17290994
Structural Determinants of Alkyne Reactivity in Copper-Catalyzed Azide-Alkyne Cycloadditions
This work represents our initial effort in identifying azide/alkyne pairs for optimal reactivity in copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions. In previous works, we have identified chelating azides, in particular 2-picolyl azide, as “privileged” azide substrates with high CuAAC reactivity. In the current work, two types of alkynes are shown to undergo rapid CuAAC reactions under both copper(II)- (via an induction period) and copper(I)-catalyzed conditions. The first type of the alkynes bears relatively acidic ethynyl C-H bonds, while the second type contains an N-(triazolylmethyl)propargylic moiety that produces a self-accelerating effect. The rankings of reactivity under both copper(II)- and copper(I)-catalyzed conditions are provided. The observations on how other reaction parameters such as accelerating ligand, reducing agent, or identity of azide alter the relative reactivity of alkynes are described and, to the best of our ability, explained.
11957672
Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides.
The cycloaddition of azides to alkynes is one of the most important synthetic routes to 1H-[1,2,3]-triazoles. Here a novel regiospecific copper(I)-catalyzed 1,3-dipolar cycloaddition of terminal alkynes to azides on solid-phase is reported. Primary, secondary, and tertiary alkyl azides, aryl azides, and an azido sugar were used successfully in the copper(I)-catalyzed cycloaddition producing diversely 1,4-substituted [1,2,3]-triazoles in peptide backbones or side chains. The reaction conditions were fully compatible with solid-phase peptide synthesis on polar supports. The copper(I) catalysis is mild and efficient (>95% conversion and purity in most cases) and furthermore, the X-ray structure of 2-azido-2-methylpropanoic acid has been solved, to yield structural information on the 1,3-dipoles entering the reaction. Novel Fmoc-protected amino azides derived from Fmoc-amino alcohols were prepared by the Mitsunobu reaction.
205840987
Bioorthogonal chemistry: strategies and recent developments.
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.
10702166
Transition metal-mediated bioorthogonal protein chemistry in living cells.
Considerable attention has been focused on improving the biocompatibility of Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), a hallmark of bioorthogonal reaction, in living cells. Besides creating copper-free versions of click chemistry such as strain promoted azide-alkyne cycloaddition (SPAAC), a central effort has also been made to develop various Cu(I) ligands that can prevent the cytotoxicity of Cu(I) ions while accelerating the CuAAC reaction. Meanwhile, additional transition metals such as palladium have been explored as alternative sources to promote a bioorthogonal conjugation reaction on cell surface, as well as within an intracellular environment. Furthermore, transition metal mediated chemical conversions beyond conjugation have also been utilized to manipulate protein activity within living systems. We highlight these emerging examples that significantly enriched our protein chemistry toolkit, which will likely expand our view on the definition and applications of bioorthogonal chemistry.
6368335
Bioorthogonal reactions for labeling proteins.
O the past 15 years a great deal of progress has been made on the discovery, rediscovery, and invention of bioorthogonal reactions between functional groups that do not react with biological entities under physiological conditions but selectively react with each other. Strategies for labeling different classes of biomolecules have been developed by coopting the biosynthetic machinery of cells to introduce molecules containing bioorthogonal functional groups. Tagging approaches have allowed some additional functional groups to be attached to proteins, and genetic code expansion and reprogramming have facilitated the site-specific incorporation of unnatural amino acids bearing bioorthogonal functional groups into proteins in bacteria, mammalian cells, and animals via the discovery and synthetic evolution of orthogonal aminoacyl-tRNA synthetase/tRNA pairs and orthogonal ribosomes. In addition, selective pressure incorporation and its derivatives have allowed the statistical labeling of proteins and proteomes with analogues of natural amino acids. The incorporation of unnatural amino acids bearing bioorthogonal functional groups and their chemoselective labeling has great potential for imaging and controlling individual proteins and labeling proteomes, but the ability of investigators to leverage these approaches for biological discovery will be crucially dependent on the properties of the chemical reactions used. The reactants in a bioorthogonal reaction should be kinetically, thermodynamically, and metabolically stable before the reaction takes place and not toxic to living systems. The reaction should yield stable covalent linkages with no or innocuous byproducts. Moreover, the two bioorthogonal moieties have to react selectively with each other under physiological conditions (ambient temperature and pressure, neutral pH, aqueous conditions), without either of them crossreacting with the plethora of chemical functionalities found in living cells. Despite the challenges of meeting these criteria, a number of reactions have been developed that show good biocompatibility and selectivity in living systems (see Figure 1). Some of these reactions are chemoselective with respect to many but not all biological functionalities and have been used to label proteins in vitro and on the cell surface, while other reactions have additionally been used for the more challenging task of labeling proteins inside cells or living animals. Most bioorthogonal reactions follow second-order kinetics, and their rates depend directly on the concentrations of both reaction partners as well as on the intrinsic second-order rate constant k2 [M −1 