A new bioorthogonal reactant pair, spiro[2.3]hex-1-ene (Sph) and 3,6-di(2-pyridyl)-s-tetrazine (DpTz), for the strain-promoted inverse electron-demand Diels–Alder cycloaddition, that is, tetrazine ligation, is reported. As compared to the previously reported strained alkenes such as trans-cyclooctene (TCO) and 1,3-disubstituted cyclopropene, Sph exhibits balanced reactivity and stability in tetrazine ligation with the protein substrates. A lysine derivative of Sph, SphK, was site-selectively incorporated into the extracellular loop regions (ECLs) of GCGR and GLP-1R, two members of class B G protein-coupled receptors (GPCRs) in mammalian cells with the incorporation efficiency dependent on the location. Subsequent bioorthogonal reactions with the fluorophore-conjugated DpTz reagents afforded the fluorescently labeled GCGR and GLP-1R ECL mutants with labeling yield as high as 68%. A multitude of functional assays were performed with these GPCR mutants, including ligand binding, ligand-induced receptor internalization, and ligand-stimulated intracellular cAMP accumulation. Several positions in the ECL3s of GCGR and GLP-1R were identified that tolerate SphK mutagenesis and subsequent bioorthogonal labeling. The generation of functional, fluorescently labeled ECL3 mutants of GCGR and GLP-1R should allow biophysical studies of conformation dynamics of this important class of GPCRs in their native environment in live cells.

A convenient method for the synthesis of symmetrical azobenzenes is reported. This one-step procedure involves treatment of anilines with N-chlorosuccinimide (NCS) and organic base 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). A wide range of commercially available substituted anilines readily participate in this reaction to produce the corresponding azobenzenes in moderate-to-excellent yields in minutes.

The genetically encoded photo-cross-linkers promise to offer a temporally controlled tool to map transient and dynamic protein–protein interaction complexes in living cells. Here we report the synthesis of a panel of 2-aryl-5-carboxytetrazole-lysine analogs (ACTKs) and their site-specific incorporation into proteins via amber codon suppression in Escherichia coli and mammalian cells. Among five ACTKs investigated, N-methylpyrroletetrazole-lysine (mPyTK) was found to give robust and site-selective photo-cross-linking reactivity in E. coli when placed at an appropriate site at the protein interaction interface. A comparison study indicated that mPyTK exhibits higher photo-cross-linking efficiency than a diazirine-based photo-cross-linker, AbK, when placed at the same location of the interaction interface in vitro. When mPyTK was introduced into the adapter protein Grb2, it enabled the photocapture of EGFR in a stimulus-dependent manner. The design of mPyTK along with the identification of its cognate aminoacyl-tRNA synthetase makes it possible to map transient protein–protein interactions and their interfaces in living cells.

Photoaffinity labels are powerful tools for dissecting ligand–protein interactions, and they have a broad utility in medicinal chemistry and drug discovery. Traditional photoaffinity labels work through nonspecific C–H/X–H bond insertion reactions with the protein of interest by the highly reactive photogenerated intermediate. Herein, we report a new photoaffinity label, 2-aryl-5-carboxytetrazole (ACT), that interacts with the target protein via a unique mechanism in which the photogenerated carboxynitrile imine reacts with a proximal nucleophile near the target active site. In two distinct case studies, we demonstrate that the attachment of ACT to a ligand does not significantly alter the binding affinity and specificity of the parent drug. Compared with diazirine and benzophenone, two commonly used photoaffinity labels, in two case studies ACT showed higher photo-cross-linking yields toward their protein targets in vitro based on mass spectrometry analysis. In the in situ target identification studies, ACT successfully captured the desired targets with an efficiency comparable to the diazirine. We expect that further development of this class of photoaffinity labels will lead to a broad range of applications across target identification, and validation and elucidation of the binding site in drug discovery.

