Synthetically Diversified Protein Nanopores: Resolving Click Reaction Mechanisms

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.