Tetrazine-trans-cyclooctene ligation for the rapid construction of 18F labeled probes

Article abstract of DOI:10.1039/c0cc03078c

A radiolabeling method for bioconjugation based on the Diels-Alder reaction between 3,6-diaryl-s-tetrazines and an ¹⁸F-labeled trans-cyclooctene is described. The reaction proceeds with exceptionally fast rates, making it an effective conjugation method within seconds at low micromolar concentrations.

Full text of DOI:10.1039/c0cc03078c

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Tetrazine–trans-cyclooctene ligation for the rapid construction
18
of F labeled probesw
a
b
Zibo Li,*a Hancheng Cai,z Matthew Hassink,z Melissa L. Blackman,b
Richard C. D. Brown,c Peter S. Contia and Joseph M. Fox*b
Received 5th August 2010, Accepted 6th September 2010
DOI: 10.1039/c0cc03078c
A radiolabeling method for bioconjugation based on the
Diels–Alder reaction between 3,6-diaryl-s-tetrazines and an
18F-labeled trans-cyclooctene is described. The reaction proceeds
with exceptionally fast rates, making it an effective conjugation
method within seconds at low micromolar concentrations.
that 3,6-diaryl-s-tetrazines offer an excellent combination of
fast reactivity and stability for both the conjugate and starting
material.6 In particular, 3,6-di(2-pyridyl)-s-tetrazines were
shown to display excellent characteristics.6,10 Thus, the reaction
between trans-cyclooctene and 1a proceeds with a rapid rate
(k2 E 2000 Mꢀ1 sꢀ1 in 9 : 1 MeOH : water), and is successful
in cell media and cell lysate.6 3,6-Di(2-pyridyl)-s-tetrazines can
easily be functionalized as their amido derivatives (1b),6 which
display excellent stability toward water and biological
nucleophiles.10
Positron emission tomography (PET) is
a non-invasive
imaging modality that utilizes positron-emitting radionuclides
(C-11, N-13, O-15 and F-18).1 F-18 PET has a number of
attributes that make it clinically attractive, including near
100% positron efficiency, a very high specific radioactivity,
and a short half-life of B110 min.2 However, the short
half-life of F-18 and the poor nucleophilicity of fluoride render
it difficult to incorporate F-18 in complex molecules.
Currently, radiochemistry is a major limiting factor for the
field of PET.3 Despite recent advances,4,5 challenges exist for
improving F-18 incorporation with respect to reaction rates,
efficiency, and selectivity.
After we described the TTCO-ligation, the groups of
Hilderbrand11a and Pipkorn and Braun11b described ligations
between tetrazines and less reactive dienophiles. However, the
use of trans-cyclooctene is the key to fast rates of reactivity,
and other groups have subsequently developed applications
for the TTCO-ligation.11c,d,12
Because of the fast rate of reactivity, the TTCO-ligation
offers opportunities for the rapid conjugation of radionuclides
to biomolecules, both of which are often available only at low
concentration. Very recently, Robillard and coworkers
elegantly demonstrated that our system for TTCO-ligation
could be utilized in In111-imaging of tumors in live mice using
a DOTA-derivative of 1b.12 Herein, we report the development
of an extremely fast and reactive method for generating
18F labeled probes based on the TTCO-ligation.
Recently, we introduced the tetrazine–trans-cyclooctene
ligation (‘TTCO ligation’, Fig. 1) as a method of bioconjugation
that proceeds with fast reaction rates without the need for
catalysis.6,7 trans-Cyclooctene derivatives are readily prepared
from cis-cyclooctenes using a photochemical flow-reaction
that we developed.8 While a variety of s-tetrazine derivatives
were known to react with strained alkenes,9 we have found
While a number of indirect methods for 18F-incorporation
into tetrazines or trans-cyclooctene derivatives could be
envisioned (e.g. derivatization by N-succinimidyl-4-18F-fluoro-
benzoate),13 such approaches are complicated by the need to
carry 18F through added manipulations. Thus, we focused on
the development of direct methods for 18F-incorporation via
reactions with fluoride ion, with an initial focus on the
Fig. 1 Tetrazine–trans-cyclooctene ligation (‘TTCO-ligation’).
a Molecular Imaging Center, Department of Radiology,
University of Southern California, Los Angeles 90033, USA.
E-mail: ziboli@usc.edu; Fax: +1 323-442-3252
b Brown Laboratories, Department of Chemistry and Biochemistry,
University of Delaware, Newark, DE 19803, USA.
E-mail: jmfox@udel.edu; Fax: +1 302-831-6335
c School of Chemistry, University of Southampton, Highfield,
Southampton, SO171BJ, UK. E-mail: rcb1@soton.ac.uk;
Fax: +44 (0)238-059-6805
w Electronic supplementary information (ESI) available: Experimental
procedures, NMR spectra and Radio-HPLC data. See DOI: 10.1039/
c0cc03078c
z Equal contributions were made by these authors.