The use of small, natural chemical reporters in conjunction with catalyst-free bioorthogonal reactions will greatly streamline protein labeling in a cellular environment with minimum perturbation to their function. Here we report the discovery of a 2-cyanobenzothiazole (CBT)-reactive peptide tag, CX10R7, from a cysteine-encoded peptide phage library using the phage-assisted interrogation of reactivity method. Fusion of CX10R7 with a protein of interest allows site-specific labeling of the protein with CBT both in vitro and on the surface of E. coli cells. Mutagenesis studies indicated that the reactivity and specificity of CX10R7 are attributed to the sequence environment, in which the residues surrounding cysteine help to stabilize the ligation product.

Incretin-based peptides are effective therapeutics for treating type 2 diabetes mellitus (T2DM). Oxyntomodulin (OXM), a dual agonist of GLP-1R and GCGR, has shown superior weight loss and glucose lowering effects, compared to single GLP-1R agonists. To overcome the short half-life and rapid renal clearance of OXM, which limit its therapeutic potential, both lipid and PEG modified OXM analogs have been reported. However, these approaches often result in reduced potency or PEG-associated toxicity. Herein, we report a new class of cross-linked OXM analogs that show increased plasma stability and higher potency in activating both GLP-1R and GCGR. Moreover, the extended in vivo half-life results in superior antihyperglycemic activity in mice compared to the wild-type OXM.

A series of red-shifted azobenzene amino acids were synthesized in moderate-to-excellent yields via a two-step procedure in which tyrosine derivatives were first oxidized to the corresponding quinonoidal spirolactones followed by ceric ammonium nitrate-catalyzed azo formation with the substituted phenylhydrazines. The resulting azobenzene–alanine derivatives exhibited efficient trans/cis photoswitching upon irradiation with a blue (448 nm) or green (530 nm) LED light. Moreover, nine superfolder green fluorescent protein (sfGFP) mutants carrying the azobenzene–alanine analogues were expressed in E. coli in good yields via amber codon suppression with an orthogonal tRNA/PylRS pair, and one of the mutants showed durable photoswitching with the LED light.

The merging of site-specific incorporation of small bioorthogonal functional groups into proteins via amber codon suppression with bioorthogonal chemistry has created exciting opportunities to extend the power of organic reactions to living systems. Here we show that a new alkyne amino acid can be site-selectively incorporated into mammalian proteins via a known orthogonal pyrrolysyl-tRNA synthetase/tRNACUA pair and directs an unprecedented, palladium-mediated cross-coupling reaction-driven protein labeling on live mammalian cell surface. A comparison study with the alkyne-encoded proteins in vitro indicated that this terminal alkyne is better suited for the palladium-mediated cross-coupling reaction than the copper-catalyzed click chemistry.

Reactive yet stable alkene reporters offer a facile route to studying fast biological processes via the cycloaddition-based bioorthogonal reactions. Here, we report the design and synthesis of a strained spirocyclic alkene, spiro[2.3]hex-1-ene (Sph), for an accelerated photoclick chemistry, and its site-specific introduction into proteins via amber codon suppression using the wild-type pyrrolysyl-tRNA synthetase/tRNACUA pair. Because of its high ring strain and reduced steric hindrance, Sph exhibited fast reaction kinetics (k2 up to 34 000 M–1 s–1) in the photoclick chemistry and afforded rapid (<10 s) bioorthogonal protein labeling.

Here we report the synthesis of storable N-phenylcarbamate palladacycles that showed robust reactivity in the cross-coupling reaction with an alkyne-encoded protein with a second-order rate constant approaching 19770 ± 930 M−1 s−1.

