Synthesis and evaluation of an 18F-labelled norbornene derivative for copper-free click chemistry reactions

Article abstract of DOI:10.1039/c3ob40548f

The copper-free click chemistry reaction between norbornene and tetrazine species is known to proceed in a rapid, reliable and selective manner under mild conditions. Due to these attractive properties, this reaction has recently been explored as a generally applicable method of bioconjugation. Here, we report a convenient synthetic procedure towards a novel ¹⁸F-labelled norbornene derivative ([¹⁸F]NFB) and have evaluated its ability to undergo strain-promoted copper-free click chemistry reactions with two model tetrazine species: an asymmetric dipyridyl tetrazine derivative (Tz) and a tetrazine thioureacoupled stabilised bombesin peptide (TT-BBN). In both cases, [¹⁸F]NFB was found to undergo rapid and high-yielding click chemistry reactions. Furthermore, as reactions of this type could also potentially be used in vivo to facilitate the development of a novel pretargeting approach for tumour imaging and therapy, we have also assessed the radiopharmacological profile (bioavailability, biodistribution, blood clearance and metabolic stability) of [¹⁸F]NFB in normal BALB/c mice. This radiolabelled compound exhibits both high bioavailability and metabolic stability with approximately 90% remaining intact up to 30 min following administration. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ob40548f

Organic &
Biomolecular Chemistry
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Synthesis and evaluation of an 18F-labelled norbornene
derivative for copper-free click chemistry reactions†
Cite this: Org. Biomol. Chem., 2013, 11,
3817
James C. Knight, Susan Richter, Melinda Wuest, Jenilee D. Way and Frank Wuest*
The copper-free click chemistry reaction between norbornene and tetrazine species is known to proceed
in a rapid, reliable and selective manner under mild conditions. Due to these attractive properties, this
reaction has recently been explored as a generally applicable method of bioconjugation. Here, we report
a convenient synthetic procedure towards a novel 18F-labelled norbornene derivative ([18F]NFB) and
have evaluated its ability to undergo strain-promoted copper-free click chemistry reactions with two
model tetrazine species: an asymmetric dipyridyl tetrazine derivative (Tz) and a tetrazine thiourea-
coupled stabilised bombesin peptide (TT-BBN). In both cases, [18F]NFB was found to undergo rapid and
high-yielding click chemistry reactions. Furthermore, as reactions of this type could also potentially be
used in vivo to facilitate the development of a novel pretargeting approach for tumour imaging and
therapy, we have also assessed the radiopharmacological profile (bioavailability, biodistribution, blood
clearance and metabolic stability) of [18F]NFB in normal BALB/c mice. This radiolabelled compound exhi-
bits both high bioavailability and metabolic stability with approximately 90% remaining intact up to
30 min following administration.
Received 19th March 2013,
Accepted 24th April 2013
DOI: 10.1039/c3ob40548f
www.rsc.org/obc
molecules to much larger vectors, e.g. antibodies) are currently
used in PET imaging applications. A key stage in the develop-
Introduction
The development of molecular probes for positron emission ment of each radiotracer is the rapid and site-selective incor-
tomography (PET) is an area of intense multi-disciplinary poration of the radionuclide. However due to the poor
investigation.1–4 PET offers several advantages compared to nucleophilicity of the fluoride anion and its limited reactivity
other imaging modalities, including its ability to track bio- in protic environments, this step can be problematic. These
chemical, physiological, and pharmacological events with obstacles in combination with the harsh labelling conditions
exquisite sensitivity. Consequently, this technique has been for the 18F-incorporation represent a challenge particularly for
widely used in biomedical and clinical settings to monitor the peptides and proteins which require mild reaction conditions.
progression of various malignancies, especially aggressive Therefore indirect radiolabelling via prosthetic groups9 or
cancers, with great success in some cases. Fluorine-18 exhibits novel direct radiolabelling strategies (e.g. aluminium–, boron–,
many desirable characteristics which have made it the most and silicon–18F bond formations10–14) are often required to
widely used radionuclide for PET imaging.5–8 For example, (i) facilitate the process. Furthermore, conventional conjugation
it can easily be produced on a cyclotron with high specific chemistry presents some limitations, such as the requirement
activity, (ii) it has a convenient radioactive half-life (t1/2
=
of large excesses of (often expensive) protein in order to speed
109.8 min), and (iii) it has a low β+ energy (0.64 MeV) which up reaction rates and obtain reasonable radiochemical yields.
results in a short range in tissue (max = 2.4 mm) and high
spatial resolution.
