Abstract
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.
Highlights
Nanopores are emerging as a powerful tool for the investigation of nanoscale processes at the single-molecule level
Fully assembled wild-type α-hemolysin nanopore (α-HL) nanopores have been modified in situ through direct chemical functionalization of lysine residues.[34]
While previous investigations of transient intermediates in the CuAAC reaction have required the use of stabilizing ligands, this study demonstrates the ability of synthetically functionalized nanopores to observe short-lived and dynamically exchanging reaction intermediates under typical reaction conditions
Summary
Citation for published version: Haugland, M, Borsley, S, Cairns-Gibson, D, Elmi, A & Cockroft, S 2019, 'Synthetically Diversified Protein Nanopores: Resolving Click Reaction Mechanisms', ACS Nano. The versatile pore-modification chemistry offers a useful approach for enabling future physical organic investigations of reaction mechanisms at the single-molecule level. The utility of this pore-modification approach to enable physical organic mechanistic investigations at the single-molecule level was demonstrated by application to CuAAC reactions (Figure 4). The high spatio-temporal resolution of the method enabled the assignment of several transient and dynamically exchanging reaction intermediates along the pathways of CuAAC reactions (Figures 5–8)
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