Scheme 1 Synthesis of 18F-labeled tetrazine. Attempts to prepare 3 by
nucleophilic substitution were low yielding. Fluorination of 4 (18F-fluoride,
MeCN, 85 1C, 15 min) gives 5 in only 1% radiochemical yield.
c
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Finally, we investigated the efficiency of the 18F labeling as a
function of time using 2 mg of 8 at 75 1C (Table 1,
entries 8–10). While the labeling yield was optimal (71%) in
an experiment conducted for 15 min, useful labeling yields
were also obtained after 3 min (43%) and 7 min (68%). The
conclusion of the experiments summarized in Table 1 is that
the conditions of entry 3 (7.0 mM 8, 75 1C, 15 min) are optimal
for 18F labeling.
Scheme 2 Synthesis of trans-cyclooctene nosylate 8. Reagents and
conditions: (a) NaH, a-bromoacetic acid. (b) CH2N2, 69%, 2 steps.
(c) DIBAL, 78%. (d) Methyl benzoate-sensitized photoisomerization
with active removal of trans-isomers, AgNO3, 55%. (e) NsCl, Et3N,
87%.
As derivatives of 3,6-di(2-pyridyl)-s-tetrazine (e.g. 1b,
Fig. 1) are readily accessible,6,10,12 3,6-di(2-pyridyl)-s-tetrazine
(1a) was used to test the efficiency of the 18F-labeled trans-
cyclooctene 9 in the TTCO-ligation. A ‘cold’ study with
19F–9 was initially conducted. As expected, the conjugate
19F–10 formed immediately, and then slowly isomerized
to 1,4-dihydropyrazine 19F–11 as a mixture of isomers6
(Scheme 3a). These isotopically stable conjugates served as
coinjection standards for analysis of reactions with 18F–9.
The conjugations between 1a and 18F–9 were carried out in
1 : 1 acetonitrile/water, and were analyzed within 10 seconds
of mixing. Prior to the conjugation, 18F–9 was purified by
synthesis of 18F-labeled tetrazines (Scheme 1). Our attempts to
convert nitrotetrazine derivatives 2a and 2b into 18F-labeled
substitution products
3
using 18F-fluoride/kryptofix or
18F-TBAF gave decomposition products and only traces of
radiolabeled products. We also combined mesylate 4 with
fluoride: in the most successful experiment (18F-TBAF at
85 1C for 15 min), 18F-labeled product 5 was obtained
in B1% labeling yield.
The instability of tetrazine precursors to F-labeling
conditions prompted us to consider methods for preparing
18F-labeled trans-cyclooctenes. To this end, we synthesized the
nosylate 8 as shown in Scheme 2. The key step in the synthesis
was photoisomerization of 6 to 7 using a flow reactor
that continuously removes the trans-isomers through selective
metal complexation.8 The major diastereomer of 7 was carried
forward in the synthesis of 8.
Nosylate 8 reacted efficiently with TBAF to provide 19F–9
in high yield. With this standard in hand, conditions for the
preparation of 18F–9 were optimized (Table 1). It was found
that 18F–9 could be obtained in good yield by adding nosylate
8
to a
mixture of [18F]-fluoride (100 mCi)/tetrabutyl-
ammonium bicarbonate (TBAB) in acetonitrile (0.8 mL).
We determined the effect of varying the concentration of 8 in
reactions conducted for 15 min at 75 1C (Table 1, entries 1–4).
The highest labeling yield (71%) was achieved with 7.0 mM 8,
but a useful radiochemical yield (18%) was still obtained with
0.35 mM 8. Different reaction temperatures were studied, and
75 1C was found to be optimal (Table 1, entries 3, 5–7).
Scheme 3 (a) Conjugation between 9 and 1a occurs rapidly to form
conjugate 10, which slowly rearranges to 11 as a mixture of regio- and
stereoisomers. (b) Radio-HPLC analysis of the crude product from
Table 2, entry 3.
Table 1 Optimization of synthesis of 18F-labeled trans-cyclooctene
Entry
Amount of 8
Temp/1C
Reaction time/min
Radiochemical yield (%)
1
2
3
4
100 mg (0.35 mM)
500 mg (1.8 mM)
2.0 mg (7.0 mM)
3.0 mg (11 mM)
2.0 mg (7.0 mM)
2.0 mg (7.0 mM)
2.0 mg (7.0 mM)
2.0 mg (7.0 mM)
2.0 mg (7.0 mM)
2.0 mg (7.0 mM)
75
75
75
75
40
55
90
75
75
75
15
15
15
15
15
15
15
3
18
25
71
71
24
34
71
43
68
71
5
6
7a
8
9
10
7
30
a
Comparable results (70% RCY) were obtained under similar conditions using K2CO3/K222 instead of TBAB.
c
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Table 2 The effect of concentration on the formation of 18F–10 through the conjugation between 18F–9 and 3,6-di(2-pyridyl)-s-tetrazine.