BH3 peptides are key mediators of apoptosis and have served as the lead structures for the development of anticancer therapeutics. Previously, we reported the application of a simple cysteine-based side chain cross-linking chemistry to NoxaBH3 peptides that led to the generation of the cross-linked NoxaBH3 peptides with increased cell permeability and higher inhibitory activity against Mcl-1 (Muppidi, A., Doi, K., Edwardraja, S., Drake, E. J., Gulick, A. M., Wang, H.-G., Lin, Q. (2012) J. Am. Chem. Soc.134, 14734). To deliver cross-linked NoxaBH3 peptides selectively into cancer cells for enhanced efficacy and reduced systemic toxicity, here we report the conjugation of the NoxaBH3 peptides with the extracellular ubiquitin, a recently identified endogenous ligand for CXCR4, a chemokine receptor overexpressed in cancer cells. The resulting ubiquitin-NoxaBH3 peptide conjugates showed increased inhibitory activity against Mcl-1 and selective killing of the CXCR4-expressing cancer cells. The successful delivery of the NoxaBH3 peptides by ubiquitin into cancer cells suggests that the ubiquitin/CXCR4 axis may serve as a general route for the targeted delivery of anticancer agents.

Fast and specific bioorthogonal reactions are highly desirable because they provide efficient tracking of biomolecules that are present in low abundance and/or involved in fast dynamic process in living systems. Toward this end, classic strategy involves the optimization of substrate structures and reaction conditions in test tubes, testing their compatibility with biological systems, devising synthetic biology schemes to introduce the modified substrates into living cells or organisms, and finally validating the superior kinetics for enhanced capacity in tracking biomolecules in vivo—a lengthy process often mired by unexpected results. Here, we report a streamlined approach in which the “microenvironment” of a bioorthogonal chemical reporter is exploited directly in biological systems via phage-assisted interrogation of reactivity (PAIR) to optimize not only reaction kinetics but also specificity. Using the PAIR strategy, we identified a short alkyne-containing peptide sequence showing fast kinetics (k2 = 13 000 ± 2000 M–1 s–1) in a palladium-mediated cross-coupling reaction. Site-directed mutagenesis studies suggested that the residues surrounding the alkyne moiety facilitate the assembly of a key palladium–alkyne intermediate along the reaction pathway. When this peptide sequence was inserted into the extracellular domain of epidermal growth factor receptor (EGFR), this reactive sequence directed the specific labeling of EGFR in live mammalian cells.

Here we report the design and synthesis of a panel of stapled peptides containing a distance-matching biphenyl cross-linker based upon a peptide capsid assembly inhibitor reported previously. Compared with the linear peptide, the biphenyl-stapled peptides exhibited significantly enhanced cell penetration and potent antiviral activity in the cell-based infection assays. Isothermal titration calorimetry and surface plasmon resonance experiments revealed that the most active stapled CAI peptide binds to the C-terminal domain of HIV capsid protein as well as envelop glycoprotein gp120 with low micromolar binding affinities, and as a result, inhibits both the HIV-1 virus entry and the virus assembly.

The tetrazole-based photoclick chemistry has provided a powerful tool to image proteins in live cells. To extend photoclick chemistry to living organisms with improved spatiotemporal control, here we report the design of naphthalene-based tetrazoles that can be efficiently activated by two-photon excitation with a 700 nm femtosecond pulsed laser. A water-soluble, cell-permeable naphthalene-based tetrazole was identified that reacts with acrylamide with the effective two-photon cross-section for the cycloaddition reaction determined to be 3.8 GM. Furthermore, the use of this naphthalene-tetrazole for real-time, spatially controlled imaging of microtubules in live mammalian cells via the fluorogenic, two-photon-triggered photoclick chemistry was demonstrated.

The design and synthesis of a new class of laser light activatable tetrazoles with extended π-systems is reported. Upon 405 nm laser light irradiation, these bithiophene-substituted tetrazoles underwent extremely fast 1,3-dipolar cycloaddition reactions with dimethyl fumarate with second-order rate constants approaching 4000 M–1 s–1. The resulting pyrazoline cycloadducts exhibited solvent-dependent red fluorescence, making these tetrazoles potentially useful as fluorogenic probes for detecting alkenes in vivo.

