Catalyzed Azide Alkyne Cycloaddition Research Articles

Supramolecular chemistry of pillar[n]arenes functionalised by a copper(i)-catalysed alkyne–azide cycloaddition “click” reaction

Since we discovered pillar[n]arenes in 2008, many chemists have developed a strong interest in pillar[n]arene chemistry because of the many advantages associated with these materials, including their facile and high yielding synthesis, versatile functionality, planar chirality and unique host-guest properties. In this feature article, we discuss recent advances in the field of supramolecular chemistry based on the use of pillar[n]arenes as substrates for copper(i)-catalysed alkyne-azide cycloaddition (CuAAC) "click" chemistry. The CuAAC reaction provides facile access to 1,4-disubstituted triazoles by a reaction between alkyne and azido substrates in the presence of a Cu(i) catalyst. Pillar[n]arenes bearing alkyne or azido groups can therefore be used as substrates for this reaction. Herein, we discuss not only the synthesis of pillar[n]arenes bearing alkyne or azido groups but also the application of these functionalised systems to the CuAAC reaction to construct supramolecular assemblies. We also discuss the rational molecular design and synthesis of guest compounds using the CuAAC reaction because linear alkanes sandwiched between 1,2,3-triazole moieties are good guests for cyclic pentamer pillar[5]arenes.

Click chemistry-based aptasensor for highly sensitive electrochemical detection of thrombin

In this study, a click chemistry-based electrochemical aptasensor is reported for the simple and fast detection of thrombin. Briefly, the thiol-terminated aptamer1 (Apt1) was first self-assembled on a gold electrode for the specific recognition of thrombin in the following step. After this, the azido-labeled aptamer2 (Apt2) was attached to the electrode via the formation of a sandwich structure with Apt1 and thrombin. The azido terminal was designed for the subsequent labeling of Apt2 with ethynylferrocene via electrochemically mediated Cu(I)-catalyzed azide–alkyne cycloaddition (eCuAAC), in which the active Cu(I) was electrochemically generated in situ. Finally, the quantitatively labeled ethynylferrocene probes could be detected on the basis of differential pulse voltammetry (DPV) for the electrochemical detection of thrombin. Under optimal conditions, this aptasensor could detect thrombin down to 84 pM with a good linear response over the range from 0.1 nM to 1000 nM. In addition, it exhibited excellent specificity in the quantitative detection of thrombin and also showed good detection reliability in serum samples.

Fluorescent labeling of Taqman oligonucleotide probes via Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) click chemistry

We describe an approach to the synthesis of TaqMan oligonucleotide probes that is based on Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) click chemistry when oligonucleotides containing an internal alkynyl group at the pyrimidine position are labeled post-synthetically with a f luorescent azide. TaqMan probes were constructed with f luorescein in different internal positions and a BHQ1 quencher on the 3′-end. Our previously designed alkynylated deoxyuridine or deoxycytidine phosphoramidites have been employed for the synthesis of alkynyl oligonucleotides. It was demonstrated that the synthesized TaqMan probes can detect accumulation of PCR product in real-time. The closer to the label the 3′-terminal quencher, the higher the quenching efficiency, but the efficiency of probe hybridization to DNA template is reduced in this case.

Eight-membered-ring diaminocarbenes bearing naphthalene moiety in the backbone: DFT studies, synthesis of amidinium salts, generation of free carbene, metal complexes, and solvent-free copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction.

A new type of eight-membered ring N-heterocyclic carbene (NHC) bearing a rigid naphthalene moiety in the backbone is reported for the first time. Stereoelectronic properties of 4,5-dihydro-1H-naphtho[1,8-ef][1,3]diazocin-3(2H)-ylidene (NaphtDHD) and smaller ring NHCs were theoretically studied at the DFT level. Amidinium salts were prepared from corresponding amidines and dibromides. Free carbene NaphtDHD-Dipp (Dipp = 2,6-diisopropylphenyl) was generated in solution by treatment of the corresponding salt with LiHMDS. It is stable in solution at low temperatures, while decomposing rapidly at room temperature. Silver(i) and copper(i) complexes were synthesized and structurally characterized in the solid state. The copper(i) complex [(NaphtDHD-Mes)CuBr] (Mes = mesityl, 2,4,6-trimethylphenyl) exhibits high catalytic activity in alkyne-azide cycloaddition (CuAAC) reaction under solvent-free conditions.

