Computation was used to design a trans-cyclooctene derivative that displays enhanced reactivity in the tetrazine-trans-cycloctene ligation. The optimized derivative is a (E)-bicyclo[6.1.0]non-4-ene with a cis-ring fusion, in which the eight-membered ring is forced to adopt a highly strained ‘half-chair’ conformation. Toward 3,6-dipyridyl-s-tetrazine in MeOH at 25 °C, the strained derivative is 19 and 27 times more reactive than the parent trans-cyclooctene and 4E-cyclooct-4-enol, respectively. Toward 3,6-diphenyl-s-tetrazine in MeOH at 25 °C, the strained derivative is 160 times more reactive than the parent trans-cyclooctene.

The inverse-electron-demand Diels-Alder cycloaddition between trans-cyclooctenes and tetrazines is biocompatible and exceptionally fast. We utilized this chemistry for site-specific fluorescence labeling of proteins on the cell surface and inside living mammalian cells by a two-step protocol. E. coli lipoic acid ligase site-specifically ligates a trans-cyclooctene derivative onto a protein of interest in the first step, followed by chemoselective derivatization with a tetrazinefluorophore conjugate in the second step. On the cell surface, this labeling was fluorogenic and highly sensitive. Inside the cell, we achieved specific labeling of cytoskeletal proteins with green and red fluorophores. By incorporating the Diels-Alder cycloaddition, we have broadened the panel of fluorophores that can be targeted by lipoic acid ligase.

Bioorthogonal ligation methods with improved reaction rates and less obtrusive components are needed for site-specifically labeling proteins without catalysts. Currently no general method exists for in vivo site-specific labeling of proteins that combines fast reaction rate with stable, nontoxic and chemoselective reagents. To overcome these limitations we have developed a tetrazine-containing amino acid, 1, that is stable inside living cells. We have site-specifically genetically encoded this unique amino acid in response to an amber codon allowing a single 1 to be placed at any location in a protein. We have demonstrated that protein containing 1 can be ligated to a conformationally strained trans-cyclooctene, 2, in vitro and in vivo with reaction rates significantly faster than most commonly used labeling methods.

Nature displays a remarkable ability to carry out site-selective post-translational modification of proteins, therefore enabling a dramatic increase in their functional diversity 1 . Inspired by this, chemical tools have evolved for the synthetic manipulation of protein structure and function, and have become essential to the continued advancement of chemical biology, molecular biology and medicine. However, the number of chemical transformations suitable for effective protein functionalization is limited because the stringent demands inherent to biological systems preclude the applicability of many potential processes 2 . Put simply, these chemical transformations often need to be selective at a single site on a protein, proceed with very fast reaction rates, operate under biologically ambient conditions and should provide homogeneous products with near perfect conversion 2 – 7 . While many elegant bioconjugation methods exist at cysteine, lysine and tyrosine, we reasoned that a method targeting a less explored amino acid would significantly expand the protein functionalization toolbox. Herein, we report the development of a multifaceted-approach to protein functionalization based on chemoselective labelling at methionine residues. By exploiting the unique electrophilic reactivity of a bespoke hypervalent iodine reagent, one can target the S -Me group in the side-chain of methionine. The bioconjugation reaction is fast, selective, operates at low µM concentrations and is complementary to existing bioconjugation strategies. Moreover, the new reaction produces a protein conjugate that is, itself, a high energy intermediate with reactive properties that can serve as a platform for the development of secondary, visible-light mediated bioorthogonal protein functionalization processes. Taken together, the merger of these approaches provides a versatile platform for the development of distinct transformations that can deliver versatile, information-rich protein conjugates directly from the native biomacromolecules.

Copper-catalyzed cascade reactions between alkenes or alkynes and diaryliodonium salts form carbocyclic products in a single step. Arylation of the unsaturated functional group is proposed to form a carbocation intermediate that facilitates hydride shift pathways to translocate the positive charge to a remote position and enables ring formation via a Friedel-Crafts-type reaction.