Figure ). The equatorial-TCO 16 clearly showed conjugation with tetrazine 19, but unfortunately no release of the PFP moiety. Instead, a stable tautomerized product with the intact PFP carbonate moiety was formed over time (Scheme 4). This rate is in line with previous studies showing that the equatorial isomer has a significant reactivity decrease because of the steric and possible electronic effects, as again confirmed in this study. In case of the diastereomic mixture of the endo-derivatives 17 and 18, we observed a significantly slower click reaction under the same conditions than for the exo-TCOs. Similar to the exo-derivatives, the reaction was followed via F NMR spectroscopy. The different axial and equatorial intermediates and PFP product had an isolated signal which was integrated over time. The endo-axial isomer 17 showed only minor PFP release within 120 minutes and the equatorial isomer 18 primarily afforded the tautomeric product and no PFP release (see: Figure ). Since we observed minor release of PFP with the endo-TCO derivatives 17 and 18, we focused on the exo-isomers, also taking into account that the endo-TCO (13 and 14) was obtained as an inseparable diastereomeric mixture. Besides, the precursor was synthesized in an overall lower yield as compared to the exo-one and an excess of the exo-derivative was formed in the cyclopropanation step (2, 2:1 ratio). Conversely, we showed that the mixture of isomers in 2 can be isomerized solely into the exo-isomer with 1M potassium tert-butoxide in THF and dry ethanol (data not shown). Next, the click kinetics of the axial exo-TCO 7 were investigated upon reaction with a single equivalent of tetrazine 19 or 20. The second-order rate constants were determined in acetonitrile at room temperature. For the click reaction of tetrazine 19 with 7 at 188 μM we found a K2 = 594.0 M -1 s -1 ± 12.38 and for tetrazine 20 K2 = 0.8589 M -1 s -1 ± 0.001, as also expected due to increased electron density of tetrazine 19 (see: Table ). When the click reaction was carried out at 97 μM we encountered similar values. These results seem to demonstrate that our new TCO outperforms the currently available releasing difunctional TCOs in the literature in terms of click kinetics. Next to the carbonate-linked PFP TCOs, we wanted to explore more stable carbamate linked derivatives, as these have already been reported in the literature to provide successful release. We synthesized fluorescently caged exo-TCO derivatives by coupling 7-amino-4methylcoumarin (AMC) via a carbamate linkage to the allylic alcohol on our TCO. With this approach we could directly asses the release properties of our system with straightforward fluorescence measurements, since the characteristic fluorescence of AMC can easily be quenched by modifying the free amine as also demonstrated in similar TCO-AMC release studies in the recent literature. The best synthesis results for the fluorescent TCO Moreover, we also synthesized aliphatic carbamate derivatives of the exo-TCOs to further elucidate the scope of their release properties (Scheme 5). To this extent, we coupled a glycine or sarcosine derivative to the allylic alcohol of the TCO. These compounds were chosen with the work of Carlson and co-workers in mind, who showed that an unmethylated nitrogen as in the glycine derivativecan result in formation of a dead-end product, while an Nmethylated nitrogen as in sarcosine should prevent this. All compounds were generated in a two-step synthesis, wherein hydroxy TCOs (< 3% in 22 hours, Figure ). Nonetheless, the axial analogue 21 afforded complete release of the fluorescent AMC within 23 hours upon ligation with 20, after less than three hours already more than 50% release was observed (kelim = 8.15 × 10 -5 s -1 ). As anticipated, the elimination reaction was heavily depending on the used tetrazine, as for tetrazine 19 the release was significantly slower (10% within 23 hours, kelim = 2.96 × 10 -6 s -1 ), as also confirmed in the recent literature. Hereafter, the stability of the releasing axial TCO was investigated in both PBS and mouse serum at 37 °C. The free carboxylic derivative of 7 dissolved in PBS showed no changes in the 1 H NMR spectrum after seven days (see: Figure ). TCO 7 was stable in mouse serum up to several hours and showed no isomerization into the CCO analogue, however, slow degradation of 7 over time was observed (see: Figure ).