Tetrazine-trans-Cyclooctene Ligation for the Rapid Construction of ¹⁸F Labeled Probes

Abstract

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.

Positron emission tomography (PET) is 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 100% positron efficiency, a very high specific radioactivity, and a short half-life of ~110 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.

Recently, we introduced the tetrazine-trans-cyclooctene ligation (‘TTCO ligation’, Fig. 1) as a method of bioconjugation that proceeds with fast reaction rates without 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 react with strained alkenes,⁹ we have found 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 ~ 2000 M^-1 s^-1 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

Fig. 1.

Tetrazine-trans-cyclooctene ligation (‘TTCO-ligation’)

After we described the TTCO-ligation, the groups of Hilderbrand^[11a] and Pipkorn and Braun^[11b] 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.

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-fluorobenzoate¹³), 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 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 °C for 15 min), ¹⁸F-labeled product 5 was obtained in ~1% labeling yield.

Scheme 1.

Synthesis of ¹⁸F-labeled tetrazine. Attempts to prepare 3 by nucleophilic substitution were low yielding. Fluorination of 4 (¹⁸F-fluoride, MeCN, 85 °C, 15 min) gives 5 in only 1% radiochemical 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.⁷ The major diastereomer of 7 was carried forward in the synthesis of 8.

Scheme 2.

Synthesis of trans-cyclooctene nosylate 8. Reagents and conditions: (a) NaH, α-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%.

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)/ tetrabutylammonium 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 °C (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 °C was found to be optimal (Table 1, entries 3, 5–7). Finally, we investigated the efficiency of the ¹⁸F labeling as a function of time using 2 mg of 8 at 75 °C (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 °C, 15 min) are optimal for ¹⁸F labeling.

Table 1.

Optimization of synthesis of ¹⁸F-labeled trans-cyclooctene

Entry	Amount of 8	Temp	Reaction Time	Radiochemical Yield
1	100 μg (0.35 mM)	75 °C	15 min	18%
2	500 μg (1.8 mM)	75 °C	15 min	25%
3	2.0 mg (7.0 mM)	75 °C	15 min	71%
4	3.0 mg (11 mM)	75 °C	15 min	71%
5	2.0 mg (7.0 mM)	40 °C	15 min	24%
6	2.0 mg (7.0 mM)	55 °C	15 min	34%
7a	2.0 mg (7.0 mM)	90 °C	15 min	71%
8	2.0 mg (7.0 mM)	75 °C	3 min	43%
9	2.0 mg (7.0 mM)	75 °C	7 min	68%
10	2.0 mg (7.0 mM)	75 °C	30 min	71%

^aComparable results (70% RCY) were obtained under similar conditions using K₂CO₃/K₂₂₂ instead of TBAB.

As derivatives of 3,6-di(2-pyridyl)-s-tetrazine (e.g. 1b, Figure 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.

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

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 HPLC and easily separated from unreacted precursor 8. When ¹⁸F-9 (1 mCi, 2 μM) was combined with 1a (concentrations of ≥21 μM), ¹⁸F-9 was completely consumed with 10 s, and ¹⁸F-10 had formed in 98% radiochemical yield, accompanied by ¹⁸F-11 (1%) (Table 2, entries 1-2). When the concentration of ¹⁸F-9 was decreased to 0.1 mCi (0.2 μM), the conjugate ¹⁸F-10/11 was still formed in excellent radiochemical yield (98%, entry 3). Useful radiochemical yields could also be obtained with even lower concentrations of 1a (entries 4-5). We also investigated the efficiency of the conjugation between 1a (21 μM) and ¹⁸F-9 (1 mCi, 2 μM) in PBS buffer and serum media: ¹⁸ F-10 is formed in quantitative yield within 10 seconds (entries 6-7).

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	Radiochemical Yield (18F-10 + 18F-11)
1	1 mCi (2 μM)	210 μM	MeCN/H2O	<10 s	>98 %
2	1 mCi (2 μM)	21 μM	MeCN/H2O	<10 s	>98 %
3	0.1 mCi (0.2 μM)	21 μM	MeCN/H2O	<10 s	98 %
4	0.1 mCi (0.2 μM)	2.1 μM	MeCN/H2O	<10 s	56 %
5	0.1 mCi (0.2 μM)	0.21 μM	MeCN/H2O	<10 s	15 %
6	1 mCi (2 μM)	21 μM	PBS buffer	<10 s	>98 %
7	1 mCi (2 μM)	21 μM	Serum	<10 s	>98 %

^aThe concentration of ¹⁸F-9 was estimated based on the specific activity of fluoride after bombardment (~4 Ci/μmol), taking into account a correction for the rate of radioactive decay.

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.

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.