Triazole Formation Research Articles

Cell-instructive hydrogels to assemble in biofluids

which mimic fundamental characteristics of naturally occurring extracellular microenvironments, is a key task in the rapidly progressing fields of tissue engineering and regenerative medicine (DOI: 10.1038/nature08602). Importantly, cell-instructive matrix signals – including matrix-associated growth factors, ligands of cellular adhesion receptors, intrinsic viscoelastic properties and susceptibility for cell-driven reorganization – have to be presented in a spatiotemporally adjusted manner (DOI: 10.1002/marc.201200382). For that purpose, the current practice in cell biology and biotechnology mostly relies on reconstituting non-covalently associating biopolymer meshworks (collagen fibrils, Matrigel). Although effective in numerous in vitro approaches to cell signaling, these preparations are hardly compatible with safety requirements for medical applications and are often not suitable for the mechanistic investigation of particular exogenous signals. Recent approaches have therefore aimed at forming covalent polymer hydrogel networks in the presence of cells in culture or even within living tissue. A range of poly(ethylene glycol) (PEG) conjugates with adhesive peptide ligands and/or degradable peptide crosslinkers were converted in cell-compatible crosslinking schemes, including Michael-type addition, triazole formation, enzymatic (Factor XIIIa/ transglutaminase-mediated) crosslinking, and photoinduced polymerization. Amongst these, the Michael-type addition reaction turned out to be particularly powerful, as it does not require a catalyst, has no side products, and proceeds rapidly under physiological conditions (DOI: 10.1002/adma.201103574). The thiol group of cysteine residues can participate as a nucleophile in this reaction, which massively extends the number of suitable substrates and simplifies the attachment of bioactive peptides or proteins. A very common synthetic scheme for the formation of PEG-peptide hydrogels utilizes dithiol-containing peptides to react with PEG-polymers carrying electron deficient double bonds at the terminal groups. Recently, this scheme was further extended by incorporating glycosaminoglycan building blocks for the in situ formation of biohybrid gel matrices, which allows for the versatile biomimetic functionalization of the obtained materials with a plethora of glycosaminoglycan-binding growth factors: Michaeltype reaction schemes were applied for the formation of customized, cell-embedding, PEG-glycosaminoglycan hydrogels with precisely adjusted polymer network properties and independently tunable signaling characteristics (DOI: 10.1002/adma. 201300691). These materials provide a means for the effective modulation of therapeutically relevant cell fate decisions in vivo and are thus expected to become instrumental in medical technologies aimed at regenerating diseased or injured tissues.

Application of Metal Free Click Chemistry in Biological Studies

The first reported click reaction, copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition had limited biocompatibility due to the high toxicity of copper. Since alternative bioorthogonal click reactions have been developed, they have strongly influenced the field of chemical biology. Here are summarized three main metal-free click methodologies based on cycloaddition, Staudinger and thioene reactions. This review contains the basic principles, some mechanistic considerations and a collection of reagents that can be used in each method. Firstly, Diels-Alder and strain promoted inverse electron demand Diels-Alder cycloadditions are outlined together with triazole and isoxazole formation by 1,3-dipolar cycloadditions. Secondly, Staudinger-Bertozzi ligation, a chemoselective reaction of azides and engineered triarylphosphines, is discussed. Finally, thio-click chemistries including thiol-ene, thiol-yne, thio-Michael and fluoro-thioclick reactions are reviewed. Among the most important bioapplications of these click methodologies is the labeling of glycans, proteins, lipids and DNA. Additionally, synthetic methods and surface immobilization of biomolecules and biologically useful polymeric materials are also reviewed. Keywords: Biological applications, Bioorthogonal chemistry, Click chemistry, Cycloadditions, Staudinger-Bertozzi ligation, Thio-click chemistry

A Fluorogenic Probe for the Catalyst-Free Detection of Azide-Tagged Molecules

Fluorogenic reactions in which non- or weakly fluorescent reagents produce highly fluorescent products can be exploited to detect a broad range of compounds including biomolecules and materials. We describe a modified dibenzocyclooctyne that under catalyst-free conditions undergoes fast strain-promoted cycloadditions with azides to yield strongly fluorescent triazoles. The cycloaddition products are more than 1000-fold brighter compared to the starting cyclooctyne, exhibit large Stokes shift, and can be excited above 350 nm, which is required for many applications. Quantum mechanical calculations indicate that the fluorescence increase upon triazole formation is due to large differences in oscillator strengths of the S(0) ↔ S(1) transitions in the planar C(2v)-symmetric starting material compared to the symmetry-broken and nonplanar cycloaddition products. The new fluorogenic probe was successfully employed for labeling of proteins modified by an azide moiety.

