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.     

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.     

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.     

Biomolecular DNP‐Supported NMR Spectroscopy using Site‐Directed Spin Labeling

Dynamic nuclear polarization (DNP) has been shown to greatly enhance spectroscopic sensitivity, creating novel opportunities for NMR studies on complex and large molecular assemblies in life and material sciences. In such applications, however, site‐specificity and spectroscopic resolution become critical factors that are usually difficult to control by current DNP‐based approaches. We have examined in detail the effect of directly attaching mono‐ or biradicals to induce local paramagnetic relaxation effects and, at the same time, to produce sizable DNP enhancements. Using a membrane‐embedded ion channel as an example, we varied the degree of paramagnetic labeling and the location of the DNP probes. Our results show that the creation of local spin clusters can generate sizable DNP enhancements while preserving the intrinsic benefits of paramagnetic relaxation enhancement (PRE)‐based NMR approaches. DNP using chemical labeling may hence provide an attractive route to introduce molecular specificity into DNP studies in life science applications and beyond.     

13C dynamic nuclear polarization using isotopically enriched 4‐oxo‐TEMPO free radicals

The nitroxide‐based free radical 2,2,6,6‐tetramethyl‐1‐piperidinyloxy (TEMPO) is a widely used polarizing agent in NMR signal amplification via dissolution dynamic nuclear polarization (DNP). In this study, we have thoroughly investigated the effects of ¹⁵N and/or ²H isotopic labeling of 4‐oxo‐TEMPO free radical on ¹³C DNP of 3 M [1‐¹³C] sodium acetate samples in 1 : 1 v/v glycerol : water at 3.35 T and 1.2 K. Four variants of this free radical were used for ¹³C DNP: 4‐oxo‐TEMPO, 4‐oxo‐TEMPO‐¹⁵N, 4‐oxo‐TEMPO‐d₁₆ and 4‐oxo‐TEMPO‐¹⁵N,d₁₆. Our results indicate that, despite the striking differences seen in the electron spin resonance (ESR) spectral features, the ¹³C DNP efficiency of these ¹⁵N and/or ²H‐enriched 4‐oxo‐TEMPO free radicals are relatively the same compared with ¹³C DNP performance of the regular 4‐oxo‐TEMPO. Furthermore, when fully deuterated glassing solvents were used, the ¹³C DNP signals of these samples all doubled in the same manner, and the ¹³C polarization buildup was faster by a factor of 2 for all samples. The data here suggest that the hyperfine coupling contributions of these isotopically enriched 4‐oxo‐TEMPO free radicals have negligible effects on the ¹³C DNP efficiency at 3.35 T and 1.2 K. These results are discussed in light of the spin temperature model of DNP. Copyright © 2016 John Wiley & Sons, Ltd.     

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.     

Unique Vibrational Features as a Direct Probe of Specific Antigen–Antibody Recognition at the Surface of a Solid‐Supported Hybrid Lipid Bilayer

Here, we demonstrate how sum frequency generation (SFG), a vibrational spectroscopy based on a nonlinear three‐photon mixing process, may provide a direct and unique fingerprint of bio‐recognition; This latter can be detected with an intrinsically discriminating unspecific adsorption, thanks to the high sensitivity of the second‐order nonlinear optical (NLO) response to preferential molecular orientation and symmetry properties. As a proof of concept, we have detected the biological event at the solid/liquid interface of a model bio‐active antigen platform, based on a solid‐supported hybrid lipid bilayer (ss‐HLB) of a 2,4‐dinitrophenyl (DNP) lipid, towards a monoclonal mouse anti‐DNP complementary antibody.     

Long‐Lived States of Magnetically Equivalent Spins Populated by Dissolution‐DNP and Revealed by Enzymatic Reactions

Hyperpolarization by dissolution dynamic nuclear polarization (D ‐DNP) offers a way of enhancing NMR signals by up to five orders of magnitude in metabolites and other small molecules. Nevertheless, the lifetime of hyperpolarization is inexorably limited, as it decays toward thermal equilibrium with the nuclear spin‐lattice relaxation time. This lifetime can be extended by storing the hyperpolarization in the form of long‐lived states (LLS) that are immune to most dominant relaxation mechanisms. Levitt and co‐workers have shown how LLS can be prepared for a pair of inequivalent spins by D ‐DNP. Here, we demonstrate that this approach can also be applied to magnetically equivalent pairs of spins such as the two protons of fumarate, which can have very long LLS lifetimes. As in the case of para‐hydrogen, these hyperpolarized equivalent LLS (HELLS) are not magnetically active. However, a chemical reaction such as the enzymatic conversion of fumarate into malate can break the magnetic equivalence and reveal intense NMR signals.     

