Copper-catalyzed Click Reaction Research Articles

2-Hydroxyethylcellulose and Amphiphilic Block Polymer Conjugates Form Mechanically Tunable and Nonswellable Hydrogels

Herein, we report a family of mechanically tunable, nonswellable hydrogels that are based on a 2-hydroxyethylcellulose (HEC) scaffold grafted with amphiphilic diblock copolymers. Poly[(oligo(ethylene glycol)methyl ether methacrylate]-b-poly(methyl methacrylate) (POEGMA-b-PMMA) diblock copolymers of different compositions were created via RAFT polymerization using an alkyne terminated macro chain transfer agent (CTA). 2-Hydroxyethylcellulose (HEC) was modified with azide groups and the diblock copolymers were attached to the backbone via the copper-catalyzed click reaction to yield HEC-g-(POEGMA-b-PMMA) graft terpolymers. The resulting conjugates were soluble in DMF and able to form hydrogels upon simple solvent exchange in water. By increasing the concentration of the conjugates in DMF, the storage moduli of the hydrogels increased and the pore size in the gel decreased. After hydrogel formation, the structures were also found to be nonswellable (no macroscopic volume change upon incubation in water), which is an important feature for retaining size and mechanical integrity of the gels over time. Moreover, these materials were able to be electrospun into fibers that, upon hydration, formed fibrous hydrogel structures. The nonswellable and tunable mechanical properties of these materials imply great potential for a variety of applications such as personal care, active delivery, and tissue engineering.

Modular synthesis of self-assembling Janus-dendrimers and facile preparation of drug-loaded dendrimersomes.

Materials and methods aimed at the next generation of nanoscale carriers for drugs and other therapeutics are currently in great demand. Yet, creating these precise molecular arrangements in a feasible and straightforward manner represents a remarkable challenge. Herein we report a modular synthetic route for amphiphilic Janus-dendrimers via a copper-catalyzed click reaction (CuAAC) and a facile procedure, using simple injection, to obtain highly uniform dendrimersomes with efficient loading of the model drug compound propranolol. The resulting assemblies were analyzed by dynamic light scattering and cryogenic transmission electron microscopy revealing the formation of unilamellar and multilamellar dendrimersomes. The formation of a bilayer structure was confirmed using cryo-TEM and confocal microscopy visualization of an encapsulated solvatochromic dye (Nile Red). The dendrimersomes reported here are tunable in size, stable over time and display robust thermal stability in aqueous media. Our results expand the scope of dendrimer-based supramolecular colloidal systems and offer the means for one-step fabrication of drug-loaded dendrimersomes in the size range of 90-200 nm, ideal for biomedical applications.

Copper-Catalyzed Click Reaction on/in Live Cells.

We demonstrated that copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction could be performed inside live mammalian cells without using a chelating azide. Under optimized conditions, the reaction was performed in human ovary cancer cell line OVCAR5 in which newly synthesized proteins were metabolically modified with homopropargylglycine (HPG). This model system allowed us to estimate the efficiency of the reaction on the cell membranes and in the cytosol using mass spectrometry. We found that the reaction was greatly promoted by a tris(triazolylmethyl)amine CuI ligand tethering a cell-penetrating peptide. Uptake of the ligand, copper, and a biotin-tagged azide in the cells was determined to be 69 ± 2, 163 ± 3 and 1.3 ± 0.1 µM, respectively. After 10 minutes of reaction, the product yields on the membrane and cytosolic proteins were higher than 18% and 0.8%, respectively, while 75% cells remained viable. By reducing the biothiols in the system by scraping or treatment with N-ethylmalemide, the reaction yield on the cytosolic proteins was greatly improved to ~9% and ~14%, respectively, while the yield on the membrane proteins remained unchanged. The results indicate that out of many possibilities, deactivation of the current copper catalysts by biothiols is the major reason for the low yield of CuAAC reaction in the cytosol. Overall, we have improved the efficiency for CuAAC reaction on live cells by 3-fold. Despite the low yielding inside live cells, the products that strongly bind to the intracellular targets can be detected by mass spectrometry. Hence, the in situ CuAAC reaction can be potentially used for screening of cell-specific enzyme inhibitors or biomarkers containing 1,4-substituted 1,2,3-triazoles.

