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 was 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.