Polarising Agents and Spin Tags Used in DNP-ssNMR Studies on Biological Samples: An Update

The present study primarily focuses on "Polarising Agents and Spin Tags for Dynamic Nuclear Polarisation (DNP)-Enhanced Solid-State Nuclear Magnetic Resonance (ssNMR) Analysis of Biological Samples". For biological materials that are frozen or have a solid-like consistency, ssNMR spectroscopy can provide structural, functional, and ligand-binding information. ssNMR spectra can be greatly improved with the application of dynamic nuclear polarisation (DNP). Through microwave irradiation at or near the electron Larmor frequency, polarisation transfer from high-gyromagnetic ratio (Y) unpaired electrons to neighboring nuclei occurs in DNP. This produces an absolute increase in the signal-to-noise ratio and allows experiments on much smaller quantities of sample and/or using much shorter acquisition times. Along with necessary instrumentation an essential requirement for DNP-ssNMR is a sample with an endogenous free radical or an exogenous free radical polarising agent must be added to the sample. The polarising agent must be soluble in the sample matrix and compatible with the biological sample. The free radical(s) of the polarising agent also must be stable for the lifetime of DNP-ssNMR experiments. Nitroxides have been most used as polarising agents, including the biradical compounds TOTAPOL and AMUPol with a wide range of biological samples to produce DNP enhancement factors (ε) of up to 250. Derivatives of TOTAPOL and AMUPol and many other different polarising agents have also been used. Whilst conventional polarising agents are mixed throughout the sample, others are targeted at specific sites to provide a more localised signal enhancement. Here we review the different polarising agents and spin tags that have been used in DNP-ssNMR studies on biological samples. Targeted polarising agents enable use of matrix-free samples to concentrate the sample, whilst others can be covalently bound to provide signal enhancement at highly specific sites. The continued development of novel polarising agents and labelling and sample preparation strategies for DNP-ssNMR can open many biological samples to NMR studies that were not previously possible.