Versatile synthesis of end-reactive polyrotaxanes applicable to fabrication of supramolecular biomaterials.

Cyclodextrin (CD)-threaded polyrotaxanes (PRXs) with reactive functional groups at the terminals of the axle polymers are attractive candidates for the design of supramolecular materials. Herein, we describe a novel and simple synthetic method for end-reactive PRXs using bis(2-amino-3-phenylpropyl) poly(ethylene glycol) (PEG-Ph-NH2) as an axle polymer and commercially available 4-substituted benzoic acids as capping reagents. The terminal 2-amino-3-phenylpropyl groups of PEG-Ph-NH2 block the dethreading of the α-CDs after capping with 4-substituted benzoic acids. By this method, two series of azide group-terminated polyrotaxanes (benzylazide: PRX-Bn-N3, phenylazide: PRX-Ph-N3,) were synthesized for functionalization via click reactions. The PRX-Bn-N3 and PRX-Ph-N3 reacted quickly and efficiently with p-(tert-butyl)phenylacetylene via copper-catalyzed click reactions. Additionally, the terminal azide groups of the PRX-Bn-N3 could be modified with dibenzylcyclooctyne (DBCO)-conjugated fluorescent molecules via a copper-free click reaction; this fluorescently labeled PRX was utilized for intracellular fluorescence imaging. The method of synthesizing end-reactive PRXs described herein is simple and versatile for the design of diverse functional PRXs and can be applied to the fabrication of PRX-based supramolecular biomaterials.

Synthesis and click reaction of tubulin polymerization inhibitor 9-azido-α-noscapine

An efficient protocol for the synthesis of tubulin polymerization inhibitor, 9-azido-α-noscapine 2h from 9-amino-α-noscapine 2g is developed using mild reaction conditions (t-butyl nitrite/trimethylsilyl azide in acetonitrile at room temperature). Operational simplicity, high product yield without formation of any side products are the advantages of this protocol. Further copper catalyzed click reactions of 9-azido-α-noscapine 2h with alkynes 6a–f resulted 9-triazolyl noscapinoids 7a–f resulted in excellent yields. Developed an amicable protocol for the synthesis of 9-azido-α-noscapine from 9-amino-α-noscapine under mild reaction conditions; was further derivatized to triazoles using click chemistry.

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy. The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

Synthesis and Biological Evaluation of Triazolyl 13α-Estrone-Nucleoside Bioconjugates.

2′-Deoxynucleoside conjugates of 13α-estrone were synthesized by applying the copper-catalyzed alkyne–azide click reaction (CuAAC). For the introduction of the azido group the 5′-position of the nucleosides and a propargyl ether functional group on the 3-hydroxy group of 13α-estrone were chosen. The best yields were realized in our hands when the 3′-hydroxy groups of the nucleosides were protected by acetyl groups and the 5′-hydroxy groups were modified by the tosyl–azide exchange method. The commonly used conditions for click reaction between the protected-5′-azidonucleosides and the steroid alkyne was slightly modified by using 1.5 equivalent of Cu(I) catalyst. All the prepared conjugates were evaluated in vitro by means of MTT assays for antiproliferative activity against a panel of human adherent cell lines (HeLa, MCF-7 and A2780) and the potential inhibitory activity of the new conjugates on human 17β-hydroxysteroid dehydrogenase 1 (17β-HSD1) was investigated via in vitro radiosubstrate incubation. Some protected conjugates displayed moderate antiproliferative properties against a panel of human adherent cancer cell lines (the protected cytidine conjugate proved to be the most potent with IC50 value of 9 μM). The thymidine conjugate displayed considerable 17β-HSD1 inhibitory activity (IC50 = 19 μM).