ChemInform Abstract: Traceless Tosyhydrazone‐Based Triazole Formation: A Metal‐Free Alternative to Strain‐Promoted Azide—Alkyne Cycloaddition.

AbstractThe Sakai reaction as an alternative strategy for metal‐free triazole formation is presented.

Arylsilanated SiOx Surfaces for Mild and Simple Two-Step Click Functionalization with Small Molecules and Oligonucleotides

The conversion of surface-bound aminophenyl groups to azidophenyl moieties on SiOx surfaces was investigated as part of a mild, simple two-step strategy for “click”-based” surface functionalization with acetylene-functionalized reagents. Small terminal alkynes (phenylacetylene, 1-hexyne) and acetylene-modified single-stranded DNA 20-mers (T20) were then used as model compounds to test the efficiency of the 1,3-dipolar cycloaddition reaction. The identities of surface species were verified, and their coverages were quantified using X-ray photoelectron spectroscopy in the C 1s, N 1s, F 1s, Cl 2p, and P 2p regions. Depending on conditions, the yield of the azidification was in the 30–90% range, and the efficiency of triazole formation depended significantly on the rigidity of the acetylene reactant. Vibrational sum frequency generation was applied to probe the C–H stretching region and test the platform’s viability for minimizing spectral interference in the C–H stretching region. Fluorescence spectroscopy w...

Resin-Supported Catalysts for CuAAC Click Reactions in Aqueous or Organic Solvents

The copper-catalyzed azide-alkyne cycloaddition click reaction is a valuable process for the synthesis of libraries of drug candidates, derivatized polymers and materials, and a wide variety of other functional molecules. In some circumstances, the removal of the copper catalyst is both necessary and inconvenient. We describe here two immobilized forms of a Cu-binding ligand that has been shown to accelerate triazole formation under many different conditions, using different resin supports that are appropriate for aqueous or organic solvents. Copper leaching from these resins was modest, allowing them to be reused in many reaction/filtration cycles without recharging with metal ion. The utility of this catalyst form was demonstrated in the convenient synthesis of 20 N-acetylgalactosamine derivatives for biological testing.

Preparation of ‘click’ hydrogels from polyaspartamide derivatives

AbstractLately, copper‐assisted azide–alkyne cycloaddition (CuAAC) has become a very interesting tool for synthesizing biocompatible polymer‐based materials such as hydrogels or microgels, which can be used as biomaterials for tissue engineering and drug delivery. Novel poly(2‐hydroxyethyl aspartamide)s (PHEAs) functionalized with pendent acetylene or azide groups were prepared from polysuccinimide, which is the thermal polycondensation product of aspartic acid, through successful ring‐opening reactions using propargylamine, 1‐azido‐2‐aminoethane and ethanolamine. The composition of the prepared copolymers was analyzed using 1H NMR spectroscopy. Clickable PHEA derivatives were crosslinked by mixing together in water with a catalyst system of Cu(I) and N, N, N′, N′, N″‐pentamethyldiethylenetriamine, a type of Huisgen's 1,3‐dipolar azide‐alkyne cycloaddition. The reaction of the polymers resulted in a chemoselective coupling between alkynyl and azido functional groups with multiple formation of triazole crosslinks to give hydrogels. The triazole linkages in the hydrogels are highly stable and may also play a role in swelling behavior. PHEA‐based hydrogels were also obtained by the crosslinking of azide‐ or alkyne‐modified PHEA with a small‐molecule crosslinker. The hydrogels prepared using these two methods were characterized by their degree of swelling and the morphology of the hydrogels was confirmed using scanning electron microscopy. The approach we describe here presents a promising alternative to common chemical hydrogel preparation techniques, and these hydrogels seem to possess structures having potential for a variety of industrial and biomedical applications. © 2012 Society of Chemical Industry

