Click‐to‐Release from trans‐Cyclooctenes: Mechanistic Insights and Expansion of Scope from Established Carbamate to Remarkable Ether Cleavage

Abstract

The bioorthogonal cleavage of allylic carbamates from trans-cyclooctene (TCO) upon reaction with tetrazine is widely used to release amines. We disclose herein that this reaction can also cleave TCO esters, carbonates, and surprisingly, ethers. Mechanistic studies demonstrated that the elimination is mainly governed by the formation of the rapidly eliminating 1,4-dihydropyridazine tautomer, and less by the nature of the leaving group. In contrast to the widely used p-aminobenzyloxy linker, which affords cleavage of aromatic but not of aliphatic ethers, the aromatic, benzylic, and aliphatic TCO ethers were cleaved as efficiently as the carbamate, carbonate, and esters. Bioorthogonal ether release was demonstrated by the rapid uncaging of TCO-masked tyrosine in serum, followed by oxidation by tyrosinase. Finally, tyrosine uncaging was used to chemically control cell growth in tyrosine-free medium.

Innovative Linker Strategies for Tumor‐Targeted Drug Conjugates

“…The crucial steps consist in the first cycloaddition to a 4,5‐dihydropyridazine intermediate ( 59 ), followed by conversion to the 2,5‐ and 1,4‐tautomers (compounds 60 and 61 ). Among these, only the latter can undergo the subsequent electronic cascade, allowing the release of different functional groups (carbamic acids, carboxylic acids, phenols and alcohols) . Interestingly, fast click reaction does not necessarily lead to highly efficient drug release.…”

“… A) Reaction mechanism of “click and release” strategy with the TCO‐Tetrazine pair. Substituent effects of the fundamental IEDDA and tautomerization steps are indicated, together with the leaving groups that have been described so far . B) Molecular structure of the TCO‐Tetrazine pair used for targeted delivery of MMAE in tumor‐bearing mice: the drug is connected to a monoclonal antibody in diabody format through a TCO linker (ADC 65 ) and released by reaction with tetrazine 66 , 48 h post ADC injection.…”

The Emerging Role of Tetrazines in Drug‐Activation Chemistries

“…This work demonstrated that TCO‐based prodrug systems offer a new approach for controlled drug release by targeting non‐internalising receptors; although the activation was relatively slow, this could well be beneficial for controlled and longer‐term dosing . Recently, the authors showed that this TCO‐based release mechanism could also be used for masking esters and alcohols, while Pluth reported a thiocarbamate linkage that allowed the release of carbonyl sulfide as a gastrotransmitter . Finally, by linking tetrazines to an alginate‐containing hydrogel, TCO–doxorubicin conjugates were successfully activated in a mouse model .…”

Tetrazine‐Triggered Release of Carboxylic‐Acid‐Containing Molecules for Activation of an Anti‐inflammatory Drug

“…[19] Al imitation of the click-toreleases trategy is the need for delivery,a nd therefore optimisation of the pharmacokinetic properties, of both the prodrug and the tetrazine. [25] Previous examples of drug releaseh ave so far been limited to the release of amine-( Figure 1A)o ra lcohol-containing (Figure 1B,C )a nticancer drugs.T hese groups, although often found in small-molecule drugs, are not alwaysp resent and might notb ev ital for the function of the drug;t his means that chemical modification at this site to form ap rodrug does not lead to reduced activity.F or this reason, it is important to [a] S. Our group [22] and the groups of Bradley [23] and Devaraj [24] independently reported using the vinyl ether protecting group, which could be cleaved by reaction with tetrazines to release alcohols ( Figure 1B).…”

“…Our group [22] and the groups of Bradley [23] and Devaraj [24] independently reported using the vinyl ether protecting group, which could be cleaved by reaction with tetrazines to release alcohols ( Figure 1B). [25] Previous examples of drug releaseh ave so far been limited to the release of amine-( Figure 1A)o ra lcohol-containing (Figure 1B,C )a nticancer drugs.T hese groups, although often found in small-molecule drugs, are not alwaysp resent and might notb ev ital for the function of the drug;t his means that chemical modification at this site to form ap rodrug does not lead to reduced activity.F or this reason, it is important to [ expand the scope of bioorthogonal decaging reactions to include other functional groups. In addition, Robillard recently reported bioorthogonal cleavage of ethers, carbonates and esters from TCO to release alcohols ( Figure 1C)o rc arboxylic acids ( Figure 1D), respectively.H owever,t he reported TCO-protected carboxylic acids proved highlyu nstable ( % 90 %f ragmentation in 50 %m ouse serum at 37 8C).…”