Bioorthogonal reactions, including the strain-promoted azide–alkyne cycloaddition (SPAAC) and inverse electron demand Diels–Alder (iEDDA) reactions, have become increasingly popular for live-cell imaging applications. However, the stability and reactivity of reagents has never been systematically explored in the context of a living cell. Here we report a universal, organelle-targetable system based on HaloTag protein technology for directly comparing bioorthogonal reagent reactivity, specificity, and stability using clickable HaloTag ligands in various subcellular compartments. This system enabled a detailed comparison of the bioorthogonal reactions in live cells and informed the selection of optimal reagents and conditions for live-cell imaging studies. We found that the reaction of sTCO with monosubstituted tetrazines is the fastest reaction in cells; however, both reagents have stability issues. To address this, we introduced a new variant of sTCO, Ag-sTCO, which has much improved stability and can be used directly in cells for rapid bioorthogonal reactions with tetrazines. Utilization of Ag complexes of conformationally strained trans-cyclooctenes should greatly expand their usefulness especially when paired with less reactive, more stable tetrazines.

Enantiomerically enriched cyclobutanes are constructed by a three-component process in which t-butyl (E)-2-diazo-5-arylpent-4-enoates are treated with Rh2(S-NTTL)4 to provide enantiomerically enriched bicyclobutanes, which can subsequently engage in homoconjugate addition/enolate trapping sequence to give densely functionalized cyclobutanes with high diastereoselectivity. This three-component, two-catalyst procedure can be carried out in a single flask. Rh2(S-NTTL)4–catalyzed reaction of t-butyl (Z)-2-diazo-5-phenylpent-4-enoate gives the Buchner cyclization product in excellent enantioselectivity.

Since tetrazines are important tools to the field of bioorthogonal chemistry, there is a need for new approaches to synthesize unsymmetrical and 3‐monosubstituted tetrazines. Described here is a general, one‐pot method for converting (3‐methyloxetan‐3‐yl)methyl carboxylic esters into 3‐thiomethyltetrazines. These versatile intermediates were applied to the synthesis of unsymmetrical tetrazines through Pd‐catalyzed cross‐coupling and in the first catalytic thioether reduction to access monosubstituted tetrazines. This method enables the development of new tetrazine compounds possessing a favorable combination of kinetics, small size, and hydrophilicity. It was applied to a broad range of aliphatic and aromatic ester precursors and to the synthesis of heterocycles including BODIPY fluorophores and biotin. In addition, a series of tetrazine probes for monoacylglycerol lipase (MAGL) were synthesized and the most reactive one was applied to the labeling of endogenous MAGL in live cells.

Described is a general method for the installation of a minimal 6-methyltetrazin-3-yl group via the first example of a Ag-mediated Liebeskind–Srogl cross-coupling. The attachment of bioorthogonal tetrazines on complex molecules typically relies on linkers that can negatively impact the physiochemical properties of conjugates. Cross-coupling with arylboronic acids and a new reagent, 3-((p-biphenyl-4-ylmethyl)­thio)-6-methyltetrazine (b-Tz), proceeds under mild, PdCl2(dppf)-catalyzed conditions to introduce minimal, linker-free tetrazine functionality. Safety considerations guided our design of b-Tz which can be prepared on decagram scale without handling hydrazine and without forming volatile, high-nitrogen tetrazine byproducts. Replacing conventional Cu­(I) salts used in Liebeskind–Srogl cross-coupling with a Ag2O mediator resulted in higher yields across a broad library of aryl and heteroaryl boronic acids and provides improved access to a fluorogenic tetrazine-BODIPY conjugate. A covalent probe for MAGL incorporating 6-methyltetrazinyl functionality was synthesized in high yield and labeled endogenous MAGL in live cells. This new Ag-mediated cross-coupling method using b-Tz is anticipated to find additional applications for directly introducing the tetrazine subunit to complex substrates.

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