Traceless Tosylhydrazone‐Based Triazole Formation: A Metal‐Free Alternative to Strain‐Promoted Azide–Alkyne Cycloaddition

Triple-T trick! Traceless tosylhydrazone-based triazole formation is readily achieved by reacting primary amines with functional α,α-dichlorotosylhydrozones under ambient conditions. This fast and efficient alternative affords exclusively 1,4-substituted triazole “click products” with complete retention of configuration. Primary amines, inherent to many natural products, can be modified in this way without protecting group manipulations. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Generation of Candidate Ligands for Nicotinic Acetylcholine Receptors via in situ Click Chemistry with a Soluble Acetylcholine Binding Protein Template

Nicotinic acetylcholine receptors (nAChRs), which are responsible for mediating key physiological functions, are ubiquitous in the central and peripheral nervous systems. As members of the Cys loop ligand-gated ion channel family, neuronal nAChRs are pentameric, composed of various permutations of α (α2 to α10) and β (β2 to β4) subunits forming functional heteromeric or homomeric receptors. Diversity in nAChR subunit composition complicates the development of selective ligands for specific subtypes, since the five binding sites reside at the subunit interfaces. The acetylcholine binding protein (AChBP), a soluble extracellular domain homologue secreted by mollusks, serves as a general structural surrogate for the nAChRs. In this work, homomeric AChBPs from Lymnaea and Aplysia snails were used as in situ templates for the generation of novel and potent ligands that selectively bind to these proteins. The cycloaddition reaction between building-block azides and alkynes to form stable 1,2,3-triazoles was used to generate the leads. The extent of triazole formation on the AChBP template correlated with the affinity of the triazole product for the nicotinic ligand binding site. Instead of the in situ protein-templated azide-alkyne cycloaddition reaction occurring at a localized, sequestered enzyme active center as previously shown, we demonstrate that the in situ reaction can take place at the subunit interfaces of an oligomeric protein and can thus be used as a tool for identifying novel candidate nAChR ligands. The crystal structure of one of the in situ-formed triazole-AChBP complexes shows binding poses and molecular determinants of interactions predicted from structures of known agonists and antagonists. Hence, the click chemistry approach with an in situ template of a receptor provides a novel synthetic avenue for generating candidate agonists and antagonists for ligand-gated ion channels.

ChemInform Abstract: Synthesis of a Fluorogenic Cyclooctyne Activated by Cu‐Free Click Chemistry.

AbstractThe coumarin‐conjugated cyclooctyne coumBARAC (X) is only weakly fluorescent.

Dye-incorporated water-soluble polymer via click triazole formation

A click triazole-polymerization is proposed for the synthesis of a water-soluble polymer dye. 4,4′-Diazidostilbene-2,2′-disulfonate (DASS) sodium salt reacted with a diacetylenic derivative of para-methyl red (PMR) in water, which was catalyzed by copper (I) ion or accelerated under ultrasound irradiation. DASS formed a soluble polymer with a carboxylate salt of PMR showing a water solubility of 120 g/100 mL but it generated an insoluble polymer with an alkyl ammonium salt. The prepared polymeric dye underwent color transition at pH 5–6, which is higher than that of PMR and similar to that of ortho-methyl red (OMR). The quinonoid canonical structure of PMR generated a purple water solution and was dominant in the polymer solution at pH below 5. Intra-molecular hydrogen bonding between the triazoles and azo nitrogens was presumed to have induced the formation of the quinonoid structure. The color change of the polymer was reproducible during successive up/down pH cycles.