s−1] of the reaction. Rapid reactions with high second-order rate constants are therefore advantageous for labeling during biological processes that occur on a very short time scale or for the labeling of low abundance proteins. Lower abundance proteins can sometimes be labeled with a large excess of labeling reagent, but this strategy may be practically limited by solubility, off target reactions and toxicity. Bioorthogonal reactions for which one partner can be installed into proteins are summarized in Figure 1. Their second-order rate constants span 9 orders of magnitude with the fastest bioorthogonal labeling reactions reaching rates up to 10 M−1 s−1, which approaches the rate constants for many enzymatic labeling approaches. Here we briefly introduce the bioorthogonal chemistries used for labeling proteins and comment on their utility for protein labeling before providing a perspective on future directions. Amongst the first functionalities to be explored as bioorthogonal reporters were ketones and aldehydes. Under acidic conditions (pH 4−6) their carbonyl groups react with strong α-effect nucleophiles such as hydrazines and alkoxyamines. Ketone/aldehyde condensations show rather slow kinetics with second-order rate constants in the range of 10−4 to 10−3 M−1 s−1, necessitating high concentrations of labeling reagent in order to achieve good labeling, which might be problematic in terms of toxicity and background signal. In general ketone/aldehyde condensations are best suited for in vitro or cell-surface labeling, because the reaction requires an acidic pH, which is difficult to obtain inside most cellular compartments. Furthermore, inside living cells, α-effect nucleophiles may undergo side-reactions with carbonyl-bearing metabolites. A functionality that is essentially absent from biological systems and truly orthogonal in its reactivity to the majority of biological functionalities is the azide group. Azide-bearing unnatural amino acids have been incorporated into proteins and used in a variety of chemical reactions. One potential limitation of the use of azides for protein labeling is that some unnatural amino acids bearing azides appear to be reduced in some proteins examined. Azide-modified proteins have been reacted with phosphines in Staudinger ligations. This reaction has been used to label biomolecules in living cells and animals. The Staudinger ligation, however, has slow kinetics: the reaction proceeds with second-order rate constants in the low 10−3 M−1 s−1 range. In addition many of the phosphine reagents are oxidized by air or metabolic enzymes. Azides can also react with terminal alkynes in [3 + 2] cycloadditions, catalyzed by Cu salts. The CuAAC (Cucatalyzed alkyne−azide cycloaddition) reaction proceeds considerably faster than the Staudinger ligation in physiological settings. However, its reliance on the Cu catalyst is not without problems, since Cu may be toxic to living systems, and decreasing the copper concentration is generally accompanied by a large decrease in reaction rate. The development of tailored water-soluble Cu ligands and/or
8892285
Glycans in pathogenic bacteria--potential for targeted covalent therapeutics and imaging agents.
A substantial obstacle to the existing treatment of bacterial diseases is the lack of specific probes that can be used to diagnose and treat pathogenic bacteria in a selective manner while leaving the microbiome largely intact. To tackle this problem, there is an urgent need to develop pathogen-specific therapeutics and diagnostics. Here, we describe recent evidence that indicates distinctive glycans found exclusively on pathogenic bacteria could form the basis of targeted therapeutic and diagnostic strategies. In particular, we highlight the use of metabolic oligosaccharide engineering to covalently deliver therapeutics and imaging agents to bacterial glycans.
23919852
Click chemistry in complex mixtures: bioorthogonal bioconjugation.
The selective chemical modification of biological molecules drives a good portion of modern drug development and fundamental biological research. While a few early examples of reactions that engage amine and thiol groups on proteins helped establish the value of such processes, the development of reactions that avoid most biological molecules so as to achieve selectivity in desired bond-forming events has revolutionized the field. We provide an update on recent developments in bioorthogonal chemistry that highlights key advances in reaction rates, biocompatibility, and applications. While not exhaustive, we hope this summary allows the reader to appreciate the rich continuing development of good chemistry that operates in the biological setting.
30215037
Finding the right (bioorthogonal) chemistry.
Bioorthogonal chemistries can be used to tag diverse classes of biomolecules in cells and other complex environments. With over 20 unique transformations now available, though, selecting an appropriate reaction for a given experiment is challenging. In this article, we compare and contrast the most common classes of bioorthogonal chemistries and provide a framework for matching the reactions with downstream applications. We also discuss ongoing efforts to identify novel biocompatible reactions and methods to control their reactivity. The continued expansion of the bioorthogonal toolkit will provide new insights into biomolecule networks and functions and thus refine our understanding of living systems.
206900939
Advances in chemical protein modification.
O.B. thanks the European Commission (Marie Curie CIG) and Ministerio de Ciencia e Innovacion, Spain (Juan de la Cierva Fellowship). G.J.L.B. thanks his generous sources of funding: Royal Society, FCT Portugal (FCT Investigator), European Commission (Marie Curie CIG), and the EPSRC. G.J.L.B. is a Royal Society University Research Fellow. The authors thank Paula Boutureira Regla and Francisco Pinteus da Cruz Lopes Bernardes for inspiration.
35112198
Orthogonal bioorthogonal chemistries.