The use of covalent chemistry to track biomolecules in their native environment—a focus of bioorthogonal chemistry—has received considerable interest recently among chemical biologists and organic chemists alike. To facilitate wider adoption of bioorthogonal chemistry in biomedical research, a central effort in the last few years has been focused on the optimization of a few known bioorthogonal reactions, particularly with respect to reaction kinetics improvement, novel genetic encoding systems, and fluorogenic reactions for bioimaging. During these optimizations, three strategies have emerged, including the use of ring strain for substrate activation in the cycloaddition reactions, the discovery of new ligands and privileged substrates for accelerated metal-catalysed reactions, and the design of substrates with pre-fluorophore structures for rapid “turn-on” fluorescence after selective bioorthogonal reactions. In addition, new bioorthogonal reactions based on either modified or completely unprecedented reactant pairs have been reported. Finally, increasing attention has been directed toward the development of mutually exclusive bioorthogonal reactions and their applications in multiple labeling of a biomolecule in cell culture. In this feature article, we wish to present the recent progress in bioorthogonal reactions through the selected examples that highlight the above-mentioned strategies. Considering increasing sophistication in bioorthogonal chemistry development, we strive to project several exciting opportunities where bioorthogonal chemistry can make a unique contribution to biology in the near future.

A 405 nm light-activatable terthiophene-based tetrazole was designed that reacts with a fumarate dipolarophile with the second-order rate constant k2 exceeding 103 M−1 s−1. The utility of this laser-activatable tetrazole in imaging microtubules in a spatiotemporally controlled manner in live cells was demonstrated.

We report the facile preparation of palladacycles as storable arylpalladium(II) reagents from acetanilides via cyclopalladation. The palladacycles exhibit good stability in PBS buffer and are capable of functionalizing a metabolically encoded HPG-containing protein, thus providing a new type of biocompatible organometallic reagent for selectively functionalizing the alkyne-encoded proteins.

Direct chemical modifications provide a simple and effective means to “translate” bioactive helical peptides into potential therapeutics targeting intracellular protein–protein interactions. We previously showed that distance-matching bisaryl cross-linkers can reinforce peptide helices containing two cysteines at the i and i+7 positions and confer cell permeability to the cross-linked peptides. Here we report the first crystal structure of a biphenyl-cross-linked Noxa peptide in complex with its target Mcl-1 at 2.0 Å resolution. Guided by this structure, we remodeled the surface of this cross-linked peptide through side-chain substitution and N-methylation and obtained a pair of cross-linked peptides with substantially increased helicity, cell permeability, proteolytic stability, and cell-killing activity in Mcl-1-overexpressing U937 cells.

Visualization in biology has been greatly facilitated by the use of fluorescent proteins as in-cell probes. The genes coding for these wavelength-tunable proteins can be readily fused with the DNA coding for a protein of interest, which enables direct monitoring of natural proteins in real time inside living cells. Despite their success, however, fluorescent proteins have limitations that have only begun to be addressed in the past decade through the development of bioorthogonal chemistry. In this approach, a very small bioorthogonal tag is embedded within the basic building blocks of the cell, and then a variety of external molecules can be selectively conjugated to these pretagged biomolecules. The result is a veritable palette of biophysical probes for the researcher to choose from.In this Account, we review our progress in developing a photoinducible, bioorthogonal tetrazole–alkene cycloaddition reaction (“photoclick chemistry”) and applying it to probe protein dynamics and function in live cells. The work described here summarizes the synthesis, structure, and reactivity studies of tetrazoles, including their optimization for applications in biology. Building on key insights from earlier reports, our initial studies of the reaction have revealed full water compatibility, high photoactivation quantum yield, tunable photoactivation wavelength, and broad substrate scope; an added benefit is the formation of fluorescent cycloadducts. Subsequent studies have shown fast reaction kinetics (up to 11.0 M–1 s–1), with the rate depending on the HOMO energy of the nitrile imine dipole as well as the LUMO energy of the alkene dipolarophile. Moreover, through the use of photocrystallography, we have observed that the photogenerated nitrile imine adopts a bent geometry in the solid state. This observation has led to the synthesis of reactive, macrocyclic tetrazoles that contain a short “bridge” between two flanking phenyl rings.This photoclick chemistry has been used to label proteins rapidly (within ∼1 min) both in vitro and in E. coli. To create an effective interface with biology, we have identified both a metabolically incorporable alkene amino acid, homoallylglycine, and a genetically encodable tetrazole amino acid, p-(2-tetrazole)phenylalanine. We demonstrate the utility of these two moieties, respectively, in spatiotemporally controlled imaging of newly synthesized proteins and in site-specific labeling of proteins. Additionally, we demonstrate the use of the photoclick chemistry to perturb the localization of a fluorescent protein in mammalian cells.

