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Bioconjugate Chemistry Mar 2018Interrogating biological systems is often limited by access to biological probes. The emergence of "click chemistry" has revolutionized bioconjugate chemistry by... (Review)
Review
Interrogating biological systems is often limited by access to biological probes. The emergence of "click chemistry" has revolutionized bioconjugate chemistry by providing facile reaction conditions amenable to both biologic molecules and small molecule probes such as fluorophores, toxins, or therapeutics. One particularly popular version is the copper-catalyzed azide-alkyne cycloaddition (AAC) reaction, which has spawned new alternatives such as the strain-promoted azide-alkyne cycloaddition reaction, among others. This focused review highlights practical approaches to AAC reactions for the synthesis of peptide or protein bioconjugates and contrasts current challenges and limitations in light of recent advances in the field. The conical success of antibody drug conjugates has expanded the toolbox of linkers and payloads to facilitate practical applications of bioconjugation to create novel therapeutics and biologic probes. The AAC reaction in particular is poised to enable a large set of functionalized molecules as a combinatorial approach to high-throughput bioconjugate generation, screening, and honing of lead compounds.
Topics: Alkynes; Animals; Azides; Click Chemistry; Cycloaddition Reaction; Humans; Nucleic Acids; Peptides; Proteins
PubMed: 29287474
DOI: 10.1021/acs.bioconjchem.7b00633 -
Journal of the American Chemical Society Jul 2021We present a chemical approach to profile fatty acid uptake in single cells. We use azide-modified analogues to probe the fatty acid influx and surface-immobilized...
We present a chemical approach to profile fatty acid uptake in single cells. We use azide-modified analogues to probe the fatty acid influx and surface-immobilized dendrimers with dibenzocyclooctyne (DBCO) groups for detection. A competition between the fatty acid probes and BHQ2-azide quencher molecules generates fluorescence signals in a concentration-dependent manner. By integrating this method onto a microfluidics-based multiplex protein analysis platform, we resolved the relationships between fatty acid influx, oncogenic signaling activities, and cell proliferation in single glioblastoma cells. We found that p70S6K and 4EBP1 differentially correlated with fatty acid uptake. We validated that cotargeting p70S6K and fatty acid metabolism synergistically inhibited cell proliferation. Our work provided the first example of studying fatty acid metabolism in the context of protein signaling at single-cell resolution and generated new insights into cancer biology.
Topics: Azides; Cell Proliferation; Cyclooctanes; Dendrimers; Fatty Acids; Fluorescence; Glioblastoma; Humans; Molecular Structure; Single-Cell Analysis; Surface Properties
PubMed: 34266234
DOI: 10.1021/jacs.1c05103 -
Chemical Society Reviews Apr 2010Copper-catalyzed azide-alkyne cycloaddition (CuAAC) is a widely utilized, reliable, and straightforward way for making covalent connections between building blocks... (Review)
Review
Copper-catalyzed azide-alkyne cycloaddition (CuAAC) is a widely utilized, reliable, and straightforward way for making covalent connections between building blocks containing various functional groups. It has been used in organic synthesis, medicinal chemistry, surface and polymer chemistry, and bioconjugation applications. Despite the apparent simplicity of the reaction, its mechanism involves multiple reversible steps involving coordination complexes of copper(I) acetylides of varying nuclearity. Understanding and controlling these equilibria is of paramount importance for channeling the reaction into the productive catalytic cycle. This tutorial review examines the history of the development of the CuAAC reaction, its key mechanistic aspects, and highlights the features that make it useful to practitioners in different fields of chemical science.
Topics: Alkynes; Azides; Catalysis; Copper; Cyclization; Iodine; Sulfones
PubMed: 20309487
DOI: 10.1039/b904091a -
The Protein Journal Jun 2023Hydrazoic acid (HN) and its deprotonated form azide ion (N) (AHA) are toxic because they inhibit the cytochrome c oxidase complex IV (CoX IV) embedded in the inner...
