Azide-alkyne Reaction Research Articles

Double Strain‐Promoted Macrocyclization for the Rapid Selection of Cell‐Active Stapled Peptides

AbstractPeptide stapling is a method for designing macrocyclic alpha‐helical inhibitors of protein–protein interactions. However, obtaining a cell‐active inhibitor can require significant optimization. We report a novel stapling technique based on a double strain‐promoted azide–alkyne reaction, and exploit its biocompatibility to accelerate the discovery of cell‐active stapled peptides. As a proof of concept, MDM2‐binding peptides were stapled in parallel, directly in cell culture medium in 96‐well plates, and simultaneously evaluated in a p53 reporter assay. This in situ stapling/screening process gave an optimal candidate that showed improved proteolytic stability and nanomolar binding to MDM2 in subsequent biophysical assays. α‐Helicity was confirmed by a crystal structure of the MDM2‐peptide complex. This work introduces in situ stapling as a versatile biocompatible technique with many other potential high‐throughput biological applications.

Double Strain-Promoted Macrocyclization for the Rapid Selection of Cell-Active Stapled Peptides.

Peptide stapling is a method for designing macrocyclic alpha-helical inhibitors of protein-protein interactions. However, obtaining a cell-active inhibitor can require significant optimization. We report a novel stapling technique based on a double strain-promoted azide-alkyne reaction, and exploit its biocompatibility to accelerate the discovery of cell-active stapled peptides. As a proof of concept, MDM2-binding peptides were stapled in parallel, directly in cell culture medium in 96-well plates, and simultaneously evaluated in a p53 reporter assay. This in situ stapling/screening process gave an optimal candidate that showed improved proteolytic stability and nanomolar binding to MDM2 in subsequent biophysical assays. α-Helicity was confirmed by a crystal structure of the MDM2-peptide complex. This work introduces in situ stapling as a versatile biocompatible technique with many other potential high-throughput biological applications.

Copper catalyst efficiency for the CuAAC synthesis of a poly(N-isopropylacrylamide) conjugated hyaluronan.

Poly(N-isopropylacrylamide) conjugated hyaluronan (HA-pN), a brush-like copolymer system which serves as a polymer vehicle for cellular and drug delivery, has been previously synthesized via the copper catalyzed azide-alkyne reaction (CuAAC) using a combination of copper sulfate and ascorbic acid (CuAsc) as the catalytic system of choice. Bromotris(triphenylphosphine) copper(I) (CuBr(PPh3)3) is an alternative catalytic compound containing a phosphorous ligand which stabilizes copper in the +1 oxidative state in aqueous solvents and can be employed at true catalyst concentrations. CuAsc and CuBr(PPh3)3 were compared for their efficiency; 1) in the synthesis of HA-pN via CuAAC; 2) in producing thermoresponsive compositions and 3) in being extracted from the polymeric compositions. The synthesis of the brush copolymer was carried out under strict Schlenk conditions, then characterized by ATR-FTIR, 1H NMR, ICP-SFMS, and rheological measurements. CuBr(PPh3)3 catalyzed CuAAC leads to better grafting in water, at a true catalyst concentration, compared to CuAsc. Polymeric solutions exhibited similar traits of increasing mechanical stiffness with rising temperature. Despite purification via chelation and/or dialysis, residual copper was present in similar concentrations in the final polymers. In the CuAAC driven copolymer synthesis of the HA-pN, CuBr(PPh3)3 is a better catalyst than CuAsc.

Cytotoxic activity of triazole-containing alkyl β-D-glucopyranosides on a human T-cell leukemia cell line.