A mild post-functionalization method for the vanadium substituted P2W15V3 Wells-Dawson polyoxometalate based on a copper catalyzed azide-alkyne cycloaddition.

A mild post-functionalization method, based on a copper(i) catalyzed azide-alkyne cycloaddition, was developed for the fragile [P2W15V3]9- (PWV). PWV was functionalized with an azide and used as a platform to attach various alkyne functionalized compounds.

Glucose selective bis-boronic acid click-fluor.

Four novel bis-boronic acid compounds were synthesised via copper catalysed azide-alkyne cycloaddition (CuAAC) reactions. Glucose selectivity was observed for a particular structural motif. Moreover, a new glucose selective fluorescent sensor was designed and synthesised as a result.

Azidoperfluoroalkanes: Synthesis and Application in Copper(I)‐Catalyzed Azide–Alkyne Cycloaddition

AbstractWe report an efficient and scalable synthesis of azidotrifluoromethane (CF3N3) and longer perfluorocarbon‐chain analogues (RFN3; RF=C2F5, nC3F7, nC8F17), which enables the direct insertion of CF3 and perfluoroalkyl groups into triazole ring systems. The azidoperfluoroalkanes show good reactivity with terminal alkynes in copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC), giving access to rare and stable N‐perfluoroalkyl triazoles. Azidoperfluoroalkanes are thermally stable and the efficiency of their preparation should be attractive for discovery programs.

Azidoperfluoroalkanes: Synthesis and Application in Copper(I)-Catalyzed Azide-Alkyne Cycloaddition.

We report an efficient and scalable synthesis of azidotrifluoromethane (CF3 N3 ) and longer perfluorocarbon-chain analogues (RF N3 ; RF =C2 F5 , n C3 F7 , n C8 F17 ), which enables the direct insertion of CF3 and perfluoroalkyl groups into triazole ring systems. The azidoperfluoroalkanes show good reactivity with terminal alkynes in copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), giving access to rare and stable N-perfluoroalkyl triazoles. Azidoperfluoroalkanes are thermally stable and the efficiency of their preparation should be attractive for discovery programs.

Photo-Tethers for the (Multi-)Cyclic, Conformational Caging of Long Oligonucleotides.

Intramolecular circularization of DNA oligonucleotides was accomplished by incorporation of alkyne-modified photolabile nucleosides into DNA sequences, followed by a CuI -catalyzed alkyne-azide cycloaddition with bis-azido linker molecules. We determined a range of ring sizes, in which the caged circular oligonucleotides exhibit superior duplex destabilizing properties. Specific binding of a full-length 90 nt C10 aptamer recognizing human Burkitt's lymphoma cells was then temporarily inhibited by locking the aptamer in a bicircularized structure. Irradiation restored the native aptamer conformation resulting in efficient cell binding and uptake. The photo-tether strategy presented here provides a robust and versatile tool for the light-activation of longer functional oligonucleotides, noteworthy without prior knowledge on the structure and the importance of specific nucleotides within a DNA aptamer.

Kinetically Inert Lanthanide Complexes as Reporter Groups for Binding of Potassium by 18-crown-6

The barcode-like spectrum of lanthanide-centered emission has been used in imaging and to make responsive luminescent reporters. The intensities and the shapes of each line in the luminescence spectrum can also report on the coordination environment of the lanthanide ion. Here, we used lanthanide-centered emission to report on the binding of potassium in an 18-crown-6 binding pocket. The responsive systems were made by linking a crown ether to a kinetically inert lanthanide binding pocket using a molecular building block approach. Specifically, an alkyne-appended Ln.DO3A was used as a building block in a copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) "click" reaction with azide-functionalized crown ethers. The resulting complexes were investigated using NMR and optical methods. Titrations with potassium chloride in methanol observing the sensititzed europium- and terbium-centered emissions were used to investigate the response of the systems. The molecular reporters based on aliphatic crown ethers were found to have strongly inhibited binding of potassium, while the benzo-18-crown-6 derived systems had essentially the same association constants as the native crown ethers. The shape of the lanthanide emission spectra was shown to be unperturbed by the binding of potassium, while the binding was reported by an overall increased intensity of the lanthanide-centered emission. This observation was contrasted to the change in spectral shape between propargyl-Ln.DO3A and the triazolyl-Ln.DO3A complexes. The solution structure of the lanthanide complexes was found to be determining for the observed physical chemical properties of these systems.

