Inverse electron demand Diels–Alder reactions in chemical biology

Abstract

The emerging inverse electron demand Diels-Alder (IEDDA) reaction stands out from other bioorthogonal reactions by virtue of its unmatchable kinetics, excellent orthogonality and biocompatibility. With the recent discovery of novel dienophiles and optimal tetrazine coupling partners, attention has now been turned to the use of IEDDA approaches in basic biology, imaging and therapeutics. Here we review this bioorthogonal reaction and its promising applications for live cell and animal studies. We first discuss the key factors that contribute to the fast IEDDA kinetics and describe the most recent advances in the synthesis of tetrazine and dienophile coupling partners. Both coupling partners have been incorporated into proteins for tracking and imaging by use of fluorogenic tetrazines that become strongly fluorescent upon reaction. Selected notable examples of such applications are presented. The exceptional fast kinetics of this catalyst-free reaction, even using low concentrations of coupling partners, make it amenable for in vivo radiolabelling using pretargeting methodologies, which are also discussed. Finally, IEDDA reactions have recently found use in bioorthogonal decaging to activate proteins or drugs in gain-of-function strategies. We conclude by showing applications of the IEDDA reaction in the construction of biomaterials that are used for drug delivery and multimodal imaging, among others. The use and utility of the IEDDA reaction is interdisciplinary and promises to revolutionize chemical biology, radiochemistry and materials science.

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging

“…27,31–33 The recent success of IEDDA reactions for in vivo pretargeting and other chemical biology applications has been reviewed by several groups. 12,34–37 Most examples incorporate TCO on the slower clearing molecule followed by a fast clearing radiolabeled tetrazine tracer, but some groups have explored reversing the chemistry. 38,39 …”

Cycloadditions of Trans‐Cyclooctenes and Nitrones as Tools for Bioorthogonal Labelling

“…The iEDDA reaction is driven by the electrophilicity of the tetrazine, coupled with the high exothermicity of the reaction with the strained alkene . Faster kinetics can be achieved by increasing the strain of the TCO by adding cis ‐cyclopropane or increasing the electron deficiency of the tetrazine . However, the biological potential of this reaction is limited by size, aqueous stability and ease of synthesis of both the tetrazine and TCO, thus underscoring the importance of accessibility and practicality as equally important considerations to that of speed (Scheme ).…”

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

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In addition to its use for the study of biomolecules in living systems, bioorthogonal chemistry has emerged as ap romising strategyt oe nable protein or drug activation in as patially and temporally controlled manner.T his study demonstrates the application of ab ioorthogonal inverse electron-demand Diels-Alder (iEDDA) reaction to cleave trans-cyclooctene (TCO) and vinyl protecting groups from carboxylic acid-containing molecules. [9,10] Of these, the iEDDA reactionb etween transcyclooctene (TCO) and at etrazine is one of the more selective and fastestb ioorthogonal reactions to date. The anti-inflammatory activity of ketoprofen was successfully reinstated after decaging of the nontoxic TCOprodrug in live macrophages.

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“…[9,10] Of these, the iEDDA reactionb etween transcyclooctene (TCO) and at etrazine is one of the more selective and fastestb ioorthogonal reactions to date. [10] Robillard'sg roup reported the first use of the TCO-tetrazine reaction for bioorthogonal decaging to release amine-containing drugs ( Figure 1A), in which they demonstrated the release of doxorubicin (Dox) from aT CO carbamate prodrug in vitro. [12][13][14] Recently,b ioorthogonal cleavage reactions have emerged as promising strategies to control the activation of caged proteins,f luorophores, and small-molecule drugs in living systems.…”

“…[15] The TCO-tetrazine iEDDA ligation can be re-engineered into ac leavage reaction by placing a leaving group at the allylic positiono fT CO. After the initial cycloaddition and elimination of nitrogen, the 4,5-dihydropyridazine now contains an appropriatelyp laced substituent that eliminates upon tautomerisation. [10] Robillard'sg roup reported the first use of the TCO-tetrazine reaction for bioorthogonal decaging to release amine-containing drugs ( Figure 1A), in which they demonstrated the release of doxorubicin (Dox) from aT CO carbamate prodrug in vitro. [16] They then applied this "click-to-release" strategy to successfullytrigger the release of Dox and monomethyl auristatin E( MMAE) from an antibody-drug conjugate (ADC).…”