Click chemistry is now a widely applied tool in synthetic
chemistry and the reactions which fall under this category are
The use of effective molecular probes is vital in order to characterised by their speed, reliability, high yields and
maximise the imaging potential of this powerful technique selectivity.15–21 The inverse electron-demand Diels–Alder
and a wide variety of targeting vectors (ranging from small cycloadditions between (i) ring-strained norbornene or (ii)
trans-cyclooctene (TCO) species with tetrazines are excellent
examples of click chemistry reactions and are demonstrating
much recent promise as
a chemoselective method of
bioconjugation.22–25 Furthermore, unlike most other click
chemistry reactions (e.g. the azide–alkyne Huisgen cyclo-
addition26,27), the aforementioned cycloadditions do not
Department of Oncology, University of Alberta, 11560 University Ave, Edmonton,
AB T6G 1Z2, Canada. E-mail: wuest@ualberta.ca; Fax: +1780 432 8483;
Tel: +1 780 989 8150
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3ob40548f
require the presence of a copper catalyst, potentially
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[18F]fluorobenzoate ([18F]SFB),49 leading to the formation of
[18F]NFB.
The radiosynthesis of [18F]NFB was optimised based on (i)
the concentration of 1, (ii) reaction time, (iii) temperature, and
(iv) solvent (ESI; Fig. S1 and S2†). It can be concluded from
these experiments that the optimal conditions for this reaction
involve stirring at 25 °C for 20 minutes and using 1 mg mL−1
of 1. Under these conditions, the reaction proceeds with equal
efficiency (quantitative yield) in acetonitrile, ethanol, hexane,
and dimethyl sulphoxide (DMSO). When performed in metha-
nol, the reaction yield was reduced to approximately 40% and
more polar, unidentified by-products were observed via radio-
TLC which most likely arise as a result of the known instability
of [18F]SFB in methanol. For practical reasons, we chose to use
acetonitrile in our subsequent radiosyntheses of this com-
pound. Following HPLC purification, [18F]NFB was obtained in
good radiochemical yields of 60 17% (decay-corrected based
on [18F]SFB) within 52 minutes. The overall non-decay radio-
chemical yield based on starting [18F]F− was approximately
18% within 106 minutes. The radiochemical purity of [18F]NFB
routinely exceeded 99% (Fig. S3†).
Fig. 1 Radiosynthesis of [18F]NFB.
facilitating the development of a bioorthogonal pretargeting
approach for both imaging and therapeutic applications.28–32
Recently, these reactions have been used in in vitro experi-
ments to fluorescently label a variety of cancer cell lines (both
at the cell surface33–38 and intracellular regions39–42) using a
two-step pretargeting strategy.
The norbornene/TCO-tetrazine ligations have also recently
been applied as effective radiolabelling strategies in the devel-
opment of radiotracers for both PET and SPECT imaging.43
Their application has been successfully demonstrated by
Zeglis et al. in their construction of a radiometalated derivative
of the HER2 antibody, trastuzumab (Herceptin).44 Similar
strategies have been employed by Weissleder and co-
workers40,45 in the preparation of 18F-labelled PARP1 inhibitors
based on AZD2281, and also by Selvaraj et al. who developed
an 18F-labelled cyclic RGD peptide for PET imaging.46
In addition to the clear utility of such reactions as effective
methods of bioconjugation, a report by Rossin et al. in 2010
provided the first example of a copper-free click chemistry
reaction (in this case, between TCO and tetrazine species)
being used successfully for pretargeted tumour imaging in live
mice.29
Here, we report the reliable and high-yielding radiosynth-
esis of an 18F-labelled norbornene derivative ([18F]NFB; Fig. 1)
which could be a useful compound for the mild, rapid and
selective radiolabelling of a wide variety of targeting vectors for
PET imaging. We have examined its ability to function as a
prosthetic group via a copper-free click chemistry reaction
using two model tetrazine species: (i) an asymmetric dipyridyl
tetrazine (Tz),47,48 and (ii) a tetrazine-modified bombesin
peptide derivative (TT-BBN). Furthermore, we have also
assessed the suitability of [18F]NFB as a molecular probe that
could potentially facilitate the development of a novel pre-
targeting strategy by evaluating its radiopharmacological
profile (bioavailability, biodistribution, blood clearance and
metabolic stability) in normal BALB/c mice.
Copper-free click chemistry reaction between [18F]NFB and a
model tetrazine species (Tz) in various media
To assess the ability of [18F]NFB to undergo a copper-free click
chemistry reaction, an asymmetric dipyridyl tetrazine species
(Tz) was selected to provide a convenient model. This reaction
(outlined in Fig. 2) was performed in (i) acetonitrile, (ii) mouse
plasma, and (iii) mouse blood.
