 mmol) of FB-DBCO was stirred in methanol (1.5 mL)
at room temperature for 30 min. Product was purified by semi-
preparative HPLC, and formed regioisomers were collected and
analysed with HR-MS.
General procedure for copper-free click chemistry of [18F]FB-
DBCO with azides 2–6. HPLC-collected peak containing
[18F]FB-DBCO (20–50 MBq) was evaporated to dryness at
◦
40 C under reduced pressure. The residue was re-solubilized in
FB-DBCO-coupled triazole-7. Regioisomer 1 (48%), tR
=
methanol, phosphate buffer (pH 7.4), 3.5% BSA solution in water,
12.0–12.3 min.; regioisomer 2 (37%) 12.5–12.8 min m/z (ESI)
C31H30FN5O7 ([M+Na+]) calcd. 626.2021, found 626.2014 (regioi-
somer 1), 626.2013 (regioisomer 2).
water, and DMSO/water containing 1–2 mM of the respective
o
o
azide. All reactions were carried out at 25 C or 40 C. Identity
of the products was confirmed by radio-HPLC analysis through
co-injection of respective reference compounds.
The following reaction conditions were applied:
A) methanol, room temperature, 15 min.
FB-DBCO-coupled triazole-8. Regioisomer 1 (46%), tR
=
12.0–12.3 min.; regioisomer 2 (39%) 12.5–12.8 min m/z (ESI)
C31H30FN5O7 ([M+Na+]) calcd. 626.2, found 626.2 (regioisomer
1), 626.2 (regioisomer 2).
B) phosphate buffer, 40 ◦C, 30 min.
FB-DBCO-coupled triazole-9. Yield: 95%. Formation of
C) 3.5 BSA solution in water, room temperature, 60 min.
inseparable regioisomers. tR
= 15.1–15.5 min. m/z (ESI)
D) DMSO/water (1/1), 40 ◦C, 60 min.
C31H25FN6O2 ([M+Na+]) 12 calcd. 555.2, found 555.2.
E) water, 40 ◦C, 30 min.
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[18F]FB-DBCO-coupled triazole-[18F]7. Condition A: Radio-
chemical yield: 98%; Condition B: Radiochemical yield: 97%;
Condition C: Radiochemical yield: 90%, tR = 11.8–12.5 min for
both isomers.
the supernatant following a second centrifugation step (5 min
at 13,000 rpm). TLC samples from the plasma fraction were
developed and analyzed using radio-TLC as described above.
[18F]FB-DBCO-coupled triazole-[18F]8. Condition A: Radio-
chemical yield: 85%; Condition B: Radiochemical yield: 69%;
Condition C: Radiochemical yield: 75%, tR = 11.9–12.5 min for
both isomers.
Acknowledgements
The authors would like to thank John Wilson, David Clen-
dening and Jayden Sader from the Edmonton PET Center for
radionuclide production and excellent technical support. F.W.
thanks the Dianne and Irving Kipnes Foundation and the Natural
Sciences and Engineering Research Council of Canada (NSERC)
for supporting this work.
[18F]FB-DBCO-coupled triazole-[18F]9. Condition A: Radio-
chemical yield: 82%, tR = 14.9–15.2 min.
[18F]FB-DBCO-coupled triazole-[18F]10. Condition D: Radio-
chemical yield: 69%, tR = 13.1–13.4 min.
References
[18F]FB-DBCO-coupled triazole-[18F]11. Condition E: Radio-
chemical yield: 75%, tR = 18.3–18.6.
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