Development of a 11C-labeled tetrazine for rapid tetrazine-trans-cyclooctene ligation

Article abstract of DOI:10.1039/c3cc41027g

Tetrazine-trans-cyclooctene ligations are remarkably fast and selective reactions even at low micro-molar concentrations. In bioorthogonal radiochemistry, tools that enable conjugation of radioactive probes to pre-targeted vectors are of great interest. Herein, we describe the successful development of the first ¹¹C-labelled tetrazine and its reaction with trans-cyclooctenol.

Full text of DOI:10.1039/c3cc41027g

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Development of a C-labeled tetrazine for rapid
tetrazine–trans-cyclooctene ligation†
Matthias M. Herth,*abc Valdemar L. Andersen,abc Szabolcs Lehel,c Jacob Madsen,c
Gitte M. Knudsena and Jesper L. Kristensenb
Cite this: Chem. Commun., 2013,
49, 3805
Received 6th February 2013,
Accepted 18th March 2013
DOI: 10.1039/c3cc41027g
www.rsc.org/chemcomm
Tetrazine–trans-cyclooctene ligations are remarkably fast and
selective reactions even at low micro-molar concentrations.
In bioorthogonal radiochemistry, tools that enable conjugation
of radioactive probes to pre-targeted vectors are of great interest.
Herein, we describe the successful development of the first
11C-labelled tetrazine and its reaction with trans-cyclooctenol.
Bioorthogonal chemical reactions between two exogenous
moieties, without interfering with native biochemical processes
in living beings, may serve as powerful diagnostic applications
in molecular imaging.1 Potentially, these ligations could permit
in vivo long-term positron emission tomography (PET imaging)
1
using short-lived isotopes such as fluorine-18 (t B 110 min) or
2
1
carbon-11 (t B 20.4 min), which are routinely applied in non-
2
invasive clinical PET imaging. The basic principle relies on a
pre-targeting approach enabling repeatable PET imaging of
applied pre-targeting vectors (the in vivo click approach).1
Snapshots at multiple time-points provide long-term imaging
information without applying long-lived nuclides. This strategy
results in a superior image contrast compared to conventional
Scheme 1 Tetrazine–trans-cyclooctene (TTCO) ligation between two asymmetric
synthons.
long-term imaging techniques and the ability to administer Recently, Blackman introduced a rapid inverse-electron-demand
higher radioactive doses.2
Diels–Alder reaction between electron-deficient tetrazines (TTA)
Bioorthogonal conjugations, like the Staudinger ligation or and strained trans-cyclooctene (TCO) derivatives often referred to
the strain-promoted azide–alkyne cycloaddition,3 have found as tetrazine–trans-cyclooctene (TTCO) ligation (Scheme 1).
widespread applications within in vivo click chemistry.4
The fast reaction kinetics (k2 = 2000 to 22 000 MÀ1 sÀ1
)
However, the slow reaction kinetics (k2 r 40 MÀ1
s
À1) necessi- and selectivity of this catalyst-free coupling make it an ideal
tated a high dose and a large excess of secondary reagent to candidate for effective applications at low concentrations.1,5
achieve detectable ligation1 (see ESI† for further details). In addition, this ligation can be performed in water and its
conjugates are stable towards biological nucleophiles.6 There-
fore, the TTCO could be applied to quantitative and repetitive
in vivo click PET or single photon emission computed tomo-
graphy (SPECT) imaging. Compared to other imaging methods,
a Center for Integrated Molecular Brain Imaging, Rigshospitalet and University of
Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen, Denmark.
