Synthetically Diversified Protein Nanopores: Resolving Click Reaction Mechanisms

Abstract

Nanopores are emerging as a powerful tool for the investigation of nanoscale processes at the singlemolecule level. Here, we demonstrate the methionineselective 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 singlemolecule 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.

Programmable nano-reactors for stochastic sensing

“…Most biological nanopores show an oligomeric symmetry 6 and introduction of a sole reactive site therefore requires a significant effort to produce a hetero-oligomeric assembly 5 , a niche technique mastered by only few in the field. Nanopore single-molecule chemistry measurements performed by engineered homo-oligomeric porins will inevitably report simultaneous binding from multiple reactants, not suitable for event recognition and quantification [24][25][26][27] . Modifications to the pore lumen may interfere unpredictably with the assembly or the stability of the pore and, consequently, a considerable effort of protein screening cannot therefore be avoided.…”

Single molecule observation of hard–soft-acid–base (HSAB) interaction in engineeredMycobacterium smegmatisporin A (MspA) nanopores

“…31 According to HSAB theory and the demonstrated results, hard acid ions such as Ca 2+ and Mn 2+ , which don't interact strongly with amino acids belonging to so or borderline bases and barely interact with amino acids belonging to hard bases, may be monitored with difficulty in any combinatorial placement of natural amino acids within the pore lumen. However, this limitation may be overcome by the introduction of unnatural amino acids, 44 chemical modications with a high specicity 45 or protein phosphorylation 46 3). (e) The proposed coordination mechanism.…”

Regiodivergent synthesis of pyrazino-indolines vs. triazocines via α-imino carbenes addition to imidazolidines

“…N -Sulfonyl-1,2,3-triazoles 1 , readily accessible through Cu( i )-catalyzed azide alkyne cycloadditions (CuAACs), 1 are key building blocks in synthetic, biological and medicinal chemistry. 2 For the purpose of this study, they also decompose under metal-catalyzed conditions to afford α-imino carbenes.…”