In order to dissolve the tetrazine species, a small amount of
the solubilising agent propylene glycol (10% v/v) was added to
each reaction mixture. The reactions which were performed in
plasma gave excellent yields (>97%) after only 10 minutes
(Fig. S4†), and those performed in mouse blood (which were
analysed at a single time point of 20 minutes) also resulted in
very high yields (95%). The reactions conducted in acetonitrile
were less rapid and lower yielding, reaching a maximum yield
of 56% after 30 minutes (Fig. S4 and S5†). These results clearly
demonstrate the potential of [18F]NFB to function as an
effective prosthetic group for radiolabelling tetrazine-modified
species, particularly in aqueous solutions. Furthermore, the
ability of [18F]NFB to undergo high yielding copper-free click
Results and discussion
Radiosynthesis of N-[2-(bicyclo[2.2.1]hept-5-en-2-yl)ethyl]-
4-[18F]fluorobenzamide ([18F]NFB)
The amine-functionalised norbornene precursor, 2-[(1S,2S,4S)-
bicyclo[2.2.1]hept-5-en-2-yl]ethanamine (1), readily undergoes
Fig. 2 Reaction scheme depicting the click chemistry reaction between [18F]-
NFB and a tetrazine (Tz) which proceeds efficiently without the requirement of
an acylation reaction with the prosthetic group 4-succinimidyl- a copper catalyst.
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Therefore, we employed a stabilised bombesin derivative intro-
duced recently by our group53 which is comprised of the
sequence Ava-Gln-Trp-Ala-Val-Sar-His-FA01010-Tle-NH2 and
selectively targets the gastrin-releasing peptide receptor
(GRPR) with high affinity.
In
a representative radiolabelling experiment, 100 μg
(300 μL) of the tetrazine-functionalised bombesin (TT-BBN)
was incubated with 0.5 MBq [18F]NFB at room temperature
over 30 min to react in an inverse electron-demand Diels–Alder
cycloaddition. The reaction scheme is outlined in Fig. 4.
Monitoring of the reaction mixture via radio-TLC showed
ca. 30% of the desired 18F-labelled Diels–Alder ‘click’ bombe-
sin [18F]FDA-BBN after 5 min and increased to nearly 50%
after 20 min. Furthermore, aside from the product, only non-
reacted [18F]NFB was observed in the reaction mixture. The
[18F]FDA-BBN yield in the reaction mixture did not change sig-
nificantly after 20 min. The results obtained by radio-TLC
nicely reflect the radio-HPLC yields (46% [18F]FDA-BBN)
recorded after 30 min (Fig. 5).
Fig. 3 Reaction kinetics between [18F]NFB and Tz in DMSO.
chemistry reactions in complex biological media highlights
the robust and highly selective nature of this ligation and
suggests that this approach could potentially be applied within
an in vivo setting to enable the pretargeted imaging of
tumours using PET.
According to radio-HPLC data in Fig. 5, [18F]FDA-BBN
showed the expected mixture of isomers corresponding to the
endo- and exo-position of the norbornene moiety as well as the
isomeric dihydropyradazines as a result of the nitrogen
release.54 [18F]FDA-BBN was isolated using HPLC purification
in radiochemical purities exceeding 95%. Whilst a few other
reports describe copper-free, strain-promoted click chemistry
involving peptides,46,55 to the best of our knowledge this study
represents the first example of an 18F-labelled peptide pre-
pared via the inverse electron-demand Diels–Alder cyclo-
addition between norbornene and tetrazine species.
Reaction kinetics between NFB and Tz
The kinetics of the reaction between NFB and Tz were esta-
blished by following an experimental protocol reported by
Karver et al. with some minor differences (see Experimental
for details).35 Using this technique, we were able to determine
a second order rate constant of 0.04 M−1 s−1 in DMSO (Fig. 3).
This represents a rapid reaction which compares very favour-
ably with many other copper-free click chemistry reactions
such as the well-known Staudinger ligation.50–52 However, this
value is lower compared with other reported norbornene–tetra-
zine click chemistry reactions. For example, Devaraj et al.
studied the reaction between (1S,2S,4S)-bicyclo[2.2.1]hept-5-en-
2-yl acetic acid and 3-(p-benzylamino)-1,2,4,5-tetrazine which
was found to proceed with a second order rate constant (k2) of
1.9 M−1 s−1 when conducted in PBS (or 1.6 M−1 s−1 when per-
formed in FBS).33 Therefore, it is likely that the rate of this
reaction may not be sufficiently high to enable an in vivo pre-
targeting approach in the traditional manner. However, we
postulate that the reaction kinetics could be markedly
improved by the adoption of a multi-valent approach invol-
ving, for example, nanoparticles substituted with multiple
tetrazine species. In this case, the favourable in vivo radio-
pharmacological profile of [18F]NFB would make it an ideally
suited ‘chaser’ molecule for this purpose and we are currently
investigating the validity of this proposed approach.
Small animal PET imaging of [18F]NFB in BALB/c mice
Analysis of the dynamic small animal PET imaging data
revealed that [18F]NFB distributes rapidly and undergoes a
relatively fast clearance from the blood based on the low mole-
cular weight of this radiolabelled compound (Fig. 6). From the
time-activity curve of the blood (heart representing the blood
pool), a blood clearance half-life of 69 seconds was determined
(Fig. S6†). Radioactivity seen within the liver, intestines and
bladder indicates this compound undergoes both hepatobili-
ary and renal excretion.