Bioorthogonal reactions have long been used to examine individual biomolecules in living systems. Studies of multi-component networks demand not only reliable bioorthogonal chemistries, but also combinations of reactions that can be used in tandem. Such 'orthogonal bioorthogonal' transformations have been reported in recent years, and these chemistries are enabling new explorations into biology. This article highlights the development of orthogonal bioorthogonal reactions and their application in multi-target imaging and macromolecule assembly. Methods to tune and control orthogonal reactivity are also discussed, along with prospects for identifying new classes of compatible reactions.
43701807
Chemically Modifying Viruses for Diverse Applications.
Long fascinating to biologists, viruses offer nanometer-scale benchtops for building molecular-scale devices and materials. Viruses tolerate a wide range of chemical modifications including reaction conditions, pH values, and temperatures. Recent examples of nongenetic manipulation of viral surfaces have extended viruses into applications ranging from biomedical imaging, drug delivery, tissue regeneration, and biosensors to materials for catalysis and energy generation. Chemical reactions on the phage surface include both covalent and noncovalent modifications, including some applied in conjunction with genetic modifications. Here, we survey viruses chemically augmented with capabilities limited only by imagination.
35758761
Cellular consequences of copper complexes used to catalyze bioorthogonal click reactions.
Copper toxicity is a critical issue in the development of copper-based catalysts for copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions for applications in living systems. The effects and related toxicity of copper on mammalian cells are dependent on the ligand environment. Copper complexes can be highly toxic, can induce changes in cellular metabolism, and can be rapidly taken up by cells, all of which can affect their ability to function as catalysts for CuAAC in living systems. Herein, we have evaluated the effects of a number of copper complexes that are typically used to catalyze CuAAC reactions on four human cell lines by measuring mitochondrial activity based on the metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to study toxicity, inductively coupled plasma mass spectrometry to study cellular uptake, and coherent anti-Stokes Raman scattering (CARS) microscopy to study effects on lipid metabolism. We find that ligand environment around copper influences all three parameters. Interestingly, for the Cu(II)-bis-L-histidine complex (Cu(his)(2)), cellular uptake and metabolic changes are observed with no toxicity after 72 h at micromolar concentrations. Furthermore, we show that under conditions where other copper complexes kill human hepatoma cells, Cu(I)-L-histidine is an effective catalyst for CuAAC labeling of live cells following metabolic incorporation of an alkyne-labeled sugar (Ac(4)ManNAl) into glycosylated proteins expressed on the cell surface. This result suggests that Cu(his)(2) or derivatives thereof have potential for in vivo applications where toxicity as well as catalytic activity are critical factors for successful bioconjugation reactions.
255636
Measuring and Suppressing the Oxidative Damage to DNA During Cu(I)-Catalyzed Azide-Alkyne Cycloaddition.
We have used the quantitative polymerase chain reaction (qPCR) to measure the extent of oxidative DNA damage under varying reaction conditions used for copper(I)-catalyzed click chemistry. We systematically studied how the damage depends on a number of key reaction parameters, including the amounts of copper, ascorbate, and ligand used, and found that the damage is significant under nearly all conditions tested, including those commonly used for bioconjugation. Furthermore, we discovered that the addition of dimethyl sulfoxide, a known radical scavenger, into the aqueous mixture dramatically suppresses DNA damage during the reaction. We also measured the efficiency of cross-linking two short synthetic oligonucleotides via click chemistry, and found that the reaction could proceed reasonably efficiently even with DMSO present. This approach for screening both DNA damage and reactivity under a range of reaction conditions will be valuable for improving the biocompatibility of click chemistry, and should help to extend this powerful synthetic tool for both in vitro and in vivo applications.
20180441
Extent of the Oxidative Side Reactions to Peptides and Proteins During the CuAAC Reaction.
The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is a powerful tool for bioconjugation of biomolecules, particularly proteins and peptides. The major drawback limiting the use of the CuAAC reaction in biological systems is the copper-mediated formation of reactive oxygen species (ROS), leading to the oxidative degradation of proteins or peptides. From the studies on a limited number of proteins and peptides, it is known that, in general, the copper mediated oxidative damage is associated with the copper coordination environment and solvent accessibility. However, there is a lack of data to help estimate the extent of copper-mediated oxidation on a wide range of proteins and peptides. To begin to address this need, we quantitatively measured the degree of copper-mediated oxidation on libraries of 1200 tetrapeptides and a model protein (bovine serum albumin, BSA) using liquid chromatography mass spectrometry (LC-MS). The collected data will be useful to researchers planning to use the CuAAC reaction for bioconjugaton on peptides or proteins.
207240737
Chelation-assisted, copper(II)-acetate-accelerated azide-alkyne cycloaddition.