Reaction of aminobenzoate esters with N,N′-dimethylformamide azine dihydrochloride: crucial influence of intramolecular hydrogen bonding for the formation of 1,2,4-triazoles

The reaction of N,N′-dimethylformamide azine (DMFA) or its dihydrochloride derivative (DMFA·2HCl) with o-RO(O)CC6H4NH2 (R = Me, Et) leads to co-crystallization products o-RO(O)CC6H4NH2·DMFA (R = Me, Et), while the same reaction of DMFA·2HCl with m- and p-RO(O)CC6H4NH2 (R = Me, Et) provides the corresponding 4-substituted 1,2,4-triazoles (1–4). Reaction of the same m- and p-substituted amines with DMFA has failed, while addition of a few drops of HCl or HNO3 in this reaction mixture has allowed us to obtain the corresponding triazole derivatives. Thus, the direct transamination reaction with the formation of triazoles 1–4 is catalysed by acidic catalysts. Molecular structures of DMFA, o-EtO(O)CC6H4NH2·DMFA and 3 were elucidated by X-ray diffraction.

One-pot morphogen driven self-constructing films based on non-covalent host–guest interactions

The construction of films with complex architectures through one-pot reactions taking place exclusively on a surface remains a challenge. Recently, to address this problem, we introduced a concept based on morphogen-driven film buildup. We used Cu(I) as morphogen and the Huisgens click-reaction between azide and alkyne groups on polymers as film building blocks. Here, we extend this concept to films whose integrity is based exclusively on non-covalent host–guest interactions that are reversible allowing much broader tunability of the film properties. We trigger electrochemically the self-construction of films based on clickable host (cyclodextrin) and guests (ferrocene or adamantane) both functionalized by alkyne functions and poly(acrylic acid) bearing azide groups. Under voltammetry cycles, where Cu(I) is formed in situ from Cu(II), the film builds up by the non-reversible covalent grafting of the host and guest molecules to poly(acrylic acid) chains through triazole formation and by the subsequent formation of reversible host–guest interactions which entirely support the film cohesion. This process leads to the continuous self-construction of a nanometre size film whose thickness increases with the application time of the electrochemical stimulus. The growth rate of the film can be tuned by changing in the buildup solution either the relative ratio in concentration of host and guest or through the competition between clickable and non-clickable guest molecules. The effect of different stimuli leading to the dissolution of the film is also reported.

One-pot synthesis of [1,2,3]triazole-fused pyrazinopyridazindione tricycles by a ‘click and activate’ approach

Substituted [1,2,3]triazole-fused pyrazinopyridazindione tricycles were synthesized in a four-component, stepwise condensation. The key step in this one-pot process was a thermal [3+2] triazole formation which activated the adjacent position and set the stage for a subsequent tandem nucleophilic aromatic substitution/cyclization sequence.

Synthesis of a Fluorogenic Cyclooctyne Activated by Cu-Free Click Chemistry

Cyclooctyne-based probes that become fluorescent upon reaction with azides are important targets for real-time imaging of azide-labeled biomolecules. The concise synthesis of a coumarin-conjugated cyclooctyne, coumBARAC, that undergoes a 10-fold enhancement in fluorescence quantum yield upon triazole formation with organic azides is reported. The design principles embodied in coumBARAC establish a platform for generating fluorogenic cyclooctynes suited for biological imaging.

Multistep flow synthesis of vinyl azides and their use in the copper-catalyzed Huisgen-type cycloaddition under inductive-heating conditions.

The multistep flow synthesis of vinyl azides and their application in the synthesis of vinyltriazoles is reported. The synthesis relies on a stable polymer-bound equivalent of iodine azide that serves to carry out 1,2-functionalization of alkenes in a telescope flow protocol. The intermediate 2-iodo azides are subjected to a DBU-mediated polymer-supported elimination step yielding vinyl azides in good yield. The third step involves the formation of vinyl triazoles by a copper-catalyzed Huisgen-"click" cycloaddition. The required heat is generated by electromagnetic induction based on copper. Copper serves both as heatable as well as catalytically active packed-bed material inside the flow reactor.

