Synthetically Diversified Protein Nanopores: Resolving Click Reaction Mechanisms.

Marius M Haugland,Alex Elmi,Dominic F Cairns-Gibson,Stefan Borsley,Scott L Cockroft

doi:10.1021/acsnano.8b08691

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

Edinburgh Research Explorer

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)

In Situ Chemical Functionalization of Methionine Residues

Click Diversification of Nanopores

CONCLUSION

Single Channel Experiment Setup

Methionine Modification of Protein Nanopore

ASSOCIATED CONTENT