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The allylic trans-cyclooctene (TCO) functionality facilitates powerful control over the spatiotemporal activity of bio-active molecules, enabling precision targeting of druglike and imaging modalities. However, the introduction of this function onto molecules remains chemically challenging, particularly for peptides. Modification with TCOs of this important class of biomolecules remains a challenge, primarily due to the sensitivity of the TCO group to the strong acids typically used in global deprotection during solid phase peptide synthesis. Here, we present a novel synthetic approach to site-selectively introduce TCO-groups in peptides. Our approach utilizes azide groups to mask amine functions, enabling selective introduction of the TCO on a single lysine residue. Staudinger reduction of the azides back to the corresponding amines proceeds without disturbing the sensitive TCO. We show that using our method, we can produce TCO-inactivated antigenic peptides of previously unseen complexity., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Bioorthogonal deprotections are readily used to control biological function in a cell-specific manner. To further improve the spatial resolution of these reactions, we here present a lysosome-targeted tetrazine for an organelle-specific deprotection reaction. We show that trans -cyclooctene deprotection with this reagent can be used to control the biological activity of ligands for invariant natural killer T cells in the lysosome to shed light on the processing pathway in antigen presenting cells. We then use the lysosome-targeted tetrazine to show that long peptide antigens used for CD8 + T cell activation do not pass through this organelle, suggesting a role for the earlier endosomal compartments for their processing.

We report the density functional theory (DFT) guided discovery of ethynyl-triazolyl-phosphinates (ETPs) as a new class of electrophilic warheads for cysteine selective bioconjugation. By using Cu I -catalysed azide alkyne cycloaddition (CuAAC) in aqueous buffer, we were able to access a variety of functional electrophilic building blocks, including proteins, from diethynyl-phosphinate. ETP-reagents were used to obtain fluorescent peptide-conjugates for receptor labelling on live cells and a stable and a biologically active antibody-drug-conjugate. Moreover, we were able to incorporate ETP-electrophiles into an azide-containing ubiquitin under native conditions and demonstrate their potential in protein-protein conjugation. Finally, we showcase the excellent cysteine-selectivity of this new class of electrophile in mass spectrometry based, proteome-wide cysteine profiling, underscoring the applicability in homogeneous bioconjugation strategies to connect two complex biomolecules., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The inverse electron-demand Diels-Alder (IEDDA) pyridazine elimination is one of the key bioorthogonal bond-breaking reactions. In this reaction trans -cyclooctene (TCO) serves as a tetrazine responsive caging moiety for amines, carboxylic acids and alcohols. One issue to date has been the lack of synthetic methods towards TCO ethers from functionalized (aliphatic) alcohols, thereby restricting bioorthogonal utilization. Two novel reagents were developed to enable controlled formation of cis -cyclooctene (CCO) ethers, followed by optimized photochemical isomerization to obtain TCO ethers. The method was exemplified by the controlled bioorthogonal activation of the lac operon system in E. coli using a TCO-ether-modified carbohydrate inducer., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The inverse electron demand Diels-Alder pyridazine elimination reaction between tetrazines and allylic substituted trans-cyclooctenes (TCOs) is a key player in bioorthogonal bond cleavage reactions. Determining the rate of elimination of alkylamine substrates has so far proven difficult. Here, we report a fluorogenic tool consisting of a TCO-linked EDANS fluorophore and a DABCYL quencher for accurate determination of both the click and release rate constants for any tetrazine at physiologically relevant concentrations., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Toll-like receptors (TLRs) are key pathogen sensors of the immune system. Their activation results in the production of cytokines, chemokines, and costimulatory molecules that are crucial for innate and adaptive immune responses. In recent years, specific (sub)-cellular location and timing of TLR activation have emerged as parameters for defining the signaling outcome and magnitude. To study the subtlety of this signaling, we here report a new molecular tool to control the activation of TLR2 via "click-to-release"-chemistry. We conjugated a bioorthogonal trans-cyclooctene (TCO) protecting group via solid support to a critical position within a synthetic TLR2/6 ligand to render the compound unable to initiate signaling. The TCO-group could then be conditionally removed upon addition of a tetrazine, resulting in restored agonist activity and TLR2 activation. This approach was validated on RAW264.7 macrophages and various murine primary immune cells as well as human cell line systems, demonstrating that TCO-caging constitutes a versatile approach for generating chemically controllable TLR2 agonists.

The inverse-electron-demand Diels-Alder/pyridazine elimination tandem reaction, in which the allylic substituent on trans-cyclooctene is eliminated following reaction with tetrazines, is gaining interest as a versatile bioorthogonal process. One potential shortcoming of such currently used reactions is their propensity to proceed faster and more efficiently at lower pH, a feature caused by the nature of the tetrazines used. Here, we present aminoethyl-substituted tetrazines as the first pH-independent reagents showing invariably fast elimination kinetics at all biologically relevant pH values., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Activation of a cytotoxic T-cell is a complex multistep process, and tools to study the molecular events and their dynamics that result in T-cell activation in situ and in vivo are scarce. Here, we report the design and use of conditional epitopes for time-controlled T-cell activation in vivo. We show that trans-cyclooctene-protected SIINFEKL (with the lysine amine masked) is unable to elicit the T-cell response characteristic for the free SIINFEKL epitope. Epitope uncaging by means of an inverse-electron demand Diels-Alder (IEDDA) event restored T-cell activation and provided temporal control of T-cell proliferation in vivo.