Click-chemistry for surface modification of monodisperse-macroporous particles

In this study, click chemistry was proposed as a tool for tuning the surface hydrophilicity of monodisperse-macroporous particles in micron-size range. The monodisperse-porous particles carrying hydrophobic or hydrophilic molecular brushes on their surfaces were obtained by the proposed modification. Hydrophilic poly(glycidyl methacrylate-co-ethylene dimethacrylate), poly(GMA-co-EDM) particles were hydrophobized by the covalent attachment of poly(octadecyl acrylate-co-propargyl acrylate), poly(ODA-co-PA) copolymer onto the particle surface via triazole formation by click chemistry. In the second part, Hydrophobic poly(4-chloromethylstyrene-co-divinylbenzene), poly(CMS-co-DVB) particles were hydrophilized by the covalent attachment of poly(vinyl alcohol), PVA onto their surface also via triazole formation by click chemistry. The presence of PVA and poly(ODA-co-PA) copolymer on the corresponding particles was shown by FTIR-DRS. After click-coupling reactions applied for both hydrophobic poly(CMS-co-DVB) and hydrophilic poly(GMA-co-EDM) particles, the marked changes in surface polarity were shown by contact angle measurements. Protein adsorption characteristics of plain and modified particles were investigated for both materials. In the isoelectric point of albumin, the non-specific albumin adsorption decreased from 225 to 80 mg/g by grafting PVA onto the poly(CMS-co-DVB) beads. On the other hand, the non-specific albumin adsorption onto the plain poly(GMA-co-EDM) beads increased from 50 to 400 mg/g by the covalent attachment of poly(ODA-co-PA) copolymer onto the bead-surface via click chemistry. The protein adsorption behavior was efficiently regulated by the covalent attachment of appropriate molecular brushes onto the surfaces of selected particles. The results indicated that “click chemistry” was an efficient tool for controlling the polarity of monodisperse-macroporous particles.

Citrus Peel Additives for One-Pot Triazole Formation by Decarboxylation, Nucleophilic Substitution, and Azide–Alkyne Cycloaddition Reactions

This undergraduate organic laboratory experiment consists of three different reactions occurring in the same flask: a cycloaddition reaction, preceded by decarboxylation and nucleophilic substitution reactions. The decarboxylation and cycloaddition reactions occur using identical Cu(I) catalyst and conditions. Orange, lemon, and other citrus fruit peels and juices in the presence of CuSO4 lead to the Cu(I)-catalyzed decarboxylation of 3-phenyl-2-propynoic acid to give ethynylbenzene. These click chemistry conditions give Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) of 2-azido-1-phenylethanone with ethynylbenzene to afford 1-phenyl-2-(4-phenyl-1H-1,2-3-triazol-1-yl)ethanone regioselectively.

Click to Join Peptides/Proteins Together

Copper(I) is able to catalyze Huisgen 1,3-dipolar cycloaddition in a "click" fashion. This copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction presents excellent chemoselectivity and occurs over a wide-range of reaction conditions. It shows tolerance to variation in both pH and solvent polarity, thereby facilitating the ligation of peptides and proteins to produce peptidomimetics and synthetic proteins. In addition, the only product formed is a 1,4-disubstituted-1,2,3-triazole moiety, in many aspects resembling the natural peptide bond, including hydrogen-bonding capability, planarity, distance between the 1 and 4 substituents, and conformational restriction of the peptide backbone; thus the triazole-backbone-modified peptide, in which a triazole replaces the amide bond, may be anticipated to present a secondary structure similar to that of its natural counterpart. This Focus Review describes the scope and applications of copper(I)-catalyzed alkyne–azide cycloaddition in synthetic peptide/protein chemistry.

Chiral Propargyl Alcohols via the Enantioselective Addition of Terminal Di- and Triynes to Aldehydes

The enantioselective addition of di- and triynes to aldehydes is presented, including the first examples of an asymmetric triyne addition. Modification of the Carreira alkynylation protocol shows that addition of diynes and triynes to α-branched aldehydes can be complete in as little as 4 h, and these reactions give good yields and enantioselectivities (up to 98% ee) for di- and triynes tested (aryl, alkyl, and silyl). It is shown for two cases (20 and 24) that products of this asymmetric addition reaction can undergo further manipulation (desilylation and triazole formation) without affecting the enantiopurity.