E-mail: matthias.herth@nru.dk; Fax: +45 3545 6713; Tel: +45 3545 6711
b Department of Drug Design and Pharmacology, Faculty of Health and Medical
Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen,
Denmark. Fax: +45 3533 6041; Tel: +45 3533 6487
PET and SPECT bear the advantage of high sensitivity (the level
of detection approaches 10À12 M of the tracer) and isotropism
(i.e. ability to detect organ accumulation accurately regardless
of tissue depth).7
Rossin et al. proved for the first time the potential of the
tetrazine ligation approach on a pre-targeted antibody for
SPECT molecular imaging.1 PET is beneficial over SPECT,
c PET and Cyclotron Unit, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen,
Denmark. Fax: +45 3545 3545; Tel: +45 3545 8621
† Electronic supplementary information (ESI) available: Comparison of bio-
orthogonal reaction kinetics, experimental procedures, HPLC conditions and
spectroscopic data of selected compounds. See DOI: 10.1039/c3cc41027g
c
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because PET results in higher spatial and temporal resolution
and better quantification.7 Therefore, Devaraj et al. extended
this TTCO approach to PET by in vivo clicking an 18F-labeled
polymer to a TCO modified antibody.8 However, this strategy is
disadvantaged by violating the tracer principle, i.e., that radio-
active compounds’ kinetics reflect physiological processes with-
out interfering with the physiology itself. This requirement is
typically met at concentrations in the nanomolar range. In the
aforementioned approach, pharmacological doses (mM) of the
labeled polymer are used because the precursor cannot be
separated from the final labeled product. As a result, toxicology
and approval concerns might arise. Labeling of a small, well
defined molecule reduces these concerns substantially since
the precursor and the labeled product are easily separated at
nanomolar concentrations. In 2010, Li et al. described the first
synthesis of a small molecule (an 18F-labeled trans-cyclooctene
derivative) for the TTCO.6 However, this approach has one
Scheme 2 Synthesis and functionalization of the tetrazine matrix: (a) hydrazine
(98%), H2O, 90 1C, 12 h, 31%; (b) DDQ, toluene, 12 h, 50%; (c) p-anisoyl chloride,
pyridine, 130 1C, MW, 5 min, 98%; (d) p-(benzoyloxy)benzoyl chloride, pyridine,
130 1C, MW, 5 min, 92%; (e) Pd/C, ammonium formate, isopropanol, 70 1C, MW,
20 min, 90%,‡ (f) various conditions (see ESI† for details)
drawback as the 18F-label was attached to the TCO instead of to conduct repeat PET studies in the same subject within hours
the TTA.6 This requires that the TTA be attached to the pre- or usually under simpler insertion conditions.
targeting vector. Although several advances have been made
Initially, we focused on the introduction of carbon-11 on
recently, TTA chemistry is still in its infancy.9 Furthermore, aniline 6 and later—since phenols are more easily labeled than
asymmetric TTAs with suitable chemical handles for conjuga- anilines13—we used a novel developed phenol derivative 9.
tion with biomolecules are challenging to create.9 However, the The necessary tetrazine matrix was synthesized according to
biggest drawback of attaching the TTA-unit to a vector is the Blackman (Scheme 2).6 Purification processes were optimized
very poor solubility and possible low long-term in vivo stability and 6 was isolated. In contrast to the reported procedures,5,6
of the highly nitrogen rich TTAs. In addition, TTAs are the amino moiety of 6 could not be easily functionalized as the
explosive,10 a cause for concern when handled in micromolar amido, alkyl or amidosulfonyl derivatives (see ESI† for details),
quantities. In contrast, the strategy to conjugate easy, commer- and all attempts to introduce a 11C-labeled methyl group
cially available and stable trans-cyclooctene derivatives to the directly on the aniline failed (Scheme 2).
pre-targeting vector and subsequently to image them via a
labeled TTA is very promising and the labeled TTA can then synthesised. The synthesis of the amide derivatives succeeded
be applied in nanomolar quantities. with microwave heating, whereas conventional heating of the
Besides the aforementioned possibility to conduct in vivo corresponding acid chloride with 6 failed (Scheme 2).
click chemistry using the TTCO, this fast and selective ligation With the precursor 9 and its reference 7 in hand, we optimized
Afterwards, phenol 9 and its corresponding reference 7 were
could also enable traditional labeling of complex molecules the radiolabeling conditions for [11C]7 (log D7.4 = 3.37) (see ESI† for
such as proteins, antibodies or nanoparticles. Even small details). The highest RCY (33 Æ 5%, n = 5, EOS) was obtained using
molecules such as peptides could benefit from an effective 0.3 mg precursor (0.8 mmol) dissolved in 0.3 mL DMF, 0.8 mL
and prompt labeling approach. A broader set of secondary 2 N NaOH (2 equiv.) and [11C]MeI at 80 1C for 2 min. [11C]7 could
labeling synthons is still required to address the chemical be synthesized, separated via semi-preparative HPLC and formu-
diversity of targeting vectors. Today, the largest and most lated in a radiochemical purity of >98% (Scheme 3) in a synthesis
promising potential to develop novel PET molecular imaging
tracers is innovative radiochemistry.6,11 The use of the TTCO
for labelling small molecules is challenging as several isomers
are formed in the reaction, which complicates subsequent
characterisation (Scheme 1). Initially, a mixture of kinetic
products is formed, which then isomerises over time to the
thermodynamically more stable ones.6 Thus, complete charac-
terisation of the product mixture is very difficult, if not impos-
sible. Nevertheless, Selvaraj et al. used this tetrazine ligation
approach to label and evaluate successfully a small cyclic RGD
peptide without separating the isomers.12 Likewise, Li et al.