All reactions were performed at room temperature
Entry
18F–9a
1a
Solvent
Reaction time/s
Radiochemical yield (18F–10 + 18F-11) (%)
1
2
3
4
5
6
7
1 mCi (2 mM)
1 mCi (2 mM)
0.1 mCi (0.2 mM)
0.1 mCi (0.2 mM)
0.1 mCi (0.2 mM)
1 mCi (2 mM)
210 mM
21 mM
21 mM
2.1 mM
0.21 mM
21 mM
21 mM
MeCN/H2O
MeCN/H2O
MeCN/H2O
MeCN/H2O
MeCN/H2O
PBS buffer
Serum
o10
o10
o10
o10
o10
o10
o10
>98
>98
98
56
15
>98
>98
1 mCi (2 mM)
a
The concentration of 18F–9 was estimated based on the specific activity of fluoride after bombardment (B4 Ci mmolꢀ1), taking into account a
correction for the rate of radioactive decay.
HPLC and easily separated from unreacted precursor 8.
When 18F–9 (1 mCi, 2 mM) was combined with 1a (concentrations
of Z 21 mM), 18F–9 was completely consumed within 10 s, and
18F–10 had formed in 98% radiochemical yield, accompanied
by 18F–11 (1%) (Table 2, entries 1 and 2). When the
concentration of 18F–9 was decreased to 0.1 mCi (0.2 mM),
the conjugate 18F–10/11 was still formed in excellent radio-
chemical yield (98%, entry 3). Useful radiochemical yields
could also be obtained with even lower concentrations of 1a
(entries 4 and 5). We also investigated the efficiency of the
conjugation between 1a (21 mM) and 18F–9 (1 mCi, 2 mM) in
PBS buffer and serum media: 18F–10 is formed in quantitative
yield within 10 seconds (entries 6 and 7).
with instrumentation supported by NSF CRIF:MU grants:
CHE 0840401 and CHE-0541775.
Notes and references
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The Diels–Alder conjugate 10 was found to be stable in
water, and the benign isomerization to 11 was the only side
reaction. Thus, 19F–10 was the only product detected by
1H NMR analysis immediately after the conjugation. In
CD3CN/H2O, the rearrangement of 19F–10 to 19F–11
proceeded to 11% conversion after 4 hours, and >95%
conversion after 48 hours. The stability of the radiolabeled
conjugation product was monitored in PBS buffer and serum
media for 4 hours, and no degradation products of 18F–10
were observed.
7 For reviews of Cu-free bioconjugation chemistry: J. C. Jewett and
C. R. Bertozzi, Chem. Soc. Rev., 2010, 39, 1272–1279; C. R. Becer,
R. Hoogenboom and U. S. Schubert, Angew. Chem., Int. Ed., 2009,
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8 M. Royzen, G. P. A. Yap and J. M. Fox, J. Am. Chem. Soc., 2008,
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9 (a) F. Thalhammer, U. Wallfahrer and J. Sauer, Tetrahedron Lett.,
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10 Acetamido-substituted dipyridyltetrazines (1b) display fast
kinetics,12 and are stable for prolonged periods (>24 h) in water,
and toward amines and thiols in MeOH: R. Selvaraj, M. L.
Blackman, J. M. Fox, unpublished results.
11 (a) N. K. Devaraj, R. Weissleder and S. A. Hilderbrand,
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W. Waldeck, B. Didinger, M. Koch, G. Mueller, M. Wiessler
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In conclusion, a new radiolabeling method for bioconjugation
based on the TTCO-ligation has been described. The method
utilizes a novel F-18 labeled trans-cyclooctene probe, and the
efficiency of the reaction has been demonstrated with 3,6-di-
(2-pyridyl)-s-tetrazine, which can be readily functionalized via
amido derivatives. The reaction proceeds with exceptionally
fast rates, making it an effective conjugation method at low
concentration. We anticipate that the TTCO-ligation will
provide the foundation for a new class of reliable methods
for 18F-labeling of biomolecules in PET applications.
12 R. Rossin, P. R. Verkerk, S. M. v. d. Bosch, R. C. M. Vulders,
I. Verel, J. Lub and M. S. Robillard, Angew. Chem., Int. Ed., 2010,
49, 3375–3378.
13 G. Vaidyanathan and M. R. Zalutsky, Nat. Protoc., 2006, 1,
1655–1661.
This work was supported by NIH Grant Number P20
RR017716 from the COBRE Program of the NCRR, and by
the USC Department of Radiology. Spectra were obtained
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 8043–8045 8045