Fluorescence biosensor for inorganic pyrophosphatase activity.

A highly sensitive and selective fluorescence biosensor for inorganic pyrophosphatase (PPase) activity has been developed based on special click ligation trigger hyperbranched rolling circle amplification (CLT-HRCA). Pyrophosphate ion (PPi) can coordinate with Cu2+ to form stable PPi/Cu2+ complex and Cu2+ in the complex cannot be reduced to Cu+. The addition of PPase causes the hydrolysis of PPi into orthophosphate (Pi) and therefore induces the releasing of Cu2+ from the stable PPi/Cu2+ complex, and the free Cu2+ is easily reduced to Cu+ by sodium ascorbate. Then Cu+ catalyzes the cyclization reaction between the specially designed 5'-azide and 3'-alkyne tagged padlock probes through Cu+ catalyzed azide-alkyne cycloaddition (CuAAC), which in turn initiates the hyperbranched rolling circle amplification (HRCA). Given that the CLT-HRCA products contain large amounts of double-stranded DNAs (dsDNAs), the addition of SYBR Green I resulted in the enhanced fluorescence signal. There was a linear relationship between the enhanced fluorescence intensity and the logarithm PPase activity ranging from 0.05 to 25mU with a detection limit of 0.02mU. Such proposed biosensor has been successfully applied to screen the potential PPase inhibitors and has accessed the related inhibit ability with high efficiency.

Characterization of non-specific protein adsorption induced by triazole groups on the chromatography media using Cu (I)-catalyzed alkyne-azide cycloaddition reaction for ligand immobilization

As an efficient and facile reaction, click chemistry has been growingly used in the preparation of chromatography media for immobilizing varying types of ligands. For the widely used Cu (I)-catalyzed alkyne-azide click reaction, a 1, 2, 3-triazole group will be inevitably introduced in the molecular linkage, which could give rise to unexpected non-specific adsorption especially for the media employing small compound ligands or high ligand density. Triazole-induced non-specific protein adsorption on sepharose resins was evaluated systematically in this work, by considering the effects of triazole content, length of spacer arm, and solution conditions. We found that triazole content of a resin played the key role. Protein adsorption became significant when the media was coupled with triazole at a medium density (about 60μmol/mL gel), and the binding amount further increased with triazole density. The resin with triazole content of about 100μmol/mL gel could adsorb human IgG, bovine serum albumin and lysozyme at the amount of 13.6, 30.0, and 5.1mg/mL respectively. Proteins tended to be adsorbed at higher amount as the pH of solution approached their isoelectric points, and increasing salt concentration could reduce triazole-induced adsorption but only within limited extent. This study can facilitate reasonable application of click chemistry in the synthesis of chromatography media, by providing some basic principles for optimizing structural properties of separation media and choosing suitable solution conditions.

Engineering an Affinity-Enhanced Peptide through Optimization of Cyclization Chemistry.