Distribution of [18F]NFB in the blood and metabolic stability
The majority of [18F]NFB was found to distribute evenly
between the blood cells and supernatant plasma (Fig. 7). We
observed only minimal binding to plasma proteins for the dura-
tion of the experiment which indicates that the radiolabelled
compound has excellent bioavailability. The results were
largely uniform across all recorded time points.
The rapid and high-yielding reaction between [18F]NFB and
the model tetrazine species, Tz, under mild conditions pro-
vides a clear indication of its utility in the preparation of
targeting vectors containing short-lived positron emitters like
fluorine-18.
Furthermore, metabolic profiling experiments revealed that
[18F]NFB has good in vivo stability with approximately 90% of
the original compound remaining intact up to 30 minutes
Copper-free click chemistry reaction between [18F]NFB and a
tetrazine-functionalised bombesin peptide (TT-BBN)
These promising results encouraged us to examine the copper- post-injection (Fig. 7). After 30 minutes, the radiolabelled com-
free click chemistry reaction between [18F]NFB and a tetrazine- pound was found to undergo rapid degradation and was
functionalised model peptide in a proof-of-concept study. almost entirely metabolised at 60 minutes post-injection.
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Fig. 4 Reaction scheme representing the click chemistry reaction of 19F/[18F]NFB with tetrazine-functionalised bombesin derivative TT-BBN.
Fig. 5 Radio-HPLC traces displaying the reaction mixture (top) and the quality control (bottom) of [18F]NFB click chemistry labelling of tetrazine-functionalised
bombesin derivative TT-BBN yielding in Diels–Alder peptide [18F]FDA-BBN.
Bioavailability and resistance to metabolic degradation are ethanamine) and the use of the common prosthetic group
important requirements for an in vivo pretargeting strategy [18F]SFB make the 18F-labelled building block [18F]NFB
using PET radiopharmaceuticals. These results indicate that an attractive species for copper-free, strain-promoted click
[18F]NFB has favourable radiopharmacological properties chemistry reactions involving tetrazine-modified targeting
which would be suitable for such an approach.
vectors.
The presented experiments clearly indicate the feasibility of
using [18F]NFB for the efficient radiolabelling of tetrazine-
modified peptides which was successfully exemplified by radio-
labelling a tetrazine-functionalised bombesin-like peptide with
Conclusions
We have developed a reliable synthetic procedure towards a good efficacy. Furthermore, this approach is also attractive for
novel 18F-labelled compound, [18F]NFB, which is capable of the radiolabelling of proteins and antibodies with the short-
undergoing click chemistry reactions with tetrazine species. lived positron emitter fluorine-18 due to the ability to perform
The commercial availability of the amine-functionalised these reactions in the absence of copper and under mild
norbornene starting material (2-(bicyclo[2.2.1]hept-5-en-2-yl)- conditions.
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The ability of [18F]NFB to undergo click chemistry reactions benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluoro-
with a model tetrazine species in both plasma and blood high- phosphate (HBTU) and ethyl cyanoglyoxylate-2-oxime (Oxyma)
lights the robust and highly selective nature of this reaction were purchased from Novabiochem (Darmstadt, Germany).
and indicates the potential of this approach to facilitate an The reactive amino acid side chains were protected as trityl
in vivo pretargeting strategy. However, while [18F]NFB revealed (Trt) for Gln and His and tert-butyloxycarbonyl (Boc) for Trp.
a promising radiopharmacological profile (e.g. high metabolic N,N-Diisopropylethylamine
(DIPEA),
dimethylformamide
stability, rapid blood clearance and high bioavailability) in (DMF), N-methyl-2-pyrrolidone (NMP), methanol, dichloro-
normal BALB/c mice, the kinetics of the reaction between [18F]- methane, acetonitrile, ethanol, diethyl ether, thioanisole, 1,2-
NFB and Tz indicate this approach is likely to be too slow for ethanedithiol (EDT), trifluoroacetic acid (TFA) and triethyl-
this purpose. We are currently optimising this particular amine (TEA) were ordered from Sigma-Aldrich (Oakville,
ligation in order to obtain higher rates of reaction which will Canada). Piperidine was purchased from Caledon (George-
hopefully move the project in this direction.
town, Canada) and 3-(4-benzylamino)-1,2,4,5-tetrazine from
In summary, we have demonstrated that [18F]NFB has great Jena Bioscience (Jena, Germany). 3-(5-Aminopyridin-2-yl)-6-
potential to function as a generally applicable tool for the (pyridine-2-yl)-s-tetrazine (Tz) was prepared according to litera-
efficient radiolabelling of tetrazine-modified peptides (and poten- ture procedure.47,48
tially other targeting vectors) via copper-free click chemistry.
