Towards highly strained trans-cyclooctene linkers for rapid bioorthogonal cleavage

Abstract

In the field of bioorthogonal chemistry the trans-cyclooctene (TCO)/ tetrazine (Tz) ligation has established as the state-of-the-art method. In the beginnings the purely click-based method has developed through improvements of the TCO-scaffold. These improvements opened the gateway for a new chemistry, which enables reactions after the initial click step. The foundation was made by the release-TCO (rTCO), which possesses an alcohol functionality at the allylic position. The alcohol can be transformed into a carbamate and upon click reaction with a tetrazine this carbamate moiety can be released.The post-click mechanism was elucidated quite recently and gave insight into a complex equilibrium of different isomers. Only one isomer is able to perform release, while others lead to the formation of non-released dead-end products. Thus, some key requirements have to be fulfilled to enable efficient release. Most importantly, it has been shown that acid-functionalized tetrazines can accelerate the elimination step and improve the overall release yield.However, since the orientation of the initial click reaction cannot be controlled, the overall efficiency is limited. To circumvent this issue, we have recently prepared a C2-symmetric cleavable TCO (c2TCO) with two allylic modifications. This strategy not only allows for efficient release independent of the click orientation, but also deals successfully with the limitations of the rTCO. A possible application would be the concept of turn-off, which is an inactivation of a biological function through release.In the course of the synthesis the metathesis has become the key-step due to several limitations: the disadvantageous thermodynamics due to the ring-size, the unfavorable substitution pattern with the allylic alcohol moieties and the additional strain of the cyclopropane making the metathesis an ambitious endeavor.