Spin‐Labeled Heparins as Polarizing Agents for Dynamic Nuclear Polarization

A potentially biocompatible class of spin‐labeled macromolecules, spin‐labeled (SL) heparins, and their use as nuclear magnetic resonance (NMR) signal enhancers are introduced. The signal enhancement is achieved through Overhauser‐type dynamic nuclear polarization (DNP). All presented SL‐heparins show high ¹H DNP enhancement factors up to E=?110, which validates that effectively more than one hyperfine line can be saturated even for spin‐labeled polarizing agents. The parameters for the Overhauser‐type DNP are determined and discussed. A striking result is that for spin‐labeled heparins, the off‐resonant electron paramagnetic resonance (EPR) hyperfine lines contribute a non‐negligible part to the total saturation, even in the absence of Heisenberg spin exchange (HSE) and electron spin‐nuclear spin relaxation (T_1ne). As a result, we conclude that one can optimize the use of, for example, biomacromolecules for DNP, for which only small sample amounts are available, by using heterogeneously distributed radicals attached to the molecule.     

Dynamic Nuclear Polarization with a Rigid Biradical

A new polarizing agent with superior performance in dynamic nuclear polarization experiments is introduced, and utilizes two TEMPO (2,2,6,6‐tetramethylpiperidine‐1‐oxyl) moieties connected through a rigid spiro tether (see structure). The observed NMR signal intensities were enhanced by a factor of 1.4 compared to those of TOTAPOL, a previously described TEMPO‐based biradical with a flexible tether.

     

Strategies for 1H-Detected Dynamic Nuclear Polarization Magic-Angle Spinning NMR Spectroscopy

Combining dynamic nuclear polarization with proton detection significantly enhances the sensitivity of magic-angle spinning NMR spectroscopy. Herein, the feasibility of proton-detected experiments with slow (10 kHz) magic angle spinning was demonstrated. The improvement in sensitivity permits the acquisition of indirectly detected ¹⁴N NMR spectra allowing biomolecular structures to be characterized without recourse to isotope labelling. This provides a new tool for the structural characterization of environmental and medical samples, in which isotope labelling is frequently intractable.     

Natural Abundance 15N NMR by Dynamic Nuclear Polarization: Fast Analysis of Binding Sites of a Novel Amine‐Carboxyl‐Linked Immobilized Dirhodium Catalyst

A novel heterogeneous dirhodium catalyst has been synthesized. This stable catalyst is constructed from dirhodium acetate dimer (Rh₂(OAc)₄) units, which are covalently linked to amine‐ and carboxyl‐bifunctionalized mesoporous silica (SBA‐15?NH₂?COOH). It shows good efficiency in catalyzing the cyclopropanation reaction of styrene and ethyl diazoacetate (EDA) forming cis‐ and trans‐1‐ethoxycarbonyl‐2‐phenylcyclopropane. To characterize the structure of this catalyst and to confirm the successful immobilization, heteronuclear solid‐state NMR experiments have been performed. The high application potential of dynamic nuclear polarization (DNP) NMR for the analysis of binding sites in this novel catalyst is demonstrated. Signal‐enhanced ¹³C CP MAS and ¹⁵N CP MAS techniques have been employed to detect different carboxyl and amine binding sites in natural abundance on a fast time scale. The interpretation of the experimental chemical shift values for different binding sites has been corroborated by quantum chemical calculations on dirhodium model complexes.     

Efficient Dynamic Nuclear Polarization at 800 MHz/527 GHz with Trityl‐Nitroxide Biradicals

Cross‐effect (CE) dynamic nuclear polarization (DNP) is a rapidly developing technique that enhances the signal intensities in magic‐angle spinning (MAS) NMR spectra. We report CE DNP experiments at 211, 600, and 800 MHz using a new series of biradical polarizing agents referred to as TEMTriPols, in which a nitroxide (TEMPO) and a trityl radical are chemically tethered. The TEMTriPol molecule with the optimal performance yields a record ¹H NMR signal enhancement of 65 at 800 MHz at a concentration of 10 mM in a glycerol/water solvent matrix. The CE DNP enhancement for the TEMTriPol biradicals does not decrease as the magnetic field is increased in the manner usually observed for bis‐nitroxides. Instead, the relatively strong exchange interaction between the trityl and nitroxide moieties determines the magnetic field at which the optimum enhancement is observed.     

Highly Efficient Trityl-Nitroxide Biradicals for Biomolecular High-Field Dynamic Nuclear Polarization

Dynamic nuclear polarization (DNP) is a powerful method to enhance the sensitivity of solid-state magnetic nuclear resonance (ssNMR) spectroscopy. However, its biomolecular applications at high magnetic fields (preferably>14 T) have so far been limited by the intrinsically low efficiency of polarizing agents and sample preparation aspects. Herein, we report a new class of trityl-nitroxide biradicals, dubbed SNAPols that combine high DNP efficiency with greatly enhanced hydrophilicity. SNAPol-1, the best compound in the series, shows DNP enhancement factors at 18.8 T of more than 100 in small molecules and globular proteins and also exhibits strong DNP enhancements in membrane proteins and cellular preparations. By integrating optimal sensitivity and high resolution, we expect widespread applications of this new polarizing agent in high-field DNP/ssNMR spectroscopy, especially for complex biomolecules.     

A Method for Dynamic Nuclear Polarization Enhancement of Membrane Proteins

Dynamic nuclear polarization (DNP) magic‐angle spinning (MAS) solid‐state NMR (ssNMR) spectroscopy has the potential to enhance NMR signals by orders of magnitude and to enable NMR characterization of proteins which are inherently dilute, such as membrane proteins. In this work spin‐labeled lipid molecules (SL‐lipids), when used as polarizing agents, lead to large and relatively homogeneous DNP enhancements throughout the lipid bilayer and to an embedded lung surfactant mimetic peptide, KL₄. Specifically, DNP MAS ssNMR experiments at 600 MHz/395 GHz on KL₄ reconstituted in liposomes containing SL‐lipids reveal DNP enhancement values over two times larger for KL₄ compared to liposome suspensions containing the biradical TOTAPOL. These findings suggest an alternative sample preparation strategy for DNP MAS ssNMR studies of lipid membranes and integral membrane proteins.     

Implementation and Characterization of Flow Injection in Dissolution Dynamic Nuclear Polarization NMR Spectroscopy

The use of dissolution dynamic nuclear polarization (D ‐DNP) offers substantially increased signals in liquid‐state NMR spectroscopy. A challenge in realizing this potential lies in the transfer of the hyperpolarized sample to the NMR detector without loss of hyperpolarization. Here, the use of a flow injection method using high‐pressure liquid leads to improved performance compared to the more common gas‐driven injection, by suppressing residual fluid motions during the NMR experiment while still achieving a short injection time. Apparent diffusion coefficients are determined from pulsed field gradient echo measurements, and are shown to fall below 1.5 times the value of a static sample within 0.8 s. Due to the single‐scan nature of D ‐DNP, pulsed field gradients are often the only choice for coherence selection or encoding, but their application requires stationary fluid. Sample delivery driven by a high‐pressure liquid will improve the applicability of these types of D‐DNP advanced experiments.