General methods
1H NMR and 13C NMR spectra were recorded on a 500 MHz
Experimental
Materials
Nα-Fmoc-protected amino acids, the 4-(2′,4′-dimethoxyphenyl-
Fmoc-aminomethyl)phenoxy (Rink Amide MBHA) resin, 2-(1H-
(Varian Unity) spectrometer. Chemical shifts are given in ppm
referenced to internal standards. Mass spectra were recorded
using a Micromass ZabSpec Hybrid Sector-TOF by positive
mode electrospray ionisation. Thin layer chromatography
(TLC) was monitored using HF254 silica gel. Crude reaction
mixtures were analysed by TLC and HPLC. HPLC purification
was performed on a semi-preparative Luna C18 column (100 Å,
10 μm, 250 × 10 mm). 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 14 min at 70/30. UV
detection was performed at 210 nm and 254 nm. Radioactivity
detection was performed using a well-scintillation NaI (Tl)
detector.
Chemical syntheses
N-[2-(Bicyclo[2.2.1]hept-5-en-2-yl)ethyl]-4-fluorobenzamide
(NFB). 2-(Bicyclo[2.2.1]hept-5-en-2-yl)ethanamine
(30
μL,
0.21 mmol) was dissolved in dichloromethane (1 mL) then
added dropwise to a solution of N-succinimidyl-4-fluorobenzo-
ate (64 mg, 0.27 mmol, 1.3 eq.) in dichloromethane (4 mL).
The reaction was stirred for 60 min at 25 °C. The reaction
mixture was concentrated under reduced pressure at room
Fig. 6 Representative small animal PET images (maximum intensity projection) of a
normal BALB/c mouse after single intravenous administration of 5 MBq of [18F]NFB.
Fig. 7 Distribution of [18F]NFB in the blood (left) revealing only minimal binding to plasma proteins and therefore good bioavailability, and evaluation of the meta-
bolic stability of [18F]NFB (right) following injection in BALB/c mice (n = 3). The radiotracer remains largely intact at 30 minutes p.i.
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temperature and the resulting yellow oil was then purified by synthesis followed by washing steps with DMF, MeOH, dichloro-
column chromatography (ethyl acetate–hexane, gradient methane and diethyl ether. The completeness of each coup-
5–50%). The collected fractions were analysed by TLC (ethyl ling step was monitored via Kaiser and TNBS (2,4,6-
acetate–hexane, 1 : 1; Rf (NFB) = 0.73). The pure fractions were trinitrobenzene-sulfonic acid) tests. Cleavage and deprotection
combined and the solvent was evaporated under reduced of the amino acid side chains was achieved by treatment of the
pressure yielding a pale yellow oil. This was left overnight at peptidyl resin with an acidic cocktail of 92.5% TFA, 5% water,
4 °C and formed a white crystalline residue (18.7 mg, 34%). 1H 5% thioanisole, and 2.5% 1,2-ethanedithiol (EDT) for 4 h at
NMR (500 MHz, CDCl3): δ 0.56 (m, 1H), 1.23 (m, 1H), room temperature. The peptide was separated from the resin
1.32–1.49 (m, 3H), 1.89 (m, 1H), 2.06 (m, 1H), 2.79 (bs, 1H), by passing it through a syringe filter and precipitated by the
2.81 (bs, 1H), 3.42 (m, 2H), 5.94 (dd, 1H), 6.07 (bs, 1H), 6.14 addition of ice-cold diethyl ether. The crude peptide was
(dd, 1H), 7.09 (m, 2H), 7.76 (m, 2H). 13C (150.8 MHz, CDCl3): δ obtained by removing the residual ether using a syringe filter.
32.2, 34.7, 36.4, 39.5, 42.5, 45.5, 49.6, 115.4, 115.6, 129.0, Purification was performed by semi-preparative HPLC using a
129.1, 131.1, 132.1, 137.4, 164.6 (d, 13C–19F, 1J = 251 Hz), 166.3. Phenomenex Jupiter 10 μm, Proteo 90 Å , 250 × 10 mm fol-
m/z (ESI) C16H18FNO ([M + H+]) calcd 260.1445, found lowed by subsequent lyophilisation, giving the sufficiently
260.1445.
pure peptide as white powder.
N-{2-[3-(5-Amino-pyridin-2-yl)-6-pyridin-2-yl-4,5-diaza-tricyclo-
Ava-Gln-Trp-Ala-Val-Sar-His-FA01010-Tle-NH2
(BBN). BBN
[6.2.1.02,7]undeca-3,6-dien-9-yl]-ethyl}-4-fluoro-benzamide was obtained as a white powder (17 mg, 15.9 μmol, 27%) start-
(NFB-Tz). NFB (14 mg, 54 μmol) in acetonitrile (2 mL) was ing from 100 mg Rink amide MBHA resin and after purifi-
added dropwise to a solution of 3-(5-aminopyridin-2-yl)-6-(pyri- cation using semi-preparative HPLC at a flow rate of 2 mL
dine-2-yl)-s-tetrazine (Tz, 19 mg, 76 μmol) in acetonitrile min−1 and a gradient of 0–5 min 30% eluent B, at 15 min 35%
(8 mL). The reaction mixture was stirred at 25 °C for 60 min eluent B, at 35 min 55% eluent B, at 40 min 70% eluent B. tR =
and then concentrated under reduced pressure. The crude 11.8 min. MW C52H82N14O10 calculated 1062.6, found HR-MS
product was purified using semi-preparative HPLC (tR
=
(ESI) 1063.6 [M + H]+, 1085.6 [M + Na]+.