BackgroundSimple glycoside surfactants represent a class of chemicals that are produced from renewable raw materials. They are considered to be environmentally safe and, therefore, are increasingly used as pharmaceuticals, detergents, and personal care products. Although they display low to moderate toxicity in cells in culture, the underlying mechanisms of surfactant-mediated cytotoxicity are poorly investigated.ResultsWe synthesized a series of triazole-linked (fluoro)alkyl β-glucopyranosides using the copper-catalyzed azide-alkyne reaction, one of many popular “click” reactions that enable efficient preparation of structurally diverse compounds, and investigate the toxicity of this novel class of surfactant in the Jurkat cell line. Similar to other carbohydrate surfactants, the cytotoxicity of the triazole-linked alkyl β-glucopyranosides was low, with IC50 values decreasing from 1198 to 24 μM as the hydrophobic tail length increased from 8 to 16 carbons. The two alkyl β-glucopyranosides with the longest hydrophobic tails caused apoptosis by mechanisms involving mitochondrial depolarization and caspase-3 activation.ConclusionsTriazole-linked, glucose-based surfactants 4a-g and other carbohydrate surfactants may cause apoptosis, and not necrosis, at low micromolar concentrations via induction of the intrinsic apoptotic cascade; however, additional studies are needed to fully explore the molecular mechanisms of their toxicity.Graphical Triazole-linked, glucose-based surfactants cause apoptosis, and not necrosis, at low micromolar concentrations via induction of the intrinsic apoptotic cascade.Electronic supplementary materialThe online version of this article (doi:10.1186/s13065-014-0072-1) contains supplementary material, which is available to authorized users.

Reversible Control over Molecular Recognition in Surface‐Bound Photoswitchable Hydrogen‐Bonding Receptors: Towards Read–Write–Erase Molecular Printboards

The synthesis of an anthracene-bearing photoactive barbituric acid receptor and its subsequent grafting onto azide-terminated alkanethiol/Au self-assembled monolayers by using an Cu(I) -catalyzed azide-alkyne reaction is reported. Monolayer characterization using contact-angle measurements, electrochemistry, and spectroscopic ellipsometry indicate that the monolayer conversion is fast and complete. Irradiation of the receptor leads to photodimerization of the anthracenes, which induces the open-to-closed gating of the receptor by blocking access to the binding site. The process is thermally reversible, and polarization-modulated IR reflection-absorption spectroscopy indicates that photochemical closure and thermal opening of the surface-bound receptors occur in 70 and 100 % conversion, respectively. Affinity of the open and closed surface-bound receptor was characterized by using force spectroscopy with a barbituric-acid-modified atomic force microscope tip.

A Generic Strategy for Co‐Presentation of Heparin‐Binding Growth Factors Based on CVD Polymerization

A multifunctional copolymer with both aldehyde and alkyne groups is synthesized by chemical vapor deposition (CVD) for orthogonal co-immobilization of biomolecules. Surface analytical methods including FTIR and XPS are used to confirm the surface modification. Heparin-binding growth factors [basic fibroblast growth factor (bFGF) in this study] can be immobilized through interaction with heparin, which was covalently attached to the CVD surface through an aldehyde-hydrazide reaction. In parallel, an alkyne-azide reaction is used to orthogonally co-immobilize an adhesion peptide as the second biomolecule.

A "Mix-and-Click" Approach to Double Core-Shell Micelle Functionalization.

A micellar scaffold formed by self-assembly of a reversible addition-fragmentation chain transfer (RAFT)-synthesized amphiphilic diblock copolymer has been prepared to contain two orthogonal click-compatible functionalities in the core and shell. These functionalities (norbornenes in the core and terminal alkynes in the shell) have been used as handles to modify the micellar assembly in the core using tetrazine-norbornene chemistry or the shell using the copper-catalyzed azide-alkyne reaction. Additionally, both core and shell modifications were carried out in a tandem, one-pot process using the orthogonal chemistries mentioned above. In all cases the reactions were found to be highly efficient, requiring little excess of the modifying small molecule and very simple to perform under ambient conditions.

ChemInform Abstract: Power Ultrasound in Metal‐Assisted Synthesis: From Classical Barbier‐like Reactions to Click Chemistry

AbstractReview: 52 refs.

