Improved Metabolic Stability for ¹⁸F PET Probes Rapidly Constructed via Tetrazine trans-Cyclooctene Ligation

Abstract

The fast kinetics and bioorthogonal nature of the tetrazine trans-cyclooctene (TCO) ligation makes it a unique tool for PET probe construction. In this study, we report the development of an 18F-labeling system based on a CF3-substituted diphenyl-s-tetrazine derivative with the aim of maintaining high reactivity while increasing in vivo stability. c(RGDyK) was tagged by a CF3-substituted diphenyl-s-tetrazine derivative via EDC-mediated coupling. The resulting tetrazine-RGD conjugate was combined with a 19F-labeled TCO derivative to give HPLC standards. The analogous 18F-labeled TCO derivative was combined with the diphenyl-s-tetrazine-RGD at μM concentration. The resulting tracer was subjected to in vivo metabolic stability assessment, and microPET studies in murine U87MG xenograft models. The diphenyl-s-tetrazine-RGD combines with an 18F-labeled TCO in high yields (>97% decay-corrected on the basis of TCO) using only 4 equiv of tetrazine-RGD relative to the 18F-labeled TCO (concentration calculated based on product’s specific activity). The radiochemical purity of the 18F-RGD peptides was >95% and the specific activity was 111 GBq/μmol. Noninvasive microPET experiments demonstrated that 18F-RGD had integrin-specific tumor uptake in subcutaneous U87MG glioma. In vivo metabolic stability of 18F-RGD in blood, urine and major organs showed two major peaks: one corresponded to the Diels-Alder conjugate and the other was identified as the aromatized analog. A CF3-substituted diphenyl-s-tetrazine displays excellent speed and efficiency in 18F-PET probe construction, providing nearly quantitative 18F labeling within minutes at low micromolar concentrations. The resulting conjugates display improved in vivo metabolic stability relative to our previously described system.

Computationally guided discovery of a reactive, hydrophilic trans-5-oxocene dienophile for bioorthogonal labeling

“…32,33 However, the hydrophobicity of TCO and s-TCO has recently been linked to high levels of non-specific background fluorescence during imaging experiments, necessitating lengthy washout protocols (>2 h) to dissociate the excess reagent from the cell. 34,35 While d-TCO displays reduced lipophilicity, the compound is relatively bulky compared to the parent TCO system.…”

“…From a kinetic standpoint, trans -cyclooctene derivatives are excellent dienophiles for studies where high reactivity is essential such as in cellular imaging and nuclear medicine 32,33. However, the hydrophobicity of TCO and s-TCO has recently been linked to high levels of non-specific background fluorescence during imaging experiments, necessitating lengthy washout protocols (>2 h) to dissociate the excess reagent from the cell 34,35.…”

18F‐Labeling of Sensitive Biomolecules for Positron Emission Tomography

“…developed a fluorine-18 labeling system for PET probe construction based on a CF 3 -substituted diphenyl-s-tetrazine derivative of biomolecules. [136] In this work, c(RGDyK) was tagged by a fluorine-18 labeled TCO via reacting with the tetrazine moiety. The resulting tracer has shown improved metabolic stability.…”

Tetrazine‐Responsive Self‐immolative Linkers

“…Tetrazines have recently been exploited in a variety of inverse electron‐demand Diels–Alder reactions (DA INV ) as a means of conjugating reporters, such as fluorophores and PET isotopes, to a variety of biological entities, such as DNA, targeting peptides and antibodies . Typically, these bioconjugations are performed between a tetrazine and a strained dienophile, such as trans ‐cyclooctene (TCO), thereby resulting in a fast DA INV reaction .…”

“…[14,15,16] Even thought hese triggersa nd materials show huge potentialt here are still major challenges, including problems associatedw ith light-stimulated materials due to high tissuea bsorbance, neurotoxicity forc ommon acrylate and acrylamide-based thermally responsive materials [17] and the lack of tissuespecificity with respect to pH. Te trazines have recently been exploited in av ariety of inverse electron-demand Diels-Alder reactions (DA INV )a sa means of conjugating reporters, such as fluorophores [18,19,20] and PET isotopes, [21,22] to av ariety of biological entities, such as DNA, [23] targeting peptides [24] and antibodies. [25,26] Typically, these bioconjugations are performed between at etrazine and as trained dienophile, such as trans-cyclooctene (TCO), thereby resultingi nafast DA INV reaction.…”