We described in a previous communication a variant of the popular Cu(I)-catalyzed azide-alkyne cycloaddition (AAC) process where 5 mol % of Cu(OAc)(2) in the absence of any added reducing agent is sufficient to enable the reaction. 2-Picolylazide (1) and 2-azidomethylquinoline (2) were found to be by far the most reactive carbon azide substrates that convert to 1,2,3-triazoles in as short as a few minutes under the discovered conditions. We hypothesized that the abilities of 1 and 2 to chelate Cu(II) contribute significantly to the observed high reaction rates. The current work examines the effect of auxiliary ligands near the azido group other than pyridyl for Cu(II) on the efficiency of the Cu(OAc)(2)-accelerated AAC reaction. The carbon azides capable of binding to the catalytic copper center at the alkylated azido nitrogen in a chelatable fashion were indeed shown to be superior substrates under the reported conditions. The chelation between carbon azide 11 and Cu(II) was demonstrated in an X-ray single-crystal structure. In a limited set of examples, the ligand tris(benzyltriazolylmethyl)amine (TBTA), developed by Fokin et al. for assisting the original Cu(I)-catalyzed AAC reactions, also dramatically enhances the Cu(OAc)(2)-accelerated AAC reactions involving nonchelating azides. This observation leads to the hypothesis of an additional effect of chelating azides on the efficiencies of Cu(OAc)(2)-accelerated AAC reactions, which is to facilitate the rapid reduction of Cu(II) to highly catalytic Cu(I) species. Mechanistic studies on the AAC reactions with particular emphasis on the role of carbon azide/copper interactions will be conducted based on the observations reported in this work. Finally, the immediate utility of the product 1,2,3-triazole molecules derived from chelating azides as multidentate metal coordination ligands is demonstrated. The resulting triazolyl-containing ligands are expected to bind with transition metal ions via the N(2) nitrogen of the 1,2,3-triazolyl group to form nonplanar coordination rings. The Cu(II) complexes of bidentate T1 and tetradentate T6 and the Zn(II) complex of T6 were characterized by X-ray crystallography. The structure of [Cu(T1)(2)(H(2)O)(2)](ClO(4))(2) reveals the interesting synergistic effect of hydrogen bonding, π-π stacking interactions, and metal coordination in forming a one-dimensional supramolecular construct in the solid state. The tetradentate coordination mode of T6 may be incorporated into designs of new molecule sensors and organometallic catalysts.
207068516
Experimental investigation on the mechanism of chelation-assisted, copper(II) acetate-accelerated azide-alkyne cycloaddition.
A mechanistic model is formulated to account for the high reactivity of chelating azides (organic azides capable of chelation-assisted metal coordination at the alkylated azido nitrogen position) and copper(II) acetate (Cu(OAc)(2)) in copper(II)-mediated azide-alkyne cycloaddition (AAC) reactions. Fluorescence and (1)H NMR assays are developed for monitoring the reaction progress in two different solvents, methanol and acetonitrile. Solvent kinetic isotopic effect and premixing experiments give credence to the proposed different induction reactions for converting copper(II) to catalytic copper(I) species in methanol (methanol oxidation) and acetonitrile (alkyne oxidative homocoupling), respectively. The kinetic orders of individual components in a chelation-assisted, copper(II)-accelerated AAC reaction are determined in both methanol and acetonitrile. Key conclusions resulting from the kinetic studies include (1) the interaction between copper ion (either in +1 or +2 oxidation state) and a chelating azide occurs in a fast, pre-equilibrium step prior to the formation of the in-cycle copper(I)-acetylide, (2) alkyne deprotonation is involved in several kinetically significant steps, and (3) consistent with prior experimental and computational results by other groups, two copper centers are involved in the catalysis. The X-ray crystal structures of chelating azides with Cu(OAc)(2) suggest a mechanistic synergy between alkyne oxidative homocoupling and copper(II)-accelerated AAC reactions, in which both a bimetallic catalytic pathway and a base are involved. The different roles of the two copper centers (a Lewis acid to enhance the electrophilicity of the azido group and a two-electron reducing agent in oxidative metallacycle formation, respectively) in the proposed catalytic cycle suggest that a mixed valency (+2 and +1) dinuclear copper species be a highly efficient catalyst. This proposition is supported by the higher activity of the partially reduced Cu(OAc)(2) in mediating a 2-picolylazide-involved AAC reaction than the fully reduced Cu(OAc)(2). Finally, the discontinuous kinetic behavior that has been observed by us and others in copper(I/II)-mediated AAC reactions is explained by the likely catalyst disintegration during the course of a relatively slow reaction. Complementing the prior mechanistic conclusions drawn by other investigators, which primarily focus on the copper(I)/alkyne interactions, we emphasize the kinetic significance of copper(I/II)/azide interaction. This work not only provides a mechanism accounting for the fast Cu(OAc)(2)-mediated AAC reactions involving chelating azides, which has apparent practical implications, but suggests the significance of mixed-valency dinuclear copper species in catalytic reactions where two copper centers carry different functions.
32653875
Tridentate complexes of 2,6-bis(4-substituted-1,2,3-triazol-1-ylmethyl)pyridine and its organic azide precursors: an application of the copper(II) acetate-accelerated azide-alkyne cycloaddition.