One-pot synthesis of oxoisoindoline-1,2,3-triazole hybrid by a Ugi–click reaction

ABSTRACT1,2,3-Triazole-3-oxoisoindoline-1-carboxamide system was successfully synthesized by using a combination of Ugi and click reactions. This two-step, one-pot synthesis was started by the reaction of 2-formyl benzoic acid, propargyl amine, and cyclohexyl isocyanide in ethanol. The resultant Ugi adduct underwent a copper-catalyzed click reaction, producing the desired products in good yields.

Safe Generation and Direct Use of Chlorine Azide in Flow Chemistry: 1,2-Azidochlorination of Olefins and Access to Triazoles

A safe, fast procedure for the formation of chlorine azide and its trapping by 1,2-addition reaction on olefins is described. ClN₃ was generated in situ from NaN₃ and NaOCl in the presence of acetic acid, hosted in an organic phase to avoid decomposition and exposed to various alkenes. A copper-catalyzed click reaction then afforded triazoles from the resulting addition products. Telescoping of both reactions was enabled by an in-line workup and subsequent liquid–liquid separation.

One-pot modification of 5′-capped RNA based on methionine analogs

This paper outlines chemically and enzymatically synthesized S-adenosylmethionine (AdoMet) analogs and their use in the site-specific modification of RNA by methyltransferases, enabling the facile attachment of clickable moieties to the nucleic acid. We then focus on methodological aspects of setting up a methyltransferase-based enzymatic cascade reaction starting from methionine analogs. This strategy is applied to the one-pot modification of the mRNA cap which is subsequently derivatized in copper-free and copper-catalyzed click reactions. We show that high transfer efficiencies to the cap are obtained using Se-propargyl-, hexenynyl- and azido-bearing methionine analogs. By switching to other methyltransferases our one-pot modification approach should be directly applicable to the regiospecific modification of other target molecules including nucleic acids, proteins and small molecules.

Efficient synthesis of novel 1,2,3-triazole-linked quinoxaline scaffold via copper-catalyzed click reactions

New 1,2,3-triazoles-linked quinoxaline, were prepared by the reaction of 2-chloro-3-(prop-2-ynyloxy)quinoxaline or 2,3-bis(prop-2-ynyloxy)quinoxaline with aromatic azides via copper-catalyzed click reactions in the presence of the Schiff base ligands.

Biotinylation of Deoxyguanosine at the Abasic Site in Double-Stranded Oligodeoxynucleotides.

Biotinylation of deoxyguanosine at an abasic site in double-stranded oligodeoxynucleotides was studied. The biotinylation of deoxyguanosine is achieved by copper-catalyzed click reaction after the conjugation of the oligodeoxynucleotide with 2-oxohex-5-ynal. The biotinylation enables visualization of the biotinylated oligodeoxynucleotides by chemiluminescence on a nylon membrane. In order to investigate the biotinylated site, the biotinylated oligodeoxynucleotides were amplified by the DNA polymerase chain reaction. Replacement of guanine opposing the abasic site with adenine generated by the activity of the terminal deoxynucleotidyl transferase of DNA polymerase was detected by DNA sequencing analysis and restriction endonuclease digestion. This study suggests that 2-oxohex-5-ynal may be useful for the detection of the unpaired deoxyguanosine endogenously generated at abasic sites in genomic DNA.