(4-[1,2,4,5]Tetrazin-3-yl-benzyl)-thiourea-Ava-Gln-Trp-Ala-Val-
Sar-His-FA01010-Tle-NH2 (TT-BBN). Peptide BBN (2.0 mg,
14.0 min) (5 mg, 19%). m/z (ESI) C28H28FN6O ([M + H+]) calcd
483.2303, found 483.2300.
3-(4-Isothiocyanatomethyl-phenyl)-[1,2,4,5]tetrazine (TT-NCS). 1.9 μmol, 1 eq.) dissolved in DMF (100 μL) was reacted with
A
solution of 3-(4-benzylamino)-1,2,4,5-tetrazine (51 mg, 3-(4-benzyl-isothiocyano)-1,2,4,5-tetrazine TT-NCS (0.9 mg,
267 μmol, 1 eq.) in chloroform (2 mL) was cooled to 0 °C and 3.8 μmol, 2 eq.) in DMF (110 μL) by adding DIPEA (0.7 μL,
treated with thiophosgene (81.9 μL, 1068 μmol, 4 eq.) forming 3.8 μmol, 2 eq.). The reaction solution was incubated at 50 °C
a red-orange coloured suspension. Addition of TEA (125 μL) for 3 h followed by semi-preparative HPLC purification at a
led to the release of HCl gas. The reaction mixture was allowed flow rate of 2 mL min−1 using the following gradient:
to warm to room temperature and was stirred for 16 h at 25 °C. 0–10 min 20% eluent B, at 25 min 50% eluent B, at 30–40 min
Deionised water (100 mL) was added to the reaction mixture 80% eluent B, at 40–50 min 90% eluent B (tR = 32.8 min).
and the crude product was extracted with chloroform (4 × TT-BBN was isolated as a pink solid (0.65 mg, 0.503 μmol,
30 mL). The organic layer was dried with sodium sulphate and 24%). MW C62H89N19O10S calculated 1291.7, found LR-MS
concentrated under reduced pressure. The residue was purified (ESI) 1292.7 [M + H]+, 646.8 [M + 2H]2+.
by flash column chromatography in 100% dichloromethane
4-Fluoro-N-{2-[3-(4-thioureidomethyl-phenyl)-4,5-diaza-tricyclo-
(Rf = 0.5) forming a pink-coloured solid (22.4 mg, 38%). 1H [6.2.1.02,7]undeca-3,6-dien-9-yl]-ethyl}-benzamide-Ava-Gln-Trp-
NMR (500 MHz, CD3CN): δ 4.93 (s, 2H), 7.63–7.64 (d, 2 Ala-Val-Sar-His-FA01010-Tle-NH2 (FDA-BBN). NFB (0.1 mg,
aromatic-H), 8.59–8.61 (m, 2 aromatic-H). 13C NMR (150.9 MHz, 0.503 μmol, 1 eq.) dissolved in acetonitrile (100 μL) was added
CD3CN): δ 48.8, 128.7, 129.2, 132.9, 140.6, 158.8, 166.8.
to a solution of TT-BBN (0.65 mg, 0.503 μmol, 1 eq.) in DMF
(200 μL). The combined solutions were allowed to sit overnight
at room temperature resulting in the disappearance of the
Peptide syntheses
The stabilised bombesin derivative was synthesised on the pink colour. The reaction mixture was purified via semi-
basis of Fmoc-orthogonal solid-phase peptide synthesis (SPPS) preparative HPLC at a flow rate of 2 mL min−1 using a gradient
via a combination of manual coupling procedures and auto- of 0–10 min 20% eluent B, at 25 min 50% eluent B, at 30–40 min
mated synthesis performed with a peptide synthesiser (Syro I, 80% eluent B, at 40–50 min 90% eluent B (tR = 35.0 min).