did not separate all possible isomers when analyzing their
18F-labeled TTCO adduct.6
Herein, we report the development of a rapid TTCO between
a 11C-labeled TTA and a trans-cyclooctenol (TCO). This develop-
ment based carbon-11 was prioritized as it displays some advantages
over fluorine-18 during development research, e.g. the possibility chromatogram of [11C]7 and its standard 7 (UV at 254 nm: black; radio: grey).
Scheme 3 11C-radiolabeling of TTA derivative: (a) radiosynthesis of
[
11C]7
succeeded in 33% RCY: [11C]CH3I, NaOH (2 equiv.), DMF, 80 1C; (b) analytical HPLC
c
3806 Chem. Commun., 2013, 49, 3805--3807
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0.1% phosphate buffer or in the HPLC eluent (MeCN: 0.1%
phosphate buffer 1 : 1). Analysis of the final 11C-TTCO conjugate
was started within 20 s, and again 4 major peaks were observed
(as judged from the radioactive trace). They corresponded to the
UV-trace (Scheme 4c). In addition, the HPLC chromatogram
showed complete consumption of [11C]7.
In conclusion, we describe here the development of a
11C-labeled tetrazine for rapid tetrazine–trans-cyclooctene ligation.
Labeling of [11C]7 succeeded in a 33% RCY and the final click
ligation proceeded very rapidly to produce various isomers of
[
11C]10 within 20 s. The secondary 11C-labeling synthon [11C]7
is the first described 11C-labeled TTA which could be used as a
click agent for conventional or bioorthogonal labeling of nano-
particles such as polymers or antibodies.
The authors wish to thank the staff at the PET and Cyclotron
unit for expert technical assistance, Peter Brøsen and François
Crestey for helping with HRMS measurements. Financial sup-
port from the Intra European Fellowship (MC-IEF-275329), the
Faculty of Health and Medical Sciences at the University
of Copenhagen and the Lundbeck Foundation is gratefully
acknowledged.
Scheme 4 (a) Tetrazine ligation of [11C]7 (radio: light grey) and trans-cyclooctenol
resulting in the Diels–Alder conjugates
(c) radio and UV-HPLC diagram of [11C]10, 10 and [11C]7 (UV at 254 nm: black;
[
11C]10; (b) LC-MS trace of 10;
Notes and references
radio: dark grey).
‡ 7 and 9 were isolated as salts, only 9 was stable under basic conditions
and could be isolated as its free base.
time of 50–60 min (EOB to EOS) with an average specific activity of
60 GBq mmolÀ1 (range 40–80 GBq mmolÀ1). Thereby, 0.4–0.8 GBq
was typically produced.
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cyclooctenol 11 (Scheme 4a). As indicated by an instant colour
change from pink to yellow, the conjugation proceeded imme-
diately in DMSO, DMF, H2O and phosphate buffer. Only one UV
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showed that the double bond of 11 disappeared suggesting
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part of the NMR spectrum hints the formation of several
isomers, as outlined in Scheme 1. Therefore, an analytical
method was pursued to try and separate the possible isomers.
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100 Å column (Phenomenex Inc.), 150 Â 4.6 mm, 50 : 50
acetonitrile : IP-buffer (5 mM Na-decanesulfonate, 25 mM
phosphate buffer); pH = 2.6 adjusted with H3PO4, flow rate:
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standards for determination of the radiolabeled product
¨
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¨
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c
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Chem. Commun., 2013, 49, 3805--3807 3807