Functionalization of graphene with self-doped conducting polypyrrole by click coupling

The synthesis of self-doped conducting polypyrrole-grafted graphene sheets (GS-PPy) for non-volatile memory applications is reported. First, the alkyne-modified graphene sheets (GS-alkyne) were covalently functionalized with a water-soluble polymer containing numerous anionic SO3− dopants by a copper-catalyzed click reaction. Then, polypyrrole was covalently grafted onto the functionalized graphene sheets by chemical oxidative polymerization to produce GS-PPy hybrids. The GS-PPy hybrids showed a uniform coating of PPy on the GS sheets, good dispersion in aqueous solutions, high electrical conductivity, and red-shifted absorption peak in the UV/Visible spectra. The non-volatile memory device composed of a Al/(GS-PPy/poly(vinyl alcohol))/Al structure, produced by spin coating of the aqueous GS-PPy/poly(vinyl alcohol) solution, showed a good write-once read-many times memory behavior, which was due to good electrical and optical absorption properties of the GS-PPy hybrids. The findings of this study provide a potential solution for the fabrication of water-soluble graphene-based hybrids for non-volatile resistive-memory-based applications.

18F-AmBF3-MJ9: A novel radiofluorinated bombesin derivative for prostate cancer imaging

A novel radiofluorinated derivative of bombesin, 18F-AmBF3-MJ9, was synthesized and evaluated for its potential to image prostate cancer by targeting the gastrin releasing peptide receptor (GRPR). AmBF3-MJ9 was prepared from an ammoniomethyl-trifluoroborate (AmBF3) conjugated alkyne 2 and azidoacetyl-MJ9 via a copper-catalyzed click reaction, and had good binding affinity for GRPR (Ki=0.5±0.1nM). The 18F-labeling was performed via a facile one-step 18F–19F isotope exchange reaction, and 18F-AmBF3-MJ9 was obtained in 23±5% (n=3) radiochemical yield in 25min with >99% radiochemical purity and 100±32GBq/μmol specific activity. 18F-AmBF3-MJ9 was stable in mouse plasma, and was partially (22–30%) internalized after binding to GRPR. Positron emission tomography (PET) imaging and biodistribution studies in mice showed fast renal excretion and good uptake of 18F-AmBF3-MJ9 by GRPR-expressing pancreas and PC-3 prostate cancer xenografts. Tumor uptake was 1.37±0.25%ID/g at 1h, and 2.20±0.13%ID/g at 2h post-injection (p.i.) with low background uptake and excellent tumor visualization (tumor-to-muscle ratios of 75.4±5.5). These data suggest that 18F-AmBF3-MJ9 is a promising PET tracer for imaging GRPR-expressing prostate cancers.

Continuous metal scavenging and coupling to one-pot copper-catalyzed azide-alkyne cycloaddition click reaction in flow

Increasing usage of catalytic chemistry calls for efficient removal of metal traces. This paper describes the development and optimization of a scavenger-based extraction in flow to remove metal catalysts. It enables liquid–liquid extraction with slug flow and phase separation with a porous fluoropolymer membrane. The use of this unit for copper scavenging of various copper sources from organic solvent was studied. The effects of scavenger type (EDTA, DTPA, EDDS), concentration and pH were also investigated. Such analysis allowed to achieve extraction performance as high as 99% at pH of the scavenger solution adjusted to 9.4 and molar ratio of scavenger to copper of 10. Process integration is achieved by coupling this unit downstream to a flow reaction using homogeneous metal-based catalysis, i.e. presenting a continuous uninterrupted metal scavenging unit. The copper-catalyzed azide-alkyne cycloaddition click reaction is studied in one-pot eliminating the need to isolate and handle potentially explosive azide. The triazole product is attained in flow with high yield of up to 92% with 30min residence time. The level of purity requirement for pharmaceuticals is met in one stage of extraction.

Application of meta‐ and para‐Phenylenediamine as Enhanced Oxime Ligation Catalysts for Protein Labeling, PEGylation, Immobilization, and Release

Meta- and para-phenylenediamines have recently been shown to catalyze oxime and hydrazone ligation reactions at rates much faster than aniline, a commonly used catalyst. Here, we demonstrate how these new catalysts can be used in a generally applicable procedure for fluorescent labeling, PEGylation, immobilization, and release of aldehyde- and ketone- functionalized proteins. The chemical orthogonality of phenylenediamine-catalyzed oxime ligation versus copper-catalyzed click reaction has also been harnessed for simultaneous dual labeling of bifunctional proteins containing both aldehyde and alkyne groups in high yield.

