The structures and properties of nitroxide radicals can be readily tailored for use as spin labels in biomolecular EPR spectroscopy.

Bicyclo[1.1.0]butanes (BCBs) are valuable precursors to four-membered rings and bicyclo[1.1.1]pentanes, and useful bioconjugation agents. We describe a versatile approach to access 1,3-disubstituted BCBs, which are otherwise challenging to prepare.

EPR spectroscopy is a powerful method to elucidate the structure of molecules through the measurement of distances between conformationally well-defined spin labels. Here we report a sequence-flexible approach to the synthesis of double spin-labeled DNA duplexes, where 2′-alkynylnucleosides are incorporated at terminal and internal positions on complementary strands. Post-DNA synthesis copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions with a variety of spin labels enable the use of double electron-electron resonance experiments to measure a number of distances on the duplex, thus affording a high level of detailed structural information.

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.

Spin labels containing nitroxyl radicals possess many properties that render them useful for electron paramagnetic resonance (EPR) spectroscopy. This review describes the relationships between the structure and properties of nitroxide spin labels, methods for their synthesis, advances in methods for their incorporation into biomolecules, and selected examples of applications in biomolecule structural investigations.