Optically Enhanced Solid-State 1H NMR Spectroscopy.

Low sensitivity is the primary limitation to extending nuclear magnetic resonance (NMR) techniques to more advanced chemical and structural studies. Photochemically induced dynamic nuclear polarization (photo-CIDNP) is an NMR hyperpolarization technique where light is used to excite a suitable donor-acceptor system, creating a spin-correlated radical pair whose evolution drives nuclear hyperpolarization. Systems that exhibit photo-CIDNP in solids are not common, and this effect has, up to now, only been observed for 13C and 15N nuclei. However, the low gyromagnetic ratio and natural abundance of these nuclei trap the local hyperpolarization in the vicinity of the chromophore and limit the utility for bulk hyperpolarization. Here, we report the first example of optically enhanced solid-state 1H NMR spectroscopy in the high-field regime. This is achieved via photo-CIDNP of a donor-chromophore-acceptor molecule in a frozen solution at 0.3 T and 85 K, where spontaneous spin diffusion among the abundant strongly coupled 1H nuclei relays polarization through the whole sample, yielding a 16-fold bulk 1H signal enhancement under continuous laser irradiation at 450 nm. These findings enable a new strategy for hyperpolarized NMR beyond the current limits of conventional microwave-driven DNP.

Heterogeneous Overhauser-DNP on 1 H dominated by scalar coupling in aqueous solution.

The Overhauser Dynamic Nuclear Polarization (O-DNP) of 1 H nuclei usually involves a dipolar coupling with the polarizing agent, whereas scalar coupling via hyperfine interactions are more common with 13 C nuclei. Here, we show a scalar-coupling dominated 1 H O-DNP, using polyaniline as a heterogeneous polarizing agent in an aqueous solution.

Electrochemical Overhauser dynamic nuclear polarization.

Nuclear Magnetic Resonance (NMR) spectroscopy suffers from low sensitivity due to the low nuclear spin polarization obtained within practically achievable external magnetic fields. Dynamic Nuclear Polarization (DNP) refers to techniques that increase the NMR signal intensity by transferring spin polarization from electrons to the nuclei. Until now, a common method of introducing unpaired electrons to a sample has been to add to it a radical such as TEMPOL or trityl. The alternative we address here is to use electrochemical oxidation and/or reduction of a redox mediator to generate radical species that can be used for DNP. Surprisingly, the potential of electrochemically-generated radicals as a source of hyperpolarization for DNP has not been investigated so far. In this communication, we show the proof of principle of performing an in situ DNP experiment at a low magnetic field in a solution phase, with electrochemically generated methyl viologen cation radicals. Electrochemistry as a source of radicals can offer exciting prospects for DNP. The electrode may be one that generates radicals with a high spin polarization. The concentration of radicals in the sample can be adjusted by changing the duration and magnitude of the applied electrode potential. Removal of the radical from the sample after spin polarization transfer is also possible, thereby increasing the lifetime of the nuclear hyperpolarization.

Hybrid polarizing solids for pure hyperpolarized liquids through dissolution dynamic nuclear polarization.

Hyperpolarization of substrates for magnetic resonance spectroscopy (MRS) and imaging (MRI) by dissolution dynamic nuclear polarization (D-DNP) usually involves saturating the ESR transitions of polarizing agents (PAs; e.g., persistent radicals embedded in frozen glassy matrices). This approach has shown enormous potential to achieve greatly enhanced nuclear spin polarization, but the presence of PAs and/or glassing agents in the sample after dissolution can raise concerns for in vivo MRI applications, such as perturbing molecular interactions, and may induce the erosion of hyperpolarization in spectroscopy and MRI. We show that D-DNP can be performed efficiently with hybrid polarizing solids (HYPSOs) with 2,2,6,6-tetramethyl-piperidine-1-oxyl radicals incorporated in a mesostructured silica material and homogeneously distributed along its pore channels. The powder is wetted with a solution containing molecules of interest (for example, metabolites for MRS or MRI) to fill the pore channels (incipient wetness impregnation), and DNP is performed at low temperatures in a very efficient manner. This approach allows high polarization without the need for glass-forming agents and is applicable to a broad range of substrates, including peptides and metabolites. During dissolution, HYPSO is physically retained by simple filtration in the cryostat of the DNP polarizer, and a pure hyperpolarized solution is collected within a few seconds. The resulting solution contains the pure substrate, is free from any paramagnetic or other pollutants, and is ready for in vivo infusion.

Highly Repeatable Dissolution Dynamic Nuclear Polarization for Heteronuclear NMR Metabolomics.

At natural (13)C abundance, metabolomics based on heteronuclear NMR is limited by sensitivity. We have recently demonstrated how hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) assisted by cross-polarization (CP) provides a reliable way of enhancing the sensitivity of heteronuclear NMR in dilute mixtures of metabolites. In this Technical Note, we evaluate the precision of this experimental approach, a critical point for applications to metabolomics. The higher the repeatability, the greater the likelihood that one can detect small biologically relevant differences between samples. The average repeatability of our state-of-the-art D-DNP NMR equipment for samples of metabolomic relevance (20 mg dry weight tomato extracts) is 3.6% for signals above the limit of quantification (LOQ) and 6.4% when all the signals above the limit of detection (LOD) are taken into account. This first report on the repeatability of D-DNP highlights the compatibility of the technique with the requirements of metabolomics and confirms its potential as an analytical tool for such applications.

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((1) H)=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 (13) C-labeled metabolites such as pyruvate, acetate, and alanine.

Dissolution dynamic nuclear polarization of deuterated molecules enhanced by cross-polarization.

We present novel means to hyperpolarize deuterium nuclei in 13CD2 groups at cryogenic temperatures. The method is based on cross-polarization from 1H to 13C and does not require any radio-frequency fields applied to the deuterium nuclei. After rapid dissolution, a new class of long-lived spin states can be detected indirectly by 13C NMR in solution. These long-lived states result from a sextet-triplet imbalance (STI) that involves the two equivalent deuterons with spin I = 1. An STI has similar properties as a triplet-singlet imbalance that can occur in systems with two equivalent I = 12 spins. Although the lifetimes TSTI are shorter than T1(Cz), they can exceed the life-time T1(Dz) of deuterium Zeeman magnetization by a factor of more than 20.

Spin Noise Detection of Nuclear Hyperpolarization at 1.2†K.

We report proton spin noise spectra of a hyperpolarized solid sample of commonly used "DNP (dynamic