Hydrazoic acid (HN) and its deprotonated form azide ion (N) (AHA) are toxic because they inhibit the cytochrome c oxidase complex IV (CoX IV) embedded in the inner mitochondrial membrane that forms part of the enzyme complexes involved in cellular respiration. Critical to its toxicity is the inhibition of CoX IV in the central nervous system and cardiovascular system. Hydrazoic acid is an ionizable species and its affinity for membranes, and the associated permeabilities, depend on the pH values of aqueous media on both sides of the membranes. In this article, we address the permeability of AHA through the biological membrane. In order to understand the affinity of the membrane for the neutral and ionized form of azide, we measured the octanol/water partition coefficients at pH values of 2.0 and 8.0, which are 2.01 and 0.00034, respectively. Using a Parallel Artificial Membrane Permeability Assay (PAMPA) experiment, we measured the effective permeability through the membrane, which is logP - 4.97 and - 5.26 for pH values of 7.4 and pH 8.0, respectively. Experimental permeability was used to validate theoretical permeability, which was estimated by numerically solving a Smoluchowski equation for AHA diffusion through the membrane. We demonstrated that the rate of permeation through the cell membrane of 8.46·10 s is much higher than the rate of the chemical step of CoX IV inhibition by azide of 200 s. The results of this study show that transport through the membrane does not represent the rate-limiting step and therefore does not control the rate of CoX IV inhibition in the mitochondria. However, the observed dynamics of azide poisoning is controlled by circulatory transport that takes place on a time scale of minutes.
Topics: Azides; Membranes, Artificial; Cell Membrane; Octanols; Permeability; Hydrogen-Ion Concentration
PubMed: 37289420
DOI: 10.1007/s10930-023-10127-3 -
Molecules (Basel, Switzerland) Mar 2016Organic azides are key motifs in compounds of relevance to chemical biology, medicinal chemistry and materials science. In addition, they also serve as useful building... (Review)
Review
Organic azides are key motifs in compounds of relevance to chemical biology, medicinal chemistry and materials science. In addition, they also serve as useful building blocks due to their remarkable reactivity. Therefore, the development of efficient protocols to synthesize these compounds is of great significance. This paper reviews the major applications and development of azidation in difunctionalization of olefins using azide reagents.
Topics: Alkenes; Azides; Catalysis; Models, Chemical
PubMed: 26999085
DOI: 10.3390/molecules21030352 -
Biomolecules Apr 2020The copper-catalyzed alkyne-azide cycloaddition (CuAAC) is one of the most powerful chemical strategies for selective fluorescent labeling of biomolecules in in vitro or...
The copper-catalyzed alkyne-azide cycloaddition (CuAAC) is one of the most powerful chemical strategies for selective fluorescent labeling of biomolecules in in vitro or biological systems. In order to accelerate the ligation process and ensure efficient formation of conjugates under diluted conditions, external copper(I) ligands or sophisticated copper(I)-chelating azides are used. This latter strategy, however, increases the bulkiness of the triazole linkage, thus perturbing the biological function or dynamic behavior of the conjugates. In a proof-of-concept study, we investigated the use of an extremely compact fluorophore-based copper(I) chelating azide in order to accelerate the CuAAC with concomitant fluorescence labeling; in our strategy, the fluorophore is able to complex copper(I) species while retaining its photophysical properties. It is believed that this unprecedented approach which was applied for the labeling of a short peptide molecule and the fluorescent labeling of live cells, could be extended to other families of nitrogen-based fluorophores in order to tune both the reaction rate and photophysical characteristics.
Topics: Animals; Azides; Chelating Agents; Click Chemistry; Copper; Fluorescence; Fluorescent Dyes; Kinetics; Ligands; PC12 Cells; Rats
PubMed: 32316290
DOI: 10.3390/biom10040619 -
Journal of the American Chemical Society Nov 2011New additions to the bioorthogonal chemistry compendium can advance biological research by enabling multiplexed analysis of biomolecules in complex systems. Here we...
New additions to the bioorthogonal chemistry compendium can advance biological research by enabling multiplexed analysis of biomolecules in complex systems. Here we introduce the quadricyclane ligation, a new bioorthogonal reaction between the highly strained hydrocarbon quadricyclane and Ni bis(dithiolene) reagents. This reaction has a second-order rate constant of 0.25 M(-1) s(-1), on par with fast bioorthogonal reactions of azides, and proceeds readily in aqueous environments. Ni bis(dithiolene) probes selectively labeled quadricyclane-modified bovine serum albumin, even in the presence of cell lysate. We have demonstrated that the quadricyclane ligation is compatible with, and orthogonal to, strain-promoted azide-alkyne cycloaddition and oxime ligation chemistries by performing all three reactions in one pot on differentially functionalized protein substrates. The quadricyclane ligation joins a small but growing list of tools for the selective covalent modification of biomolecules.
