7 Tetrazine-Based Cycloadditions in Click Chemistry

The spontaneous cycloaddition of tetrazines with a number of different dienophiles has become a powerful tool in chemical biology, in particular for the biocompatible conjugation and modification of (bio)molecules. The exceptional reaction kinetics made these bioorthogonal ligations the methods of choice for time-critical processes at very low concentrations, facilitating controlled molecular transformations in complex environments and even in vivo. The emerging concept of bond-cleavage reactions triggered by tetrazine-based cycloadditions enabled the design of diagnostic and therapeutic strategies. The tetrazine-triggered activation of prodrugs represents the first bioorthogonal reaction performed in humans, marking the beginning of the era of clinical translation of bioorthogonal chemistry. This chapter provides an overview of the synthesis and reactivity of tetrazines, their cycloadditions with various dienophiles, and transformations triggered by these reactions, focusing on reaction mechanisms, kinetics and efficiency, and selected applications.

A Genetically Encoded Picolyl Azide for Improved Live Cell Copper Click Labeling

Bioorthogonal chemistry allows rapid and highly selective reactivity in biological environments. The copper-catalyzed azide–alkyne cycloaddition (CuAAC) is a classic bioorthogonal reaction routinely used to modify azides or alkynes that have been introduced into biomolecules. Amber suppression is an efficient method for incorporating such chemical handles into proteins on the ribosome, in which noncanonical amino acids (ncAAs) are site specifically introduced into the polypeptide in response to an amber (UAG) stop codon. A variety of ncAA structures containing azides or alkynes have been proven useful for performing CuAAC chemistry on proteins. To improve CuAAC efficiency, biologically incorporated alkyne groups can be reacted with azide substrates that contain copper-chelating groups. However, the direct incorporation of copper-chelating azides into proteins has not been explored. To remedy this, we prepared the ncAA paz-lysine (PazK), which contains a picolyl azide motif. We show that PazK is efficiently incorporated into proteins by amber suppression in mammalian cells. Furthermore, PazK-labeled proteins show improved reactivity with alkyne reagents in CuAAC.

Artificial metalloenzymes in a nutshell: the quartet for efficient catalysis

Artificial metalloenzymes combine the inherent reactivity of transition metal catalysis with the sophisticated reaction control of natural enzymes. By providing new opportunities in bioorthogonal chemistry and biocatalysis, artificial metalloenzymes have the potential to overcome certain limitations in both drug discovery and green chemistry or related research fields. Ongoing advances in organometallic catalysis, directed evolution, and bioinformatics are enabling the design of increasingly powerful systems that outperform conventional catalysis in a growing number of cases. Therefore, this review article collects challenges and opportunities in designing artificial metalloenzymes described in recent review articles. This will provide an equitable insight for those new to and interested in the field.

Catalytic Activation of Bioorthogonal Chemistry with Light (CABL) Enables Rapid, Spatiotemporally-controlled Labeling and No-Wash, Subcellular 3D-Patterning in Live Cells using Long Wavelength Light

Described is the spatiotemporally controlled labeling and patterning of biomolecules in live cells through the catalytic activation of bioorthogonal chemistry with light, referred to as “CABL”. Here, an unreactive dihydrotetrazine (DHTz) is photocatalytically oxidized in the intracellular environment by ambient O2 to produce a tetrazine that immediately reacts with a trans-cyclooctene (TCO) dieno-phile. 6-(2-Pyridyl)-dihydrotetrazine-3-carboxamides were developed as stable, cell permeable DHTz reagents that upon oxidation pro-duce the most reactive tetrazines ever used in live cells with Diels-Alder kinetics exceeding k2 106 M-1s-1. CABL photocatalysts are based on fluorescein or silarhodamine dyes with activation at 470 or 660 nm. Strategies for limiting extracellular production of singlet oxygen are described that increase the cytocompatibility of photocatalysis. The HaloTag self-labeling platform was used to introduce DHTz tags to proteins localized in the nucleus, mitochondria, actin or cytoplasm, and high-yielding subcellular activation and labeling with a TCO-fluorophore was demonstrated. CABL is light-dose dependent, and 2-photon excitation promotes CABL at the sub-organelle level to selectively pattern live cells under no-wash conditions. CABL was also applied to spatially resolved live-cell labeling of an endogenous pro-tein target by using TIRF microscopy to selectively activate intracellular monoacylglycerol lipase tagged with DHTz-labeled small mole-cule covalent inhibitor. Beyond spatiotemporally controlled labeling, CABL also improves the efficiency of ‘ordinary’ tetrazine ligations by rescuing the reactivity of commonly used 3-aryl-6-methyltetrazine reporters that become partially reduced to DHTzs inside cells. The spatiotemporal control and fast rates of photoactivation and labeling of CABL should enable a range of biomolecular labeling applications in living systems.

Tuning Reactivity of Micellar Nanoreactors by Precise Adjustments of the Amphiphiles and Substrates Hydrophobicity

<p>Polymeric assemblies, such as micelles, are gaining increasing attention due to their ability to serve as nanoreactors for the execution of organic reactions in aqueous media. The ability to conduct transformations, which have been limited to organic media, in water is essential for the further development of the important fields of green</p><p>catalysis and bioorthogonal chemistry, among other fields. In light of the recent progress in the expanding the scopes of reactions that can be conducted using nanoreactors, we aimed to gain deeper understanding of the roles of the hydrophobicity of both the core of micellar nanoreactors and the substrates on the reaction rates in water. Towards this goal we designed a set of metal-loaded micelles, composed of PEG-dendron amphiphiles and studied their ability to serve as nanoreactors for a palladium mediated depropargylation reaction of four substrates with different LogP values. Using dendrons as the hydrophobic block, allowed us to fine tune the lipophilicity of the dendritic end-groups and study how precise structural changes in the hydrophobicity of the amphiphiles affect the reaction rates. The kinetic data revealed linear relations between the rate constants and the hydrophobicity of the amphiphiles (estimated by the dendron’s</p><p>cLogP), while exponential dependence was obtained for the lipophilicity of the substrates (estimated by their LogP values). Our results demonstrate the vital contributions of the hydrophobicity of both the substrates and amphiphiles on the lipo-selectivity of nanoreactors, illustrating the potential of tuning hydrophobicity as a tool for optimizing</p><p>the reactivity and selectivity of nanoreactors.</p>