Bioorthogonal reactions suitable for functionalization of genetically or metabolically encoded alkynes, for example, copper-catalyzed azide–alkyne cycloaddition reaction (“click chemistry”), have provided chemical tools to study biomolecular dynamics and function in living systems. Despite its prominence in organic synthesis, copper-free Sonogashira cross-coupling reaction suitable for biological applications has not been reported. In this work, we report the discovery of a robust aminopyrimidine–palladium(II) complex for copper-free Sonogashira cross-coupling that enables selective functionalization of a homopropargylglycine (HPG)-encoded ubiquitin protein in aqueous medium. A wide range of aromatic groups including fluorophores and fluorinated aromatic compounds can be readily introduced into the HPG-containing ubiquitin under mild conditions with good to excellent yields. The suitability of this reaction for functionalization of HPG-encoded ubiquitin in Escherichia coli was also demonstrated. The high efficiency of this new catalytic system should greatly enhance the utility of Sonogashira cross-coupling in bioorthogonal chemistry.

Photoactivatable fluorescent probes are invaluable tools for the study of biological processes with high resolution in space and time. Numerous strategies have been developed in generating photoactivatable fluorescent probes, most of which rely on the photo-“uncaging” and photoisomerization reactions. To broaden photoactivation modalities, here we report a new strategy in which the fluorophore is generated in situ through an intramolecular tetrazole-alkene cycloaddition reaction (“photoclick chemistry”). By conjugating a specific microtubule-binding taxoid core to the tetrazole/alkene prefluorophores, robust photoactivatable fluorescent probes were obtained with fast photoactivation (∼1 min) and high fluorescence turn-on ratio (up to 112-fold) in acetonitrile/PBS (1:1). Highly efficient photoactivation of the taxoid–tetrazoles inside the mammalian cells was also observed under a confocal fluorescence microscope when the treated cells were exposed to either a metal halide lamp light passing through a 300/395 filter or a 405 nm laser beam. Furthermore, a spatially controlled fluorescent labeling of microtubules in live CHO cells was demonstrated with a long-wavelength photoactivatable taxoid–tetrazole probe. Because of its modular design and tunability of the photoactivation efficiency and photophysical properties, this intramolecular photoclick reaction based approach should provide a versatile platform for designing photoactivatable fluorescent probes for various biological processes.

We report the design of bisarylmethylene bromides as a new class of rigid, distance-matching cysteine cross-linkers. By cross-linking a peptide dual inhibitor of Mdm2/Mdmx containing cysteines at i,i+7 positions, dramatic enhancement in cell permeability was achieved, along with increased helicity and biological activity.

We report the first synthesis of the C-terminally spermine-conjugated stapled peptide-based inhibitors of the p53-Mdm2 interaction. Subsequent biological, biophysical and cellular uptake assays with the spermine-conjugated stapled peptides revealed that spermine conjugation minimally affects biological activity while significantly increases peptide helicity and cellular uptake without apparent cytotoxicity.

We report the first application of a photoinduced 1,3-dipolar cycloaddition reaction to ‘staple’ a peptide dual inhibitor of the p53-Mdm2/Mdmx interactions. A series of stapled peptide inhibitors were efficiently synthesized and showed excellent dual inhibitory activity in ELISA assay. Furthermore, the positively charged, stapled peptides showed enhanced cellular uptake along with modest in vivo activity.

