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    Synthetically Diversified Protein Nanopores: Resolving Click Reaction Mechanisms
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    • Marius M. Haugland
      Marius M. Haugland
      EaStCHEM School of Chemistry, University of Edinburgh, Joseph Black Building, David Brewster Road, Edinburgh EH9 3FJ, United Kingdom
      More by Marius M. Haugland
      Orcidhttp://orcid.org/0000-0001-7017-344X
    • Stefan Borsley
      Stefan Borsley
      EaStCHEM School of Chemistry, University of Edinburgh, Joseph Black Building, David Brewster Road, Edinburgh EH9 3FJ, United Kingdom
      More by Stefan Borsley
    • Dominic F. Cairns-Gibson
      Dominic F. Cairns-Gibson
      EaStCHEM School of Chemistry, University of Edinburgh, Joseph Black Building, David Brewster Road, Edinburgh EH9 3FJ, United Kingdom
      More by Dominic F. Cairns-Gibson
    • Alex Elmi
      Alex Elmi
      EaStCHEM School of Chemistry, University of Edinburgh, Joseph Black Building, David Brewster Road, Edinburgh EH9 3FJ, United Kingdom
      More by Alex Elmi
    • Scott L. Cockroft*
      Scott L. Cockroft
      EaStCHEM School of Chemistry, University of Edinburgh, Joseph Black Building, David Brewster Road, Edinburgh EH9 3FJ, United Kingdom
      *E-mail: [email protected]
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      Orcidhttp://orcid.org/0000-0001-9321-8997
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    ACS Nano

    Cite this: ACS Nano 2019, 13, 4, 4101–4110
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    https://doi.org/10.1021/acsnano.8b08691
    Published March 13, 2019
    Copyright © 2019 American Chemical Society
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    Abstract

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    Nanopores are emerging as a powerful tool for the investigation of nanoscale processes at the single-molecule level. Here, we demonstrate the methionine-selective synthetic diversification of α-hemolysin (α-HL) protein nanopores and their exploitation as a platform for investigating reaction mechanisms. A wide range of functionalities, including azides, alkynes, nucleotides, and single-stranded DNA, were incorporated into individual pores in a divergent fashion. The ion currents flowing through the modified pores were used to observe the trajectory of a range of azide–alkyne click reactions and revealed several short-lived intermediates in Cu(I)-catalyzed azide–alkyne [3 + 2] cycloadditions (CuAAC) at the single-molecule level. Analysis of ion-current fluctuations enabled the populations of species involved in rapidly exchanging equilibria to be determined, facilitating the resolution of several transient intermediates in the CuAAC reaction mechanism. The versatile pore-modification chemistry offers a useful approach for enabling future physical organic investigations of reaction mechanisms at the single-molecule level.

    Copyright © 2019 American Chemical Society

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    Supporting Information

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.8b08691.

    • Experimental setup, synthetic procedures, full nanopore current traces, experimental details, detailed data analysis, Figures S1–S74, Tables S1–S6 (PDF)

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    ACS Nano

    Cite this: ACS Nano 2019, 13, 4, 4101–4110
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acsnano.8b08691
    Published March 13, 2019
    Copyright © 2019 American Chemical Society
    Request reuse permissions

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