Fluorogenic Bifunctional trans‐Cyclooctenes as Efficient Tools for Investigating Click‐to‐Release Kinetics

Abstract

The inverse electron demand Diels-Alder pyridazine elimination reactionb etween tetrazines and allylic substituted trans-cyclooctenes (TCOs) is ak ey player in bioorthogonal bond cleavage reactions. Determining the rate of eliminationo fa lkylamine substrates has so far provend iffi-cult. Here, we report af luorogenic tool consisting of aT COlinked EDANS fluorophore and aD ABCYL quencher for accurate determination of both the click and releaser ate constants for any tetrazine at physiologically relevant concentrations.

Conditionally Controlling Human TLR2 Activity via Trans-Cyclooctene Caged Ligands

“…These data suggest that elimination of the TCO-cage occurs negligibly fast in human cells. Typical ligation kinetics are on the order of 10 3 M –1 s –1 , and consequent elimination can proceed with a reaction constant of 0.06 s –1 ( t 1/2 = 13 s) at physiological pH. , It should be noted that full recovery of agonist is likely not required for the induction of TLR2 signaling, meaning that the kinetics of NF-κB nuclear translocation may not accurately reflect the true uncaging kinetics. Still, the data illustrate the power of the Tz-induced “click-to-release” reaction for triggering the near-instantaneous activation of TLR2 in cells pretreated with TCO-P 2 TEG, making this (un)­caging strategy extremely powerful for precisely controlling TLR2 activation.…”

“…33,34,36,38 The speed of the reaction drives TCO-elimination in an almost instantaneous "switch-like" manner, which is of crucial importance when investigating the complex kinetics of receptor signaling. 31,32 Here, we set out to develop a C2R-approach for conditionally controlling the activation of TLRs. We focused on TLR2 (Figure 1), because many of its downstream signaling properties remain to be elucidated.…”

“…This is followed by a tautomerization to liberate the carbamate functionality, which quickly decarboxylates to release a free amine functional group and carbon dioxide (Figure ). The above C2R-reaction has gained popularity due to its rapid ligation and elimination kinetics − and has found use in antibody-drug conjugates, , protein activation, , RNA synthesis, and on-demand T-cell activation . Furthermore, tetrazine doses required for inducing this reaction are nontoxic, thus, permitting studies even in live animals. ,,, The speed of the reaction drives TCO-elimination in an almost instantaneous “switch-like” manner, which is of crucial importance when investigating the complex kinetics of receptor signaling. , …”

Readily Accessible Strained Difunctionalized trans‐Cyclooctenes with Fast Click and Release Capabilities**

“… [17] The authors reported the formation of a mixture of the axial and equatorial difunctionalized TCO derivatives after isomerization with UV‐light and a sensitizer as described by the Fox group (Figure 1B). [25] Currently, the synthetic accessibility of the most commonly used difunctionalized TCOs with releasing properties is rather problematic, mainly due to low yielding steps and a lengthy overall synthesis [17,24] . Ideally, a TCO should have multiple orthogonal handles, a fast click reaction with a tetrazine and a rapid release functionality.…”

Readily Accessible Strained Difunctionalized trans-Cyclooctenes with Fast Click and Release Capabilities

“…[22] Currently, the synthetic accessibility of the most commonly used difunctionalized TCOs with releasing properties is rather problematic, mainly due to low yielding steps and a lengthy overall synthesis. [16,23] Ideally, a TCO should have multiple orthogonal handles, a fast click reaction with a tetrazine and a rapid release functionality. In comparison with the difunctionalized TCO of Rossin et al, a shorter and higher yielding synthesis method of a TCO with similar properties is desired.…”