Rapid coupling reactions between 2,6-bis(azidomethyl)pyridine and terminal alkynes in the presence of 5 mol% Cu(OAc)(2)·H(2)O without the addition of a reducing agent afford tridentate ligands for first-row transition-metal ions. The chelation between Cu(II) and alkylated nitrogen atoms of the azido groups of 2,6-bis(azidomethyl)pyridine, as observed in the solid state, is credited for the acceleration of the azide-alkyne cycloaddition reactions.
7145255
Fast, cell-compatible click chemistry with copper-chelating azides for biomolecular labeling.
We report that azides capable of copper-chelation undergo much faster “Click chemistry” (copper-accelerated azide-alkyne cycloaddition, or CuAAC) than nonchelating azides under a variety of biocompatible conditions. This kinetic enhancement allowed us to perform site-specific protein labeling on the surface of living cells with only 10–40 µM CuI/II and much higher signal than could be obtained using the best previously-reported live-cell compatible CuAAC labeling conditions. Detection sensitivity was also increased for CuAAC detection of alkyne-modified proteins and RNA labeled by metabolic feeding.
2657396
Monitoring Dynamic Glycosylation in Vivo Using Supersensitive Click Chemistry
To monitor the kinetics of biological processes that take place within the minute time scale, simple and fast analytical methods are required. In this article, we present our discovery of an azide with an internal Cu(I)-chelating motif that enabled the development of the fastest protocol for Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) to date, and its application toward following the dynamic process of glycan biosynthesis. We discovered that an electron-donating picolyl azide boosted the efficiency of the ligand-accelerated CuAAC 20–38-fold in living systems with no apparent toxicity. With a combination of this azide and BTTPS, a tris(triazolylmethyl)amine-based ligand for Cu(I), we were able to detect newly synthesized cell-surface glycans by flow cytometry using as low as 1 nM of a metabolic precursor. This supersensitive chemistry enabled us to monitor the dynamic glycan biosynthesis in mammalian cells and in early zebrafish embryogenesis. In live mammalian cells, we discovered that it takes approximately 30–45 min for a monosaccharide building block to be metabolized and incorporated into cell-surface glycoconjugates. In zebrafish embryos, the labeled glycans could be detected as early as the two-cell stage. To our knowledge, this was the first time that newly synthesized glycans were detected at the cleavage period (0.75–2 hpf) in an animal model using bioorthogonal chemistry.
23838677
Copper-chelating azides for efficient click conjugation reactions in complex media.
The concept of chelation-assisted copper catalysis was employed for the development of new azides that display unprecedented reactivity in the copper(I)-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) reaction. Azides that bear strong copper-chelating moieties were synthesized; these functional groups allow the formation of azide copper complexes that react almost instantaneously with alkynes under diluted conditions. Efficient ligation occurred at low concentration and in complex media with only one equivalent of copper, which improves the biocompatibility of the CuAAC reaction. Furthermore, such a click reaction allowed the localization of a bioactive compound inside living cells by fluorescence measurements.
205560101
One‐Step Derivatization of Reducing Oligosaccharides for Rapid and Live‐Cell‐Compatible Chelation‐Assisted CuAAC Conjugation
We report a new reagent for the functionalization of unprotected oligosaccharides with a picolyl azide group at the anomeric position for chelation‐assisted copper‐catalyzed alkyne–azide cycloaddition (CuAAC) glycoconjugation. We show that oligosaccharides functionalized with this moiety react with an apparent second‐order rate constant of 193 m−1 s−1 and can be used to functionalize biomolecules bearing alkyne moieties introduced through metabolic labeling, including in live cells.
41092665
Site-specific protein labeling using PRIME and chelation-assisted click chemistry
This protocol describes an efficient method to site-specifically label cell-surface or purified proteins with chemical probes in two steps: probe incorporation mediated by enzymes (PRIME) followed by chelation-assisted copper-catalyzed azide-alkyne cycloaddition (CuAAC). In the PRIME step, Escherichia coli lipoic acid ligase (LplA) site-specifically attaches a picolyl azide (pAz) derivative to a 13-aa recognition sequence that has been genetically fused onto the protein of interest. Proteins bearing pAz are chemoselectively derivatized with an alkyne-probe conjugate by chelation-assisted CuAAC in the second step. We describe herein the optimized protocols to synthesize pAz to perform PRIME labeling and to achieve CuAAC derivatization of pAz on live cells, fixed cells and purified proteins. Reagent preparations, including synthesis of pAz probes and expression of LplA, take 12 d, whereas the procedure for performing site-specific pAz ligation and CuAAC on cells or on purified proteins takes 40 min–3 h.
93417717
1,3-Dipolar cycloaddition of azides with electron-deficient alkynes under mild condition in water
We report a simple synthetic protocol for the 1,3-dipolar cycloaddition of azides with electron-deficient alkynes. Alkyne with at least one neighboring electron-withdrawing group proceeds with the cycloaddition successfully without any catalysts at room temperature in water. Under this simple condition, we evaluated a series of small molecule model reactions and then coupled an azido-DNA molecule with electron-deficient alkynes for the formation of [1,2,3]-triazole heterocycle, providing a potential method for introducing functional groups to DNA under biological conditions.