MultiSynTech, Germany). The Rink Amide MBHA resin FDA-BBN was isolated as a white solid (0.3 mg, 0.200 μmol,
(loading 0.6 mmol g−1) provided the solid support for syn- 39%). MW C78H107FN18O11S calculated 1522.8, found HR-MS
thesis of stabilised bombesin with the sequence Ava-Gln-Trp- (ESI) 1523.6 [M + H]+, 1524.8 [M + 2H]+, 1525.8 [M + 3H]+,
Ala-Val-Sar-His-FA01010-Tle-NH2. Fmoc deprotection was 1526.8 [M + 4H]+, 1545.8 [M + Na]+, 1546.8 [M + Na + H]+,
achieved by treatment with 40% piperidine/DMF for 5 min, fol- 1547.8 [M + Na + 2H]+, 1548.8 [M + Na + 3H]+.
lowed by further incubation with 20% piperidine/DMF for
Radiosyntheses
15 min. 5 eq. of each Fmoc-protected amino acid were acti-
vated and coupled using the reagents HBTU (5 eq.), Oxyma (5 No-carrier added aqueous [18F]fluoride was produced using
eq.) and DIPEA (10 eq.) for 60 min throughout the peptide the TR19/9 (Advanced Cyclotron systems Inc.) cyclotron at the
3822 | Org. Biomol. Chem., 2013, 11, 3817–3825
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Edmonton PET Center (Ep = 17.8 MeV) by irradiation of for 20 min. The red blood cells were lysed by adding deionised
enriched [18O]water (3.0 mL, Rotem Germany, >98% enrich- water (900 μL) and leaving in a thermoshaker (500 rpm) at
ment) via the 18O(p,n)18F nuclear reaction. N-Succinimidyl- room temperature for 5 min. The sample was centrifuged for
[18F]-4-fluorobenzoate ([18F]SFB) was synthesised following the 5 min at 10 000 rpm and the clear red solution was analysed
procedure reported by Mäding et al.49 The overall synthesis by radio-TLC as described above. In this case, a single time
was usually carried out in less than 56 min, providing [18F]SFB point of 20 minutes was selected due to the additional proces-
in decay-corrected yield of 60% and with a radiochemical sing time of the sample prior to radio-TLC analysis which ren-
purity greater than 95%.
dered earlier time points meaningless. All click chemistry
N-[2-(Bicyclo[2.2.1]hept-5-en-2-yl)ethyl]-4-[18F]fluorobenzamide reactions were performed in triplicate.
([18F]NFB). 2-(Bicyclo[2.2.1]hept-5-en-2-yl)ethanamine (2 μL,
Rate constant determination between NFB and Tz
15 μmol) was dissolved in acetonitrile (1 mL) containing [18F]-
SFB (typically in the range of 272–862 MBq). The reaction was Kinetic experiments were performed in a 96-well plate format
stirred for 20 min at 25 °C. Progress of the reaction was moni- using a SpectraMax 340PC384 microplate reader and analysed
tored by radio-TLC (hexane–ethyl acetate 1/1; product Rf
=
using SpectraMax Pro software version 5.4.1 (Molecular
0.73). Upon completion, the reaction mixture was purified Devices). A dilution series of NFB and a solution 3-(5-amino-
using semi-preparative HPLC (tR = 20.0 min). [18F]NFB was iso- pyridin-2-yl)-6-(pyridine-2-yl)-s-tetrazine (Tz) were prepared in
lated in radiochemical yields of 60
based on [18F]SFB, n = 14). The radiochemical purity routinely reactions between NFB and Tz were then initiated by combin-
exceeded 99%. ing these solutions in the 96-well plate, leading to final con-
17% (decay-corrected DMSO and allowed to equilibrate to 37 °C for 10 minutes. The
N-{2-[3-(5-Amino-pyridin-2-yl)-6-pyridin-2-yl-4,5-diaza-tricyclo- centrations of 5, 6, 7, 8, 9, 10 mM for NFB and 1 mM of Tz,
[6.2.1.02,7]undeca-3,6-dien-9-yl]-ethyl}-4-[18F]fluoro-benzamide representing a 5- to 10-fold excess of the norbornene reactant.
([18F]NFB-Tz). To a solution of 3-(5-aminopyridin-2-yl)-6-(pyri- The total reaction volume in each well was 100 μL. The 96-well
dine-2-yl)-s-tetrazine (1 mg, 4 μmol) in acetonitrile (900 μL) plate was then loaded immediately into the plate reader and
was added [18F]NFB in acetonitrile (typically 25 MBq, 100 μL). the reactions were monitored by recording the intensity of the
The reaction mixture was then stirred at 25 °C for 40 min. Pro- characteristic tetrazine absorption (λmax = 540 nm) which
gress of the reaction was monitored by radio-TLC (hexane– diminished over the course of each reaction. Measurements
ethyl acetate 1/1; Rf ([18F]NFB-Tz) = 0). Upon completion, the were acquired at 30 second intervals over 1 h. The temperature
reaction mixture was purified using semi-preparative HPLC was maintained at 37 °C over the course of the experiment.
(tR ([18F]NFB-Tz) = 14.0 min).
kobs (s−1) Values (baseline corrected) were determined using
Prism 5 (GraphPad) and these values were then plotted against
[NFB]. From this graph, the second order rate constant
General procedure for copper-free click chemistry of [18F]NFB
with Tz in a variety of solvents
(k2, M−1 −1) was calculated from the gradient. Each experi-
s
Reactions performed in acetonitrile and mouse plasma. The ment was performed in triplicate.
