A Fluorogenic 1,3-Dipolar Cycloaddition Reaction of 3-Azidocoumarins and Acetylenes †

Department of Chemistry & Biochemistry, University of South Carolina, 631 Sumter Street, Columbia, SC 29208, USA.
Organic Letters (Impact Factor: 6.36). 12/2004; 6(24):4603-6. DOI: 10.1021/ol047955x
Source: PubMed


Copper(I)-catalyzed 1,3-dipolar cycloaddition reaction of nonfluorescent 3-azidocoumarins and terminal alkynes afforded intense fluorescent 1,2,3-triazole products. The mild condition of this reaction allowed us to construct a large library of pure fluorescent coumarin dyes. Since both azide and alkyne are quite inert to biological systems, this reaction has potential in bioconjugation and bioimaging applications. [reaction: see text]

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    • "A milky turbidity observed in the CuAAC reaction mixture dropped in proportion to DMSO concentration. In order to evaluate reaction efficiency, we employed fluorogenic assay using azidocoumarin (Figure 1C) which, initially non-fluorescent, becomes highly fluorescent upon its conjugation to terminal alkyne through CuAAC, thereby enabling spectroscopic tracking of the reaction progress [47]. Higher DMSO concentration allowed higher conjugation efficiency, indicating that solubility of TBTA is important to a high-yielding CuAAC reaction (Figure 4A). "
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    ABSTRACT: Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is an efficient reaction linking an azido and an alkynyl group in the presence of copper catalyst. Incorporation of a non-natural amino acid (NAA) containing either an azido or an alkynyl group into a protein allows site-specific bioconjugation in mild conditions via CuAAC. Despite its great potential, bioconjugation of an enzyme has been hampered by several issues including low yield, poor solubility of a ligand, and protein structural/functional perturbation by CuAAC components. In the present study, we incorporated an alkyne-bearing NAA into an enzyme, murine dihydrofolate reductase (mDHFR), in high cell density cultivation of Escherichia coli, and performed CuAAC conjugation with fluorescent azide dyes to evaluate enzyme compatibility of various CuAAC conditions comprising combination of commercially available Cu(I)-chelating ligands and reductants. The condensed culture improves the protein yield 19-fold based on the same amount of non-natural amino acid, and the enzyme incubation under the optimized reaction condition did not lead to any activity loss but allowed a fast and high-yield bioconjugation. Using the established conditions, a biotin-azide spacer was efficiently conjugated to mDHFR with retained activity leading to the site-specific immobilization of the biotin-conjugated mDHFR on a streptavidin-coated plate. These results demonstrate that the combination of reactive non-natural amino acid incorporation and the optimized CuAAC can be used to bioconjugate enzymes with retained enzymatic activity.
    PLoS ONE 06/2014; 9(6):e98403. DOI:10.1371/journal.pone.0098403 · 3.23 Impact Factor
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    • "The oxidized hIgG was coupled with EH following the standard hydrazide protocol. Ethynylation of hIgG was confirmed by a fluorogenic click reaction of 1 developed by Wang and coworkers (Fig. 4A) [28]. For a mild and efficient click reaction, we used a water-soluble copper ligand, THPTA, in aqueous medium . "
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    ABSTRACT: A water-soluble, dual reactive hydrazide/click crosslinker (EH) was synthesized and characterized. A model antibody, human IgG was ethynylated by conventional oxidation/hydrazide reactions with the hydrazide moiety of EH. The terminal alkyne conjugated to the glycan of human IgG was easily functionalized by quantitative and bioorthogonal copper(I)-catalyzed azide-alkyne cycloaddition. The potential of the hydrazide/click crosslinker as a reagent to functionalize antibodies was demonstrated with fluorophore labeling and antibody immobilization.
    Analytical Biochemistry 01/2013; 435(1). DOI:10.1016/j.ab.2012.12.018 · 2.22 Impact Factor
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    • "Intriguingly, LC-traces show very low fluorescence of UN3C, but solutions of the isolated compound become increasingly fluorescent upon irradiation at 365 nm (Figure 2E and F). This strongly suggests that the characteristic coumarin fluorescence is quenched by the azidofunction (51), and that fluorescence is restored by its photochemical destruction. Photochemical conversion to the fluorescent reduction product of a presumed nitrene intermediate (see Supplementary Figure S2) could be monitored in an HPLC/UV/FLD and LC/MS analysis of the irradiated UN3C (See Supplementary Figure S3). "
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    ABSTRACT: A multifunctional reagent based on a coumarin scaffold was developed for derivatization of naive RNA. The alkylating agent N3BC [7-azido-4-(bromomethyl)coumarin], obtained by Pechmann condensation, is selective for uridine. N3BC and its RNA conjugates are pre-fluorophores which permits controlled modular and stepwise RNA derivatization. The success of RNA alkylation by N3BC can be monitored by photolysis of the azido moiety, which generates a coumarin fluorophore that can be excited with UV light of 320 nm. The azidocoumarin-modified RNA can be flexibly employed in structure-function studies. Versatile applications include direct use in photo-crosslinking studies to cognate proteins, as demonstrated with tRNA and RNA fragments from the MS2 phage and the HIV genome. Alternatively, the azide function can be used for further derivatization by click-chemistry. This allows e.g. the introduction of an additional fluorophore for excitation with visible light.
    Nucleic Acids Research 06/2011; 39(16):7348-60. DOI:10.1093/nar/gkr449 · 9.11 Impact Factor
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