We report the discovery of two long-wavelength (365 nm) photoactivatable diaryltetrazoles through screening a small library of diaryltetrazoles that were designed using a ‘scaffold hopping’ strategy. A naphthalene-derived tetrazole showed excellent reactivity in the photoinduced cycloaddition reaction with methyl methacrylate under 365 nm photoirradiation in acetonitrile PBS buffer mixture. Besides, the brightly fluorescent pyrazoline cycloadducts that were formed further increase the potential utility of these new diaryltetrazoles as ‘photoclick’ reagents and as reporters in biological studies.

Light-induced chemical reactions exist in nature, regulating many important cellular and organismal functions, e.g., photosensing in prokaryotes and vision formation in mammals. Here, we report the genetic incorporation of a photoreactive unnatural amino acid, p-(2-tetrazole)phenylalanine (p-Tpa), into myoglobin site-specifically in E. coli by evolving an orthogonal tRNA/aminoacyl-tRNA synthetase pair and the use of p-Tpa as a bioorthogonal chemical “handle” for fluorescent labeling of p-Tpa-encoded myoglobin via the photoclick reaction. Moreover, we elucidated the structural basis for the biosynthetic incorporation of p-Tpa into proteins by solving the X-ray structure of p-Tpa-specific aminoacyl-tRNA synthetase in complex with p-Tpa. The genetic encoding of this photoreactive amino acid should make it possible in the future to photoregulate protein function in living systems.

The ability to use covalent chemistry to label biomolecules selectively in their native habitats has greatly enhanced our understanding of biomolecular dynamics and function beyond what is possible with genetic tools alone. To attain the exquisite selectivity that is essential in this covalent approach a “bottom-up” two-step strategy has achieved many successes recently. In this approach, a bioorthogonal chemical functionality is built into life’s basic building blocks—amino acids, nucleosides, lipids, and sugars—as well as coenzymes; after the incorporation, an array of biophysical probes are selectively appended to the tagged biomolecules via a suitable bioorthogonal reaction. While much has been accomplished in the expansion of non-natural building blocks carrying unique chemical moieties, the dearth of robust bioorthogonal reactions has limited both the scope and utility of this promising approach. Here, we summarize the recent progress in the development of bioorthogonal reactions and their applications in various biological systems. A major emphasis has been placed on the mechanistic and kinetic studies of these reactions with the hope that continuous improvements can be made with each reaction in the future. In view of the gap between the capabilities of the current repertoire of bioorthogonal reactions and the unmet needs of outstanding biological problems, we also strive to project the future directions of this rapidly developing field.

We report a new bioorthogonal ligation reaction between p-nitrodiphenylazirine and dimethyl fumarate. This photoinduced azirine–alkene cycloaddition provides a rapid (∼2 min) and highly selective route to protein conjugation at neutral pH and room temperature in biological medium.

The nonsymmetrical spatial distribution of newly synthesized proteins in animal cells plays a central role in many cellular processes. Here, we report that a simple alkene tag, homoallylglycine (HAG), was co-translationally incorporated into a recombinant protein as well as endogenous, newly synthesized proteins in mammalian cells with high efficiency. In conjunction with a photoinduced tetrazole-alkene cycloaddition reaction (“photoclick chemistry”), this alkene tag further served as a bioorthogonal chemical reporter both for the selective protein functionalization in vitro and for a spatiotemporally controlled imaging of the newly synthesized proteins in live mammalian cells. This two-step metabolic alkene tagging-photocontrolled chemical functionalization approach may offer a potentially useful tool to study the role of spatiotemporally regulated protein synthesis in mammalian cells.

We report a chemical lipidation model for the study of protein lipidations in vitro and in live mammalian cells based on a bioorthogonal, photoinduced tetrazole-alkene cycloaddition reaction.