HPLC-collected peak containing [18F]NFB was diluted with
Radiolabelling of (4-[1,2,4,5]tetrazin-3-yl-benzyl)-thiourea-
water (20 mL per 1.5 mL of HPLC eluent) and passed through Ava-Gln-Trp-Ala-Val-Sar-His-FA01010-Tle-NH2 (TT-BBN) with
a Sep-Pak cartridge (Sep-Pak Plus tC18, Waters). The trapped [18F]NFB. TT-BBN (100 μg, 77 nmol) dissolved in a 20%
[18F]NFB was eluted with diethyl ether (1 mL) which was then MeCN–PBS (pH 7.6) solution (150 μL) was treated with purified
blown dry under a stream of N2(g). [18F]NFB (ca. 5 MBq) was [18F]NFB (14 μL, as obtained from semi-preparative HPLC in
then dissolved in propylene glycol (25 μL). In a separate eppen- 70% MeCN–water) corresponding to 0.3–1.2 MBq. The overall
dorf tube, Tz (1 mg, 4 μmol) was dissolved in propylene glycol reaction volume equaled 100 μL and consisted of 20% MeCN.
(25 μL) and then made up to 475 μL with either acetonitrile or The reaction mixture was incubated at room temperature for
plasma. The tetrazine solution was then added to the [18F]NFB 30 min. Determination of the formed Diels–Alder peptide [18F]-
solution and stirred at 37 °C for 30 min. Each of these reac- FDA-BBN was monitored at 5 minute intervals via radio-TLC
tions was monitored at 0.5, 1, 2.5, 5, 10, 15, 20, 25, and (hexane–ethyl acetate 1/1; Rf ([18F]FDA-BBN) = 0.0, Rf ([18F]NFB) =
30 minutes by radio-TLC (hexane–ethyl acetate, 1/1; Rf ([18F]- 0.73). Additionally, the reaction mixture was analysed after
NFB-Tz) = 0). In each case, a control reaction was performed in 30 min via analytical radio-HPLC performed on a Luna C18
the absence of Tz which confirmed the presence of the column (100 Å, 10 μm, 250 × 4.5 mm) and using a gradient of
unreacted starting material (Rf([18F]NFB) = 0.73).
acetonitrile (solvent B) and water/0.2% TFA (solvent A) with a
Reactions performed in mouse blood. In this case, the click flow rate of 1 mL min−1 (0–10 min 80% A/20% B, 25 min 50%
chemistry reaction was performed in a smaller overall volume A/50% B, 30–40 min 20% A/80% B, 40–50 min 10% A/90% B).
to allow for subsequent cell lysis within the same eppendorf tR ([18F]FDA-BBN) = 25.8 min, tR ([18F]NFB) = 35.3 min.
tube. Dried [18F]NFB (ca. 5 MBq) was dissolved in propylene
Radiopharmacology
glycol (5 μL). In a separate eppendorf tube, Tz (0.2 mg,
0.8 μmol) was dissolved in propylene glycol (5 μL) and then
Small animal PET imaging in normal BALB/c mice. All
made up to 95 μL with mouse blood. The tetrazine solution animal experiments were carried out in accordance with guide-
was then added to the [18F]NFB solution and stirred at 37 °C lines of the Canadian Council on Animal Care (CCAC) and
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Organic & Biomolecular Chemistry
were approved by the local animal care committee of the Cross
Cancer Institute. Positron emission tomography (PET) experi-
ments were performed using normal BALB/c mice. The mice
were not fasted prior to imaging experiments. The animals
were anaesthetised through inhalation of isoflurane in 40%
oxygen/60% nitrogen (gas flow, 1 L min−1) and body tempera-
ture was kept constant at 35 °C for the entire experiment. Mice
were positioned and immobilised in the prone position with
their medial axis parallel to the axial axis of the scanner in the
centre of the field of view of the microPET® R4 scanner
(Siemens Preclinical Solutions, Knoxville, TN, USA). A trans-
mission scan for attenuation correction was not acquired.
4–6 MBq of [18F]NFB in 100–150 μL saline (0.9%) containing
10% of ethanol was injected through a needle catheter into the
tail vein. Data acquisition continued for 60 min in 3D list
mode. The frames were reconstructed using maximum a poster-
iori (MAP) reconstruction. The pixel size was 0.085 by 0.085 by
0.12 cm and the resolution in the centre field of view was
1.8 mm. No correction for partial volume effects was per-
formed. The image files were further processed using the
ROVER v 2.0.21 software (ABX GmbH, Radeberg, Germany).
Masks for defining 3D regions of interest (ROI) were set and
the ROIs were defined by thresholding. ROI time-activity
curves (TAC) were generated for subsequent data analysis.
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