Bioinspired,Size‐Tunable Self‐Assembly of Polymer–Lipid Bilayer Nanodiscs

Polymer‐based nanodiscs are valuable tools in biomedical research that can offer a detergent‐free solubilization of membrane proteins maintaining their native lipid environment. Herein, we introduce a novel ca. 1.6 kDa SMA‐based polymer with styrene:maleic acid moieties that can form nanodiscs containing a planar lipid bilayer which are useful to reconstitute membrane proteins for structural and functional studies. The physicochemical properties and the mechanism of formation of polymer‐based nanodiscs are characterized by light scattering, NMR, FT‐IR, and TEM. A remarkable feature is that nanodiscs of different sizes, from nanometer to sub‐micrometer diameter, can be produced by varying the lipid‐to‐polymer ratio. The small‐size nanodiscs (up to ca. 30 nm diameter) can be used for solution NMR spectroscopy studies whereas the magnetic‐alignment of macro‐nanodiscs (diameter of > ca. 40 nm) can be exploited for solid‐state NMR studies on membrane proteins.     

Deuterated TEKPol Biradicals and the Spin-Diffusion Barrier in MAS DNP

The sensitivity of NMR spectroscopy is considerably enhanced by dynamic nuclear polarization (DNP). In DNP polarization is transferred from unpaired electrons of a polarizing agent to nearby proton spins. In solids, this transfer is followed by the transport of hyperpolarization to the bulk via ¹H-¹H spin diffusion. The efficiency of these steps is critical to obtain high sensitivity gains, but the pathways for polarization transfer in the region near the unpaired electron spins are unclear. Here we report a series of seven deuterated and one fluorinated TEKPol biradicals to probe the effect of deprotonation on MAS DNP at 9.4 T. The experimental results are interpreted with numerical simulations, and our findings support that strong hyperfine couplings to nearby protons determine high transfer rates across the spin diffusion barrier to achieve short build-up times and high enhancements. Specifically, ¹H DNP build-up times increase substantially with TEKPol isotopologues that have fewer hydrogen atoms in the phenyl rings, suggesting that these protons play a crucial role transferring the polarization to the bulk. Based on this new understanding, we have designed a new biradical, NaphPol, which yields significantly increased NMR sensitivity, making it the best performing DNP polarizing agent in organic solvents to date.     

Phenylazide Hybrid‐Silica – Polarization Platform for Dynamic Nuclear Polarization at Cryogenic Temperatures

Hyperpolarization of NMR‐active nuclei is key to gather high quality spectra of rare species and insensitive isotopes. We have recently established that silica‐based materials containing regularly distributed nitroxyl radicals connected to the silica matrix by flexible linkers can serve as promising polarization matrices for dynamic nuclear polarization (DNP). Here we investigate the influence of the linker on the efficiency of the polarization. The materials were fully characterized and exhibit high surface areas and narrow pore size distributions with a tunable amount of phenyl azide groups over a broad range of concentrations. The phenyl azide groups can be easily functionalized via a two‐step procedure with 4‐carboxy‐2,2,6,6‐tetramethyl‐1‐oxylpiperidine (TEMPO) to give polarizing matrices with controllable radical content. The DNP efficiency was found to be similar as in materials with flexible linkers, both for magic angle spinning at 105 K and dissolution DNP at 4 K.     

Vibrational Sum‐Frequency Generation Activity of a 2,4‐Dinitrophenyl Phospholipid Hybrid Bilayer: Retrieving Orientational Parameters from a DFT Analysis of Experimental Data

