Conformationally Strained trans-Cyclooctene (sTCO) Enables the Rapid Construction of ¹⁸F-PET Probes via Tetrazine Ligation

Abstract

The bioorthogonal reaction between tetrazines and trans-cyclooctenes is a method for the rapid construction of F-18 probes for PET imaging. Described here is a second generation 18F-labeling system based on a conformationally strained trans-cyclooctene (sTCO)—a dienophile that is approximately 2 orders of magnitude more reactive than conventional TCO dienophiles. Starting from a readily prepared tosylate precursor, an 18F labeled sTCO derivative (18F-sTCO) could be synthesized in 29.3 +/- 5.1% isolated yield and with high specific activity. Tetrazine ligation was carried out with a cyclic RGD-conjugate of a diphenyl-s-tetrazine analogue (RGD-Tz) chosen from a diene class with an excellent combination of fast reactivity and stability both for the diene as well as the Diels-Alder adduct. For both the tetrazine and the sTCO, mini-PEG spacers were included to enhance solubility and improve the in vivo distribution profile of the resulting probe. Extremely fast reactivity (up to 2.86 x 105 M-1s-1 at 25 °C in water) has been observed in kinetic studies in the reaction of sTCO with diphenyl-s-tetrazine derivatives. A kinetic study on sTCO diastereomers in 55:45 MeOH:water showed that the syn-diastereomer displayed slightly faster reactivity than the anti-diastereomer. An 18F-sTCO conjugate with RGD-Tz demonstrated prominent and persistent tumor uptake in vivo with good tumor-to-background contrast. Unlike most radiolabeled RGD peptides, the tumor uptake of this PET agent increased from 5.3 +/- 0.2% ID/g at 1 h post injection (p.i.), to 8.9 +/- 0.5% ID/g at 4 h p.i., providing evidence for prolonged blood circulation. These findings suggest that tetrazine ligations employing 18F-sTCO should serve as a powerful and general platform for the rapid construction of peptide or protein derived PET agents.

Designing Molecular Building Blocks for Functional Polymersomes

“…Fox et al . developed an approach for the synthesis of tetrazine functionalized PEG in their pursuit to develop radiolabels …”

Cycloadditions of Trans‐Cyclooctenes and Nitrones as Tools for Bioorthogonal Labelling

“…The inverse electron demand Diels–Alder (iEDDA) reaction between tetrazine and trans ‐cyclooctene (TCO) arguably provides the most significant leap in bioorthogonal chemistry in terms of rate acceleration, achieving rate constants up to 10 6 m −1 s −1 . This allows biological processes to be tracked on the minutes‐to‐seconds timescale at sub‐micromolar concentrations of reagents and holds potential for biomedical and material applications . The iEDDA reaction is driven by the electrophilicity of the tetrazine, coupled with the high exothermicity of the reaction with the strained alkene .…”

18F‐Labeling of Sensitive Biomolecules for Positron Emission Tomography

“…The [18 F]TCO derivative was further improved by the same groups to radiolabel a cyclic RGD-conjugate 162 with a TCOPEG analog 161 to improve solubility and in vivo distribution of the resulting probe (Figure 29). [137] The tracer 163 was obtained in 99% RCY relative to the fluorine-18 TCO derivative 161 with high molar activity of 33.7± 7.4 GBq/μmol (0.91 ± 0.20 Ci/μmol). PET imaging of human U87MG tumor bearing mice showed good tumor uptake of 5.3 ± 0.2% ID/g at 1h post injection of the radiotracer and increased to 8.9 ± 0.5% ID/g at 2h.…”

“…It is worth noting that structural rearrangement of adduct dihydropyridazine and aromatization, as identified by chromatography, could occur during IEDDA reactions. [137, 139-140] …”