The development of genetically encoded, wavelength-tunable fluorescent proteins has provided a powerful imaging tool to the study of protein dynamics and functions in cellular and organismal biology. However, many biological functions are not directly encoded in the protein primary sequence, e.g., dynamic regulation afforded by protein posttranslational modifications such as phosphorylation. To meet this challenge, an emerging field of bioorthogonal chemistry has promised to offer a versatile strategy to selectively label a biomolecule of interest and track their dynamic regulations in its native habitat. This strategy has been successfully applied to the studies of all classes of biomolecules in living systems, including proteins, nucleic acids, carbohydrates, and lipids. Whereas the incorporation of a bioorthogonal reporter site-selectively into a biomolecule through either genetic or metabolic approaches has been well established, the development of bioorthogonal reactions that allow fast ligation of exogenous chemical probes with the bioorthogonal reporter in living systems remains in its early stage. Here, we review the recent development of bioorthogonal reactions and their applications in various biological systems, with a detailed discussion about our own work—the development of the tetrazole based, photoinducible 1,3-dipolar cycloaddition reaction.

We report the direct observation of a bent geometry for a nonstabilized nitrile imine in a metal-coordination crystal. The photoinduced tetrazole ring rupture to release N2 appears to depend on the size of voids around the N3−N4 bond in the crystal lattice. We further observed the selective formation of the 1,3-addition product when a reactive nitrile imine was photogenerated in water. Overall, the bent nitrile imine geometry agrees with the 1,3-dipolar structure, a transient reactive species that mediates the photoinduced 1,3-dipolar cycloaddition in the aqueous medium.

Six photoreactive tetrazole amino acids were efficiently synthesized either by the de novo Kakehi tetrazole synthesis method or by alkylation of a glycine Schiff base with tetrazole-containing alkyl halides, and four of them showed excellent reactivity toward a simple alkene in the photoinduced 1,3-dipolar cycloaddition reaction in acetonitrile/PBS buffer (1:1) mixture.

We report the first use of a photoinduced 1,3-dipolar cycloaddition reaction in “stapling” peptide sidechains to reinforce a model peptide helical structure with moderate to excellent yields; the resulting pyrazoline “staplers” exhibit unique fluorescence useful in a cell permeability study.

Protein ubiquitination is a widespread protein posttranslational modification in eukaryotes that regulates essentially every aspect of cellular processes. The attachment of ubiquitin to a protein substrate is accomplished through an enzymatic cascade involving the actions of an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3). There are more than 600 E3 ligases estimated to exist in the human genome that regulate the targeting specificity of protein ubiquitination. To understand the dynamic role of protein ubiquitination in biological processes, robust tools need to be developed which can be employed to establish the substrate specificity of each of these E3 ligases. In this report, we show that the ubiquitin carboxyl-terminally derived peptide probes can serve as modest ubiquitin surrogates for the ubiquitination pathway. In the E1-catalyzed probe adenylation assay, peptide probe 3 with a RLRGG recognition sequence exhibited the highest activity, with the kcat/K1/2 determined to be 1.1 × 104 M−1 s−1, roughly 470-fold lower than that of ubiquitin. The rate of transfer from the E1 peptide probe thioesters to E2 showed clear sequence dependency, with peptide probe 4 with an LRLRGG recognition sequence showed the fastest rate (t1/2 = 0.9 min), essentially identical to that of ubiquitin (t1/2 = 0.8 min) under our assay conditions. Furthermore, peptide probes 4 and 8 also exhibited the selective, parkin-mediated labeling of tubulins in a semipurified tubulin−parkin complex. Finally, these carboxyl-terminally derived peptide probes were shown to label the ubiquitination substrates in fraction II of the rabbit reticulocyte lysate with an efficiency parallel to their substrate properties. The selective use of these ubiquitin carboxyl-terminally derived peptide probes by the ubiquitination pathway suggests that perhaps more potent peptide ubiquitination probes based on the ubiquitin C-terminal scaffold can be developed through additional structural optimization.