The vibrational nonlinear activity of films of 2,4‐dinitrophenyl phospholipid (DNP) at the solid interface is measured by sum‐frequency generation spectroscopy (SFG). Hybrid bilayers are formed by a Langmuir–Schaefer approach in which the lipid layer is physisorbed on top of a self‐assembled monolayer of dodecanethiol on Pt with the polar heads pointing out from the surface. The SFG response is investigated in two vibrational frequency domains, namely, 3050–2750 and 1375–1240 cm^?1. The first region probes the CH stretching modes of DNP films, and the latter explores the vibrational nonlinear activity of the 2,4‐dinitroaniline moiety of the polar head of the lipid. Analysis of the CH stretching vibrations suggests substantial conformational order of the aliphatic chains with only a few gauche defects. To reliably assign the detected SFG signals to specific molecular vibrations, DFT calculations of the IR and Raman activities of molecular models are performed and compared to experimental solid‐state spectra. This allows unambiguous assignment of the observed SFG vibrations to molecular modes localized on the 2,4‐dinitroaniline moiety of the polar head of DNP. Then, SFG spectra of DNP in the 1375–1240 cm^?1 frequency range are simulated and compared with experimental ones, and thus the 1,4‐axis of the 2,4‐dinitrophenyl head is estimated to have tilt and rotation angles of 45±5° and 0±30°, respectively.     

Molecular Level Characterization of the Structure and Interactions in Peptide‐Functionalized Metal–Organic Frameworks

We use density functional theory, newly parameterized molecular dynamics simulations, and last generation ¹⁵N dynamic nuclear polarization surface enhanced solid‐state NMR spectroscopy (DNP SENS) to understand graft–host interactions and effects imposed by the metal–organic framework (MOF) host on peptide conformations in a peptide‐functionalized MOF. Focusing on two grafts typified by MIL‐68‐proline ( ‐Pro ) and MIL‐68‐glycine‐proline ( ‐Gly‐Pro ), we identified the most likely peptide conformations adopted in the functionalized hybrid frameworks. We found that hydrogen bond interactions between the graft and the surface hydroxyl groups of the MOF are essential in determining the peptides conformation(s). DNP SENS methodology shows unprecedented signal enhancements when applied to these peptide‐functionalized MOFs. The calculated chemical shifts of selected MIL‐68‐NH‐ Pro and MIL‐68‐NH‐ Gly‐Pro conformations are in a good agreement with the experimentally obtained ¹⁵N NMR signals. The study shows that the conformations of peptides when grafted in a MOF host are unlikely to be freely distributed, and conformational selection is directed by strong host–guest interactions.     

Assembly and performance of a 6.4 T cryogen‐free dynamic nuclear polarization system

We report on the assembly and performance evaluation of a 180‐GHz/6.4 T dynamic nuclear polarization (DNP) system based on a cryogen‐free superconducting magnet. The DNP system utilizes a variable‐field superconducting magnet that can be ramped up to 9 T and equipped with cryocoolers that can cool the sample space with the DNP assembly down to 1.8 K via the Joule–Thomson effect. A homebuilt DNP probe insert with top‐tuned nuclear magnetic resonance coil and microwave port was incorporated into the sample space in which the effective sample temperature is approximately 1.9 K when a 180‐GHz microwave source is on during DNP operation. ¹³C DNP of [1‐¹³C] acetate samples doped with trityl OX063 and 4‐oxo‐TEMPO in this system have resulted in solid‐state ¹³C polarization levels of 58 ± 3% and 18 ± 2%, respectively. The relatively high ¹³C polarization levels achieved in this work have demonstrated that the use of a cryogen‐free superconducting magnet for ¹³C DNP is feasible and in fact, relatively efficient—a major leap to offset the high cost of liquid helium consumption in DNP experiments.     

Bis‐Gadolinium Complexes for Solid Effect and Cross Effect Dynamic Nuclear Polarization

High‐spin complexes act as polarizing agents (PAs) for dynamic nuclear polarization (DNP) in solid‐state NMR spectroscopy and feature promising aspects towards biomolecular DNP. We present a study on bis(Gd‐chelate)s which enable cross effect (CE) DNP owing to spatial confinement of two dipolar‐coupled electron spins. Their well‐defined Gd⋅⋅⋅Gd distances in the range of 1.2–3.4 nm allowed us to elucidate the Gd⋅⋅⋅Gd distance dependence of the DNP mechanism and NMR signal enhancement. We found that Gd⋅⋅⋅Gd distances above 2.1 nm result in solid effect DNP while distances between 1.2 and 2.1 nm enable CE for ¹H, ¹³C, and ¹⁵N nuclear spins. We compare 263 GHz electron paramagnetic resonance (EPR) spectra with the obtained DNP field profiles and discuss possible CE matching conditions within the high‐spin system and the influence of dipolar broadening of the EPR signal. Our findings foster the understanding of the CE mechanism and the design of high‐spin PAs for specific applications of DNP.     

