Polarizing agents and mechanisms for high-field dynamic nuclear polarization of frozen dielectric solids

Abstract

This article provides an overview of polarizing mechanisms involved in high-frequency dynamic nuclear polarization (DNP) of frozen biological samples at temperatures maintained using liquid nitrogen, compatible with contemporary magic-angle spinning (MAS) nuclear magnetic resonance (NMR). Typical DNP experiments require unpaired electrons that are usually exogenous in samples via paramagnetic doping with polarizing agents. Thus, the resulting nuclear polarization mechanism depends on the electron and nuclear spin interactions induced by the paramagnetic species. The Overhauser Effect (OE) DNP, which relies on time-dependent spin-spin interactions, is excluded from our discussion due the lack of conducting electrons in frozen aqueous solutions containing biological entities. DNP of particular interest to us relies primarily on time-independent, spin interactions for significant electron-nucleus polarization transfer through mechanisms such as the Solid Effect (SE), the Cross Effect (CE) or Thermal Mixing (TM), involving one, two or multiple electron spins, respectively. Derived from monomeric radicals initially used in DNP experiments, bi- or multiple-radical polarizing agents facilitate CE/TM to generate significant NMR signal enhancements in dielectric solids at low temperatures (< 100 K). For example, large DNP enhancements (~300 times at 5 T) from a biologically compatible biradical, 1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol (TOTAPOL), have enabled high-resolution MAS NMR in sample systems existing in submicron domains or embedded in larger biomolecular complexes. The scope of this review is focused on recently developed DNP polarizing agents for high-field applications and leads up to future developments per the CE DNP mechanism. Because DNP experiments are feasible with a solid-state microwave source when performed at <20 K, nuclear polarization using lower microwave power (< 100 mW) is possible by forcing a high proportion of biradicals to fulfill the frequency matching condition of CE (two EPR frequencies separated by the NMR frequency) using the strategies involving hetero-radical moieties and/or molecular alignment. In addition, the combination of an excited triplet and a stable radical might provide alternative DNP mechanisms without the microwave requirement.

Helium-cooling and -spinning dynamic nuclear polarization for sensitivity-enhanced solid-state NMR at 14T and 30K

“…The benchmark MAS DNP enhancements are e DNP = 170 and 120 observed at B 0 = 5 T and 9 T, respectively, under T % 90 K [25,57]. Using the 1/B 0 dependence of the CE DNP enhancement, both enhancements can be extrapolated to e DNP % 60 for B 0 = 14.1 T. Thus, the enhancement factor e DNP = 10.9 we observed at B 0 = 14.1 T and T = 90 K for a static sample is five times smaller than the expectation.…”

“…1 By this definition, the DNP enhancement factor can be obtained independently of specific polarization buildup time assuming the time constants for the buildup are similar with and without microwave irradiation [25,41]. A background 1 H spectrum recorded for a rotor without sample was subtracted at each temperature from both microwave-on and -off spectra before calculating the enhancement factor.…”

“…Originating with the seminal work of 1993 that first introduced the cyclotron resonance maser (gyrotron) as a high-power microwave source [24], DNP by irradiation of radicals in the high-field (B 0 > 5 T) NMR magnets has become a viable hyperpolarization approach. Together with the advent of biradical polarizing agents [25][26][27], this in situ DNP technique has evolved into a powerful tool for sensitizing SSNMR, and has been applied to small organic compounds [28,29], microcrystalline [30], membraneembedded [2,[31][32][33] and fibrillar proteins [34,35] as well as inorganic materials [36,37].…”

“…In fact, modern SSNMR experiments targeting large and complex systems are often conducted at very high field conditions of up to B 0 = 21 T, which offers better spectral resolution and sensitivity. A concern with DNP SSNMR at very high fields, however, centers on the fact that the efficiency of DNP via CE decreases inversely proportional to the external field strength [23,25,38,39].…”

A Method for Dynamic Nuclear Polarization Enhancement of Membrane Proteins

“…[8][9][10][11][12][13][14][15][16] The strength of the dipolar coupling between the tethered nitroxides, the relative perpendicular orientation of their two g-tensors, and the protonation of nearby substituents have all been cited as important factors impacting signal enhancements in DNP experiments. [17,18] These properties have been further exploited in the design of nextgeneration polarizing agents AMUPol and TEKPol, resulting in additional enhancements. [19,20] The work presented here demonstrates an alternative strategy for introducing polarizing reagents into membrane protein samples; specifically, one that enables increased DNP enhancement within the lipid bilayer and does not require the use of small molecule cryoprotectants, which can further dilute a sample and potentially introduce artifacts in the membrane mimetic environment.…”