Here we report the synthesis of storable N-phenylcarbamate palladacycles that showed robust reactivity in the cross-coupling reaction with an alkyne-encoded protein with a second-order rate constant approaching 19770 ± 930 M−1 s−1.

We report a new bioorthogonal ligation reaction between p-nitrodiphenylazirine and dimethyl fumarate. This photoinduced azirine–alkene cycloaddition provides a rapid (∼2 min) and highly selective route to protein conjugation at neutral pH and room temperature in biological medium.

The ability to use covalent chemistry to label biomolecules selectively in their native habitats has greatly enhanced our understanding of biomolecular dynamics and function beyond what is possible with genetic tools alone. To attain the exquisite selectivity that is essential in this covalent approach a “bottom-up” two-step strategy has achieved many successes recently. In this approach, a bioorthogonal chemical functionality is built into life’s basic building blocks—amino acids, nucleosides, lipids, and sugars—as well as coenzymes; after the incorporation, an array of biophysical probes are selectively appended to the tagged biomolecules via a suitable bioorthogonal reaction. While much has been accomplished in the expansion of non-natural building blocks carrying unique chemical moieties, the dearth of robust bioorthogonal reactions has limited both the scope and utility of this promising approach. Here, we summarize the recent progress in the development of bioorthogonal reactions and their applications in various biological systems. A major emphasis has been placed on the mechanistic and kinetic studies of these reactions with the hope that continuous improvements can be made with each reaction in the future. In view of the gap between the capabilities of the current repertoire of bioorthogonal reactions and the unmet needs of outstanding biological problems, we also strive to project the future directions of this rapidly developing field.

The use of covalent chemistry to track biomolecules in their native environment—a focus of bioorthogonal chemistry—has received considerable interest recently among chemical biologists and organic chemists alike. To facilitate wider adoption of bioorthogonal chemistry in biomedical research, a central effort in the last few years has been focused on the optimization of a few known bioorthogonal reactions, particularly with respect to reaction kinetics improvement, novel genetic encoding systems, and fluorogenic reactions for bioimaging. During these optimizations, three strategies have emerged, including the use of ring strain for substrate activation in the cycloaddition reactions, the discovery of new ligands and privileged substrates for accelerated metal-catalysed reactions, and the design of substrates with pre-fluorophore structures for rapid “turn-on” fluorescence after selective bioorthogonal reactions. In addition, new bioorthogonal reactions based on either modified or completely unprecedented reactant pairs have been reported. Finally, increasing attention has been directed toward the development of mutually exclusive bioorthogonal reactions and their applications in multiple labeling of a biomolecule in cell culture. In this feature article, we wish to present the recent progress in bioorthogonal reactions through the selected examples that highlight the above-mentioned strategies. Considering increasing sophistication in bioorthogonal chemistry development, we strive to project several exciting opportunities where bioorthogonal chemistry can make a unique contribution to biology in the near future.

We report the design of bisarylmethylene bromides as a new class of rigid, distance-matching cysteine cross-linkers. By cross-linking a peptide dual inhibitor of Mdm2/Mdmx containing cysteines at i,i+7 positions, dramatic enhancement in cell permeability was achieved, along with increased helicity and biological activity.

We report the facile preparation of palladacycles as storable arylpalladium(II) reagents from acetanilides via cyclopalladation. The palladacycles exhibit good stability in PBS buffer and are capable of functionalizing a metabolically encoded HPG-containing protein, thus providing a new type of biocompatible organometallic reagent for selectively functionalizing the alkyne-encoded proteins.

A 405 nm light-activatable terthiophene-based tetrazole was designed that reacts with a fumarate dipolarophile with the second-order rate constant k2 exceeding 103 M−1 s−1. The utility of this laser-activatable tetrazole in imaging microtubules in a spatiotemporally controlled manner in live cells was demonstrated.