Topics: Alkynes; Animals; Azides; Bridged-Ring Compounds; Cattle; Cyclization; Molecular Structure; Organometallic Compounds; Serum Albumin, Bovine
PubMed: 21962173
DOI: 10.1021/ja2072934 -
Bioorganic & Medicinal Chemistry Dec 2020As a versatile reaction for bioconjugation, Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) has enormous potential in the synthesis of antibody-drug conjugates...
As a versatile reaction for bioconjugation, Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) has enormous potential in the synthesis of antibody-drug conjugates (ADCs). In order to optimize CuAAC-based ADC synthesis, we characterized kinetically different formulation processes by mimicking ADC synthesis using small molecules and subsequently revealed unique kinetic behaviors of different combinations of alkyne and azide conditions. Our results indicate that under ADC synthesis conditions, for an alkyne-containing drug, its concentration has minimal impact on the reaction rate when an antibody has a non-metal-chelating azide but is proportional to concentration when an antibody contains a metal-chelating azide; however, for an alkyne-containing antibody, the ADC synthesis rate is proportional to the concentration of a drug with a non-metal-chelating azide but displays almost no dependence on drug concentration with a metal-chelating azide. Based on our results, we designed and tested an optimal "click" formulation strategy that allowed rapid and cost-effective synthesis of a new ADC.
Topics: Alkynes; Antibodies, Monoclonal, Humanized; Azides; Catalysis; Click Chemistry; Copper; Cycloaddition Reaction; Immunoconjugates; Pharmaceutical Preparations
PubMed: 33071032
DOI: 10.1016/j.bmc.2020.115808 -
The Journal of Organic Chemistry Mar 2018The binding interactions between the azide anion (N) and the strapped calix[4]pyrroles 2 and 3 bearing auxiliary hydrogen bonding donors on the bridging moieties, as...
The binding interactions between the azide anion (N) and the strapped calix[4]pyrroles 2 and 3 bearing auxiliary hydrogen bonding donors on the bridging moieties, as well as of normal calix[4]pyrrole 1, were investigated via H NMR spectroscopic and isothermal titration calorimetry analyses. The resulting data revealed that receptors 2 and 3 have significantly higher affinities for the azide anion in organic media as compared with the unfunctionalized calix[4]pyrrole 1 and other azide receptors reported to date. Single crystal X-ray diffraction analyses and calculations using density functional theory revealed that receptor 2 binds CsN in two distinct structural forms. As judged from the metric parameters, in the resulting complexes one limiting azide anion resonance contributor is favored over the other, with the specifics depending on the binding mode. In contrast to what is seen for 2, receptor 3 forms a CsN complex in 20% CDOD in CDCl, wherein the azide anion is bound only vertically to the NH protons of the calix[4]pyrrole and the cesium cation is complexed within the cone shaped-calix[4]pyrrole bowl. The bound cesium cation is also in close proximity to a naphthobipyrrole subunit present in a different molecule, forming an apparent cation-π complex.
Topics: Azides; Calixarenes; Hydrogen Bonding; Models, Molecular; Molecular Conformation; Pyrroles
PubMed: 29441791
DOI: 10.1021/acs.joc.7b03135 -
Angewandte Chemie (International Ed. in... Mar 2018We describe a selective and mild chemical approach for controlling RNA hybridization, folding, and enzyme interactions. Reaction of RNAs in aqueous buffer with an...
We describe a selective and mild chemical approach for controlling RNA hybridization, folding, and enzyme interactions. Reaction of RNAs in aqueous buffer with an azide-substituted acylating agent (100-200 mm) yields several 2'-OH acylations per RNA strand in as little as 10 min. This poly-acylated ("cloaked") RNA is strongly blocked from hybridization with complementary nucleic acids, from cleavage by RNA-processing enzymes, and from folding into active aptamer structures. Importantly, treatment with a water-soluble phosphine triggers a Staudinger reduction of the azide groups, resulting in spontaneous loss of acyl groups ("uncloaking"). This fully restores RNA folding and biochemical activity.
Topics: Acylation; Azides; Molecular Structure; Phosphines; RNA; RNA Folding
PubMed: 29370460
DOI: 10.1002/anie.201708696