Producing Radical‐Free Hyperpolarized Perfusion Agents for In Vivo Magnetic Resonance Using Spin‐Labeled Thermoresponsive Hydrogel

Dissolution dynamic nuclear polarization (DNP) provides a way to tremendously improve the sensitivity of nuclear magnetic resonance experiments. Once the spins are hyperpolarized by dissolution DNP, the radicals used as polarizing agents become undesirable since their presence is an additional source of nuclear spin relaxation and their toxicity might be an issue. This study demonstrates the feasibility of preparing a hyperpolarized [1‐¹³C]2‐methylpropan‐2‐ol (tert‐butanol) solution free of persistent radicals by using spin‐labeled thermoresponsive hydrophilic polymer networks as polarizing agents. The hyperpolarized ¹³C signal can be detected for up to 5 min before the spins fully relax to their thermal equilibrium. This approach extends the applicability of spin‐labeled thermoresponsive hydrogel to the dissolution DNP field and highlights its potential as polarizing agent for preparing neat slowly relaxing contrast agents. The hydrogels are especially suited to hyperpolarize deuterated alcohols which can be used for in vivo perfusion imaging.

     

Filterable Agents for Hyperpolarization of Water,Metabolites, and Proteins

Hyperpolarization is generated by dissolution dynamic nuclear polarization (d‐DNP) using a polymer‐based polarizing agent dubbed FLAP (filterable labeled agents for polarization). It consists of a thermo‐responsive poly(N‐isopropylacrylamide), also known as pNiPAM‐COOH, labeled with nitroxide radicals. The polymer powder is impregnated with an arbitrary solution of interest and frozen as is. Dissolution is followed by a simple filtration, leading to hyperpolarized solutions free from any contaminants. We demonstrated the use of FLAP to hyperpolarize partially deuterated water up to P(¹H)=6 % with a long relaxation T₁ >36 s characteristic of high purity. Water hyperpolarization can be transferred to drugs, metabolites, or proteins that are waiting in an NMR spectrometer, either by exchange of labile protons or through intermolecular Overhauser effects. We also show that FLAPs are suitable polarizing agents for ¹³C‐labeled metabolites such as pyruvate, acetate, and alanine.     

Chemical‐Shift‐Resolved 19F NMR Spectroscopy between 13.5 and 135 MHz: Overhauser–DNP‐Enhanced Diagonal Suppressed Correlation Spectroscopy

Overhauser–DNP‐enhanced homonuclear 2D ¹⁹F correlation spectroscopy with diagonal suppression is presented for small molecules in the solution state at moderate fields. Multi‐frequency, multi‐radical studies demonstrate that these relatively low‐field experiments may be operated with sensitivity rivalling that of standard 200–1000 MHz NMR spectroscopy. Structural information is accessible without a sensitivity penalty, and diagonal suppressed 2D NMR correlations emerge despite the general lack of multiplet resolution in the 1D ODNP spectra. This powerful general approach avoids the rather stiff excitation, detection, and other special requirements of high‐field ¹⁹F NMR spectroscopy.     

Heteronuclear Cross‐Relaxation Effects in the NMR Spectroscopy of Hyperpolarized Targets

Dissolution dynamic nuclear polarization (DNP) enables high‐sensitivity solution‐phase NMR experiments on long‐lived nuclear spin species such as ¹⁵N and ¹³C. This report explores certain features arising in solution‐state ¹H NMR upon polarizing low‐γ nuclear species. Following solid‐state hyperpolarization of both ¹³C and ¹H, solution‐phase ¹H NMR experiments on dissolved samples revealed transient effects, whereby peaks arising from protons bonded to the naturally occurring ¹³C nuclei appeared larger than the typically dominant ¹²C‐bonded ¹H resonances. This enhancement of the satellite peaks was examined in detail with respect to a variety of mechanisms that could potentially explain this observation. Both two‐ and three‐spin phenomena active in the solid state could lead to this kind of effect; still, experimental observations revealed that the enhancement originates from ¹³C→¹H polarization‐transfer processes active in the liquid state. Kinetic equations based on modified heteronuclear cross‐relaxation models were examined, and found to well describe the distinct patterns of growth and decay shown by the ¹³C‐bound ¹H NMR satellite resonances. The dynamics of these novel cross‐relaxation phenomena were determined, and their potential usefulness as tools for investigating hyperpolarized ensembles and for obtaining enhanced‐sensitivity ¹H NMR traces was explored.