Peptide cyclization is a strategy used to improve stability and activity of peptides. The most commonly used cyclization method is disulfide bridge formation of cysteine-containing peptides, as is typically found in nature. Over the years, an increasing number of alternative chemistries for peptide cyclization with improved efficiency, kinetics, orthogonality, and stability have been reported. However, there has been less appreciation for the opportunity to fine-tune peptide activity via the diverse chemical entities introduced at the site of linkage by different cyclization strategies. Here, we demonstrate how cyclization optimization of an M2 "anti-inflammatory" macrophage-binding peptide (M2pep) resulted in a significant increase in binding affinity of the optimized analog to M2 macrophages while maintaining binding selectivity compared to M1 "pro-inflammatory" macrophages. In this study, we report synthesis and evaluation of four cyclic M2pep(RY) analogs with diverse cyclization strategies: (1) Asp-[amide]-Lys, (2) azido-Lys-[triazole(copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC))]-propargyl-Gly, (3) Cys-[decafluorobiphenyl (DFBP)]-Cys, and (4) Cys-[decafluorobiphenyl sulfone (DFS)]-Cys, whereby the chemical entity or linker at the linkage site is shown in the square bracket and is between the residues involved in cyclization. These peptides are compared to a disulfide-cyclized M2pep(RY) that we previously reported as a serum-stable, affinity-enhanced analog to the original linear M2pep. DFBP-cyclized M2pep(RY) exhibits the highest binding activity to M2 macrophages with apparent dissociation constant (KD) about 2.03 μM compared to 36.3 μM for the original disulfide-cyclized M2pep(RY) and 220 μM for the original linear peptide. DFS-cyclized M2pep(RY) also binds more strongly than the original cyclized analog, whereas amide- and triazole-cyclized M2pep(RY) analogs bind less strongly. We verified that DFBP alone has negligible binding to M2 macrophages and the incorporation of diphenylalanine to the original sequence improves binding activity at the expense of solubility and increased toxicity. In conclusion, we report development of cyclic M2pep(RY) analogs with diverse cyclization strategies leading to the discovery of DFBP-cyclized M2pep(RY) with enhanced M2 macrophage-binding activity.

Synthesis of 1,2,3-triazole functionalized hyperbranched poly(ethyleneimine) and its use as multifunctional anionic macroinitiator for diglycidyl ether of bisphenol A curing

Hyperbranched poly(ethyleneimine) (PEI) has been modified by the addition of propargyl acrylate following a Michael addition reaction. On this polymer (PEI-yne) a copper (I)-catalyzed azide alkyne cycloaddition (CuAAC) has been performed to obtain a multifunctional triazole initiator (PEI-TA). After structural and thermal characterization, this polymer has been used in different proportions as anionic multifunctional macroinitiator in diglycidyl ether of bisphenol A (DGEBA) homopolymerization. The curing process has been studied by calorimetry and the thermosets obtained have been thermally characterized and compared with thermosets prepared by using 1-methylimidazole (1-MI) as standard initiator. The electron microscopy inspection of the fracture surfaces of the new materials prepared shows the formation of submicrometer particles that should enhance toughness characteristics, changing smooth fracture surfaces in 1-MI initiated materials to multi-planar surface with tortuous and thicker cracks.

Acid-Cleavable Unimolecular Micelles from Amphiphilic Star Copolymers for Triggered Release of Anticancer Drugs.

In this contribution, amphiphilic star copolymers (H40-star-PCL-a-PEG) with an H40 hyperbranched polyester core and poly(ε-caprolactone)-a-poly(ethylene glycol) copolymer arms linked with acetal groups are synthesized using ring-opening polymerization and a copper (I)-catalyzed alkyne-azide cycloaddition click reaction. The acid-cleavable acetal groups between the hydrophilic and hydrophobic segments of the arms endow the amphiphilic star copolymers with pH responsiveness. In aqueous solution, unimolecular micelles can be formed with good stability and a unique acid degradability, as is desirable for anticancer drug carriers. For the model drug of doxorubicin, the in vitro release behavior, intracellular release, and inhibition of proliferation of HeLa cells show that the acid-cleavable unimolecular micelles with anticancer activity can be dissociated in an acidic environment and efficiently internalized by HeLa cells. Due to the acid-cleavable and biodegradable nature, unimolecular micelles from amphiphilic star copolymers are promising for applications in intracellular drug delivery for cancer chemotherapy.

Synthesis of Multivalent [Lys8]-Oxytocin Dendrimers that Inhibit Visceral Nociceptive Responses

Peptide dendrimers are a novel class of precisely defined macromolecules of emerging interest. Here, we describe the synthesis, structure, binding affinity, receptor selectivity, functional activity, and antinociceptive properties of oxytocin-related dendrimers containing up to 16 copies of [Lys8]-oxytocin or LVT. These were generated using a copper(i)-catalyzed azide–alkyne cycloaddition (CuAAc) reaction with azido-pegylated LVT peptides on an alkyne–polylysine scaffold. 2D NMR analysis demonstrated that each attached LVT ligand was freely rotating and maintained identical 3D structures in each dendrimeric macromolecule. The binding affinity Ki at the oxytocin receptor increased approximately 17-, 12-, 3-, and 1.5-fold respectively for the 2-, 4-, 8-, and 16-mer dendrimeric LVT conjugates, compared with monomer azido-pegylated LVT (Ki = 9.5 nM), consistent with a multivalency effect. A similar trend in affinity was also observed at the related human V1a, V1b, and V2 receptors, with no significant selectivity change observed across this family of receptors. All LVT dendrimers were functionally active in vitro on human oxytocin receptors and inhibited colonic nociceptors potently in a mouse model of chronic abdominal pain.