Spatial and temporal control of the alkyne–azide cycloaddition by photoinitiated Cu(II) reduction

The click reaction paradigm is focused on the development and implementation of reactions that are simple to perform while being robust and providing exquisite control of the reaction and its products. Arguably the most prolific and powerful of these reactions, the copper-catalysed alkyne-azide reaction (CuAAC) is highly efficient and ubiquitous in an ever increasing number of synthetic methodologies and applications, including bioconjugation, labelling, surface functionalization, dendrimer synthesis, polymer synthesis and polymer modification. Unfortunately, as the Cu(I) catalyst is typically generated by the chemical reduction of Cu(II) to Cu(I), or added as a Cu(I) salt, temporal and spatial control of the CuAAC reaction is not readily achieved. Here, we demonstrate catalysis of the CuAAC reaction via the photochemical reduction of Cu(II) to Cu(I), affording comprehensive spatial and temporal control of the CuAAC reaction using standard photolithographic techniques. Results reveal the diverse capability of this technique in small molecule synthesis, patterned material fabrication and patterned chemical modification.

Power ultrasound in metal-assisted synthesis: From classical Barbier-like reactions to click chemistry

The search for more efficient and greener synthetic procedures to obtain highly functionalized chemical structures has always found in metal-assisted reactions a noteworthy strategy. All these reactions fall in the main domain of sonochemistry; in fact few techniques can compete with power ultrasound in so efficiently activating a metal surface, thus enhancing and accelerating its subsequent reaction with an organic substrate. Young researchers will certainly benefit from the rich literature and past experience of several pioneers who have, since the early eighties, laid the foundations of modern sonochemical synthetic protocols. Herein we provide a concise overview that describes how ultrasound acts in such a way as to make it a fundamental tool in improving the classical one-step coupling promoted by zero-valent metal species, usually referred to as Barbier-like reactions. From early hallmarks to recent accomplishments, especially the latest Cu-catalyzed alkyne-azide reaction (the so-called Click reaction), intended to be a universal ligation in chemistry and biology; we highlight the role and crucial effects of sonication on these processes.

Polyvalent oligonucleotide iron oxide nanoparticle "click" conjugates.

We have utilized the copper-catalyzed azide-alkyne reaction to form a dense monolayer of oligonucleotides on a superparamagnetic nanoparticle core. These particles exhibit the canonical properties of materials densely functionalized with DNA, which can be controlled by modulating the density of oligonucleotides on the surface of the particles. Furthermore, like their Au analogues, these particles can easily cross HeLa (cervical cancer) cell membranes without transfection agents due to their dense DNA shell. Importantly, this approach should be generalizable to other azide-functionalized particles.

In situ click chemistry: probing the binding landscapes of biological molecules

Combinatorial approaches to the discovery of new functional molecules are well established among chemists and biologists, inspired in large measure by the modular composition of many systems and molecules in Nature. Many approaches rely on the synthesis and testing of individual members of a candidate combinatorial library, but attention has also been paid to techniques that allow the target to self-assemble its own binding agents. These fragment-based methods, grouped under the general heading of target-guided synthesis (TGS), show great promise in lead discovery applications. In this tutorial review, we review the use of the 1,3-dipolar cycloaddition reaction of organic azides and alkynes in a kinetically-controlled TGS approach, termed in situ click chemistry. The azide-alkyne reaction has several distinct advantages, most notably high chemoselectivity, very low background ligation rates, facile synthetic accessibility, and the stability and properties of the 1,2,3-triazole products. Examples of the discovery of potent inhibitors of acetylcholinesterases, carbonic anhydrase, HIV-protease, and chitinase are described, as are methods for the templated assembly of agents that bind DNA and proteins.

Functionalization of Polymer Microspheres Using Click Chemistry

We describe a new method to covalently link a wide variety of molecules to the surface of colloidal polymer microspheres using the Cu(I)-catalyzed azide-alkyne reaction, most commonly known under the class of reactions identified by the term click chemistry. The method is generic and readily applied to a spectrum of colloidal particle systems allowing surfaces to be tailored with virtually any desired functionality. To demonstrate this method, polystyrene microspheres were functionalized with two different polyethylene oxide-based polymers, and changes in hydrodynamic radii after functionalization were measured using dynamic light scattering. Control of surface functional groups was demonstrated by fluorescently labeling the colloidal microspheres using the same Cu-catalyzed azide-alkyne cycloaddition reaction.