Transferase-Mediated Labeling of Protein N-Termini with Click Chemistry Handles.

The E. coli aminoacyl transferase (AaT) can be used to transfer a variety of unnatural amino acids, including those with azide or alkyne groups, to the α-amine of a protein with an N-terminal Lys or Arg. Subsequent functionalization through either copper-catalyzed or strain-promoted click reactions can be used to label the protein with fluorophores or biotin. This method can be used to directly detect AaT substrates or in a two-step protocol to detect substrates of the mammalian ATE1 transferase.

Abstract 512: Cell-based analysis of oxidative stress, lipid peroxidation and lipid peroxidation-derived protein modifications using fluorescence microscopy

Abstract Oxidative stress plays an important role in the progression of several diseases including inflammation, atherosclerosis, aging and age-related degenerative disorders. Reactive oxygen species damage membrane bound lipids resulting in lipid peroxidation-derived protein modifications. Cell-based measurements of oxidative stress, lipid peroxidation and protein carbonylation by traditional fluorescence microscopy provide a powerful platform to quantitate oxidative stress and lipid peroxidation. Here, we used three different approaches to measure oxidative stress and lipid peroxidation in cells by fluorescence microscopy. 1) Two new fluorogenic probes, CellROX™ Orange and CellROX ™ Green Reagents to measure oxidative stress in cells, 2) Image-iT® Lipid Peroxidation Kit for a ratiometric determination of lipid peroxidation in live cells 3) Click-iT® Lipid Peroxidation Imaging Kit, a click chemistry-based approach which utilizes incorporation of an alkyne-modified unsaturated fatty acid analog, linoleamide, into the cellular membranes. The resulting oxidation products, like 9, 12-dioxo-10(E) dodecenoic acid (DODE) can readily modify proteins and these modifications were readily detected in fixed cells by the copper-catalyzed click reaction using fluorescent azides. Using these approaches, we measured oxidative stress and lipid peroxidation caused by several oxidants in cells. Increases in oxidative stress, lipid peroxidation, and protein modifications were assessed by high content imaging and analysis as well as traditional fluorescence microscopy. In the models tested, at least 2-3 fold increases were observed compared to controls was and responses were successfully inhibited by antioxidants. The three strategies described here provide powerful new tools for the assessment of oxidative stress in cells and convey distinct advantages over existing cell-based methods. Citation Format: Bhaskar S. Mandavilli, Robert Aggeler. Cell-based analysis of oxidative stress, lipid peroxidation and lipid peroxidation-derived protein modifications using fluorescence microscopy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 512. doi:10.1158/1538-7445.AM2014-512

A dicationic, podand-like, ionic liquid water system accelerated copper-catalyzed azide-alkyne click reaction

In this work, an effective, task specific, dicationic, podand-like ionic liquid was synthesized and applied to improve the capability features of click reaction. Moreover, to broaden the scope and decreasing the serious limitations of preparation methods of organic azides, a simple green procedure for the preparation of alkyl azides, the fundamental starting materials in click reactions, from alcohols under solvent-free conditions and microwave irradiation has been reported, for the first time.

Magnetically Separable Nano Fe3O4 Catalyzed Direct Azidation of Allylic and Benzylic Alcohols Followed by Copper-Catalyzed Click Reaction

A competent one-pot method comprising magnetically separable nano Fe₃O₄ catalyzed direct azidation of allylic and benzylic alcohols followed by the copper-catalyzed click reaction of the corresponding azides with alkynes is reported. This method gave a direct access to several 1,2,3-triazoles starting from various allylic and benzylic alcohols via their respective azides.