Handling Hazards Using Continuous Flow Chemistry: Synthesis of N1-Aryl-[1,2,3]-triazoles from Anilines via Telescoped Three-Step Diazotization, Azidodediazotization, and [3 + 2] Dipolar Cycloaddition Processes

The conversion of commercially available anilines into triazole products was realized using a telescoped three-reactor flow diazotization, azidodediazotization, and [3 + 2] dipolar cycloaddition process. The diazotization–azidodediazotization sequence was accelerated by means of an ultrasonic bath resulting in a degassed, segmented effluent. An automated continuous flow unit controlled by custom software created in-house was used to collect the aryl azide stream and restore it to a continuous column of the reagent. When combined with a variety of dipolarophiles, 1-aryl-[1,2,3]-triazoles were thus assembled by either copper catalyzed alkyne–azide cycloaddition (CuAAC) or Huisgen cycloaddition reactions.

Copper(II) complexes of chiral 1,2,3-triazole biheterocyclic ‘click’ ligands equipped in Cinchona alkaloid moiety

Chiral 1,4-disubstituted-1,2,3-triazole mono- and biheterocyclic ‘click’ ligands derived from Cinchona alkaloids were synthesized using copper(I) catalyzed azide–alkyne cycloaddition (CuAAC) and studied in terms of coordination chemistry towards Cu(II). In broad range of pH the studied ligands form complexes with stoichiometry ML2 in which the coordination involves two neighboring nitrogen atoms from 1,2,3-triazole (N3) and pyridine unit, respectively. The copper(II) coordination is site-selective among studied ligands (L1,2,4) apart for L3, which due to the lack of pyridine next to the triazole moiety and/or possible steric strain in formed complex turned out to be the ligand which metal binding is weaker than others under studied conditions. Thermodynamic analysis which involved potentiometric and calorimetric studies revealed relatively low stability of formed complexes, which binding events are entropy driven processes.

P-tert-Butylcalix[4]arene core based ferrocenyl dendrimers: Novel sensor for toxic Hg2+ ion even in presence of Zn2+, Cu2+ and Ag+ ions

A new class of p-tert-butylcalix[4]arene cored triazolyl ferrocene conjugates 1–3 has been synthesized in good yield using copper(I) catalyzed alkyne azide cycloaddition (CuAAC). In UV–vis spectrum the absorption intensity increases as the dendrimer generation increases though λmax remains almost constant for all the synthesized dendrimers. All the synthesized calixarene conjugated ferrocenyl dendrimers 1, 2 and 3 sense toxic Hg2+ ion at the lowest concentration of 2×10−5M even in the presence of Zn2+, Cu2+, Ag+ as revealed by Cyclic Voltammetric and through NMR titration studies.

1H-1,2,3-Triazolyl-substituted 1,3,4-oxadiazole derivatives containing structural features of ibuprofen/naproxen: Their synthesis and antibacterial evaluation

1H-1,2,3-Triazolyl-substituted 1,3,4-oxadiazole derivatives containing structural features of ibuprofen/naproxen were synthesized for the first time using a Cu catalyzed azide–alkyne cycloaddition (CuAAC) strategy. An optimized reaction condition was established for this purpose and twenty new compounds were synthesized using this methodology. Several of these compounds showed good to reasonable antibacterial activities when tested against three gram-positive and three gram-negative species. The compound 4m i.e. N-(2-chlorophenyl)-2-(4-((5-(1-(6-methoxynaphthalen-2-yl)ethyl)-1,3,4-oxadiazol-2-ylthio)methyl)-1H-1,2,3-triazol-1-yl)acetamide showed promising activities across both the species.