24060138
"Click labeling" with 2-[18f]fluoroethylazide for positron emission tomography.
As an effort in the development of more flexible (18)F-labeling chemistry, we report herein on the use of the Cu(I)-catalyzed Huisgen cycloaddition, also known as the "click reaction", to form (18)F-labeled 1,2,3-triazoles. Nucleophilic fluorination of 2-azidoethyl-4-toluenesulfonate followed by distillation provided 2-[(18)F]fluoroethylazide in 55% radiochemical yield (decay-corrected). 2-[(18)F]fluoroethylazide was reacted with a small library of terminal alkynes in the presence of excess Cu(2+)/ascorbate or copper powder. The most reactive alkyne, N-benzylpropynamide provided nearly quantitative incorporation of 2-[(18)F]fluoroethylazide after 15 min at ambient temperature, whereas the majority of the alkyne substrates provided excellent yields of the corresponding (18)F-labeled 1,2,3-triazoles following heating to 80 degrees C. Using the method described, a model peptide was obtained in 92.3 +/- 0.3% (n = 3) radiochemical yield (decay-corrected) after purification by semipreparative HPLC.
8315403
Relative performance of alkynes in copper-catalyzed azide-alkyne cycloaddition.
Copper-catalyzed azide-alkyne cycloaddition (CuAAC) has found numerous applications in a variety of fields. We report here only modest differences in the reactivity of various classes of terminal alkynes under typical bioconjugative and preparative organic conditions. Propargyl compounds represent an excellent combination of azide reactivity, ease of installation, and cost. Electronically activated propiolamides are slightly more reactive, at the expense of increased propensity for Michael addition. Certain alkynes, including tertiary propargyl carbamates, are not suitable for bioconjugation due to copper-induced fragmentation. A fluorogenic probe based on such reactivity is available in one step from rhodamine 110 and can be useful for optimization of CuAAC conditions.
17447035
Isolation of bis(copper) key intermediates in Cu-catalyzed azide-alkyne “click reaction”
Isolation of bis(copper) complexes that are kinetically more active than their mononuclear counterparts in the most popular “click chemistry” reaction. The copper-catalyzed 1,3-dipolar cycloaddition of an azide to a terminal alkyne (CuAAC) is one of the most popular chemical transformations, with applications ranging from material to life sciences. However, despite many mechanistic studies, direct observation of key components of the catalytic cycle is still missing. Initially, mononuclear species were thought to be the active catalysts, but later on, dinuclear complexes came to the front. We report the isolation of both a previously postulated π,σ-bis(copper) acetylide and a hitherto never-mentioned bis(metallated) triazole complex. We also demonstrate that although mono- and bis-copper complexes promote the CuAAC reaction, the dinuclear species are involved in the kinetically favored pathway.
22502745
Cationic 1,2,3-Triazolium Alkynes: Components To Enhance 1,4-Regioselective Azide-Alkyne Cycloaddition Reactions.
4-Alkynyl-1,2,3-triazolium cations undergo thermal [3 + 2] cycloaddition reactions with azides roughly 50- to 100-fold faster than comparable noncharged alkynes. Further, the reaction is highly 1,4-regioselective (dr up to 99:1) owing to the selective stabilization of 1,4-TS transition states via conjugative π-acceptor assistance of the alkyne triazolium ring. The novel cationic triazolium alkynes also accelerate the CuAAC reaction to provide bis(1,2,3-triazoles) in an "ultrafast" way (<5 min).
206755956
Chemoselective Sequential Click Ligations Directed by Enhanced Reactivity of an Aromatic Ynamine.
Aromatic ynamines or N-alkynylheteroarenes are highly reactive alkyne components in Cu-catalyzed Huisgen [3 + 2] cycloaddition ("click") reactions. This enhanced reactivity enables the chemoselective formation of 1,4-triazoles using the representative aromatic ynamine N-ethynylbenzimidazole in the presence of a competing aliphatic alkyne substrate. The unique chemoselectivity profile of N-ethynylbenzimidazole is further demonstrated by the sequential click ligation of a series of highly functionalized azides using a heterobifunctional diyne, dispelling the need for alkyne protecting groups.
43764692
Ligand-assisted, copper(II) acetate-accelerated azide-alkyne cycloaddition.
Polytriazole ligands such as the widely used tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), are shown to assist copper(II) acetate-mediated azide-alkyne cycloaddition (AAC) reactions that involve nonchelating azides. Tris(2-{4-[(dimethylamino)methyl]-1H-1,2,3-traizol-1-yl}ethyl)amine (DTEA) outperforms TBTA in a number of reactions. The satisfactory solubility of DTEA in a wide range of polar and nonpolar solvents, including water and toluene, renders it advantageous under copper(II) acetate-mediated conditions. The copper(II) acetate-mediated formation of the three triazolyl groups in a tris(triazolyl)-based ligand occurs sequentially with an inhibitory effect in the last step. The kinetic investigations of the ligand-assisted reactions reveal an interesting mechanistic dependence on the relative affinity of azide and alkyne to copper (II). In addition to expanding the scope of the copper(II) acetate-mediated AAC reactions to include nonchelating azides, this work offers evidence for the mechanistic synergy between the title reaction and the alkyne oxidative homocoupling reaction. The elucidation of the structural details of the polytriazole-ligand-bound reactive species in copper(I/II)-mediated AAC reactions, however, awaits further characterization of the metal coordination chemistry of polytriazole ligands.
197274384
2-Ethynylpyridine-Promoted Rapid Copper(I) Chloride Catalyzed Azide–Alkyne Cycloaddition Reaction in Water
The copper(I) chloride catalyzed reaction of azides with alkynes in water at room temperature was promoted by the addition of a catalytic amount of 2-ethynylpyridine, affording the corresponding 1,4-disubstituted 1,2,3-triazoles in good yields after a specific reaction time. The catalytic system could be successfully applied to electron-rich, electron-poor, and sterically crowded substrates. A study of the additive effect of pyridine derivatives revealed that alkynyl and 2-pyridyl groups were essential for activating the copper catalyst.
205708095
'Click' cycloaddition catalysts: copper(I) and copper(II) tris(triazolylmethyl)amine complexes.
The CuI complex of the 'click' ligand tris(benzyltriazolylmethyl)amine is an unusual dinuclear dication with one triazole unit bridging two metal centers, and is an effective catalyst for the 'click' cycloaddition reaction.
40647997
On the Mechanism of Copper(I)-Catalyzed Azide-Alkyne Cycloaddition.
The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction regiospecifically produces 1,4-disubstituted-1,2,3-triazole molecules. This heterocycle formation chemistry has high tolerance to reaction conditions and substrate structures. Therefore, it has been practiced not only within, but also far beyond the area of heterocyclic chemistry. Herein, the mechanistic understanding of CuAAC is summarized, with a particular emphasis on the significance of copper/azide interactions. Our analysis concludes that the formation of the azide/copper(I) acetylide complex in the early stage of the reaction dictates the reaction rate. The subsequent triazole ring-formation step is fast and consequently possibly kinetically invisible. Therefore, structures of substrates and copper catalysts, as well as other reaction variables that are conducive to the formation of the copper/alkyne/azide ternary complex predisposed for cycloaddition would result in highly efficient CuAAC reactions. Specifically, terminal alkynes with relatively low pKa values and an inclination to engage in π-backbonding with copper(I), azides with ancillary copper-binding ligands (aka chelating azides), and copper catalysts that resist aggregation, balance redox activity with Lewis acidity, and allow for dinuclear cooperative catalysis are favored in CuAAC reactions. Brief discussions on the mechanistic aspects of internal alkyne-involved CuAAC reactions are also included, based on the relatively limited data that are available at this point.
23840344
Muscarinic receptor binding and activation of second messengers by substituted N-methyl-N-[4-(1-azacycloalkyl)-2-butynyl]acetamides.
A series of substituted azacycloalkyl analogues of the muscarinic agonist UH 5 (N-methyl-N-[4-(1-pyrrolidinyl)-2-butynyl]acetamide, 1a) were synthesized and evaluated pharmacologically. These compounds were developed as intermediates for further derivatization leading to functionalized congeners of 1a. The compounds were synthesized by using a Mannich-type condensation of N-acetyl-N-methylpropargylamine to various substituted saturated azaheterocycles. The compounds were screened at a single concentration in competitive binding assays in rat cerebral cortical membranes against either [3H]N-methylscopolamine (at 100 microM) or [3H]oxotremorine-M (at 1 microM) labels. Candidates were then selected for further evaluation of their effect on phosphoinositide (PI) turnover in membranes from A9L cells transfected with cDNA of either m1-muscarinic cholinergic receptors (m1AChRs) or m3AChRs. The analogues were also tested for the inhibition of adenylate cyclase in NG108-15 cells expressing m4AChRs. The azetidine analogue of 1a had a Ki value of 12 nM for the inhibition of [3H]oxotremorine-M binding in rat brain and had an agonist potency at m1-,m3-, and m4AChRs comparable to 1a. The substituted 5- and 6-member ring analogues generally had lower binding affinities and were less potent than 1a in stimulating PI turnover. Several compounds were moderately effective in inhibiting cyclic AMP production in NG108-15 cells.
22708926
2-Anthryltriazolyl-containing multidentate ligands: zinc-coordination mediated photophysical processes and potential in live-cell imaging applications.
1,2,3-Triazol-4-yl (triazolyl)-containing tetradentate ligand 1 undergoes fluorescence enhancement upon binding to zinc ion (Zn(2+)) in both organic (acetonitrile) and aqueous solutions. A 1:1 complex of 1 with a trigonal bipyramidal Zn(2+) was characterized by X-ray crystallography. The cyclic voltammogram (CV) of 1 suggests that an intramolecular photoinduced electron transfer (PET) process is thermodynamically feasible which would quench the fluorescence of the 2-anthryltriazolyl fluorophore. On the basis of the X-ray and CV data, it was initially postulated that the 1:1 binding between Zn(2+) and ligand 1 shuts down the PET quenching pathway of the free ligand, which leads to the fluorescence enhancement of 1. However, the nuance of the interaction between 1 and Zn(2+) was revealed by isothermal titration calorimetry (ITC) and (1)H NMR titration experiments. A two-step binding process was observed which proceeds through an intermediate species of 2:1 (ligand/Zn(2+)) stoichiometry. Upon close examination of the fluorescence spectra of 1 during the Zn(2+) titration experiment, the fluorescence profile is in fact consistent with a two-step binding process in which a moderate fluorescence enhancement was observed during the early stage of the titration, followed by a bathochromic shift in conjunction with a more pronounced enhancement as Zn(2+) concentration increases. The studies on compounds 2-5 support the amended hypothesis that upon increasing Zn(2+) concentration, compound 1 first undergoes fluorescence enhancement because of the formation of a 2:1 (ligand to Zn(2+)) complex which slows down the PET quenching process. As Zn(2+) concentration increases, the 2:1 complex is converted into a 1:1 complex which facilitates an intramolecular exciplex formation between the excited 2-anthryltriazolyl fluorophore and the Zn(2+)-bound pyridyl moiety. Finally, the potential of compound 1 as an intracellular fluorescent indicator for Zn(2+) was evaluated. HeLa cells loaded with compound 1 grown in Zn(2+)-rich media show stronger fluorescence than those grown under Zn(2+)-deprived conditions, confirming the promise that the triazolyl-containing polyaza fluoroionophores can be developed into intracellular fluorescent indicators targeting biological Zn(2+).
5260614
Integrated and passive 1,2,3-triazolyl groups in fluorescent indicators for zinc(II) ions: thermodynamic and kinetic evaluations.
In addition to being a covalent linker in molecular conjugation chemistry, the function of a 1,2,3-triazolyl moiety resulting from the copper(I)-catalyzed azide-alkyne cycloaddition reaction as a ligand for metal ions is receiving considerable attention. In this work, we characterize the thermodynamic and kinetic effects of incorporating a 1,2,3-triazolyl group in a multidentate ligand scaffold on metal coordination in the context of fluorescent zinc(II) indicator development. Ligands L14, BrL14, and FL14 (1,4-isomers) contain the 1,4-disubstituted-1,2,3-triazolyl group that is capable of binding with zinc(II) in conjunction with a di(2-picolylamino) (DPA) moiety within a multidentate ligand scaffold. Therefore, the 1,2,3-triazolyl in the 1,4-isomers is "integrated" in chelation. The 1,5-isomers L15, BrL15, and FL15 contain 1,2,3-triazolyls that are excluded from participating in zinc(II) coordination. These 1,2,3-triazolyls are "passive linkers". Zinc(II) complexes of 2:1 (ligand/metal) stoichiometry are identified in solution using (1)H NMR spectroscopy and isothermal titration calorimetry (ITC) and, in one case, characterized in the solid state. The 1:1 ligand/zinc(II) affinity ratio of L14 over L15, which is attributed to the affinity enhancement of a 1,2,3-triazolyl group to zinc(II) over that of the solvent acetonitrile, is quantified at 18 (-1.7 kcal/mol at 298 K) using an ITC experiment. Fluorescent ligands FL14 and FL15 are evaluated for their potential in zinc(II) sensing applications under pH neutral aqueous conditions. The 1,4-isomer FL14 binds zinc(II) both stronger and faster than the 1,5-isomer FL15. Visualization of free zinc(II) ion distribution in live HeLa cells is achieved using both FL14 and FL15. The superiority of FL14 in staining endogenous zinc(II) ions in live rat hippocampal slices is evident. In summation, this work is a fundamental study of 1,2,3-triazole coordination chemistry, with a demonstration of its utility in developing fluorescent indicators.
6485404
Tailored ligand acceleration of the Cu-catalyzed azide-alkyne cycloaddition reaction: practical and mechanistic implications.
Tris(heterocyclemethyl)amines containing mixtures of 1,2,3-triazolyl, 2-benzimidazoyl, and 2-pyridyl components were prepared for ligand acceleration of the copper-catalyzed azide-alkyne cycloaddition reaction. Two classes of ligands were identified: those that give rise to high reaction rates in coordinating solvents but inhibit the process when used in excess relative to copper and those that provide for fast catalysis in water and are not inhibitory in excess. Several "mixed" ligands were identified that performed well under both types of conditions. Kinetics measurements as a function of ligand:metal ratio and catalyst concentration were found to be consistent with an active Cu(2)L formulation. Since strongly bound chelating agents are not always the most effective, achieving optimal rates requires an assessment of the potential donor molecules in the reaction mixture. Simple rules are provided to guide the user in the choice of effective ligands and reaction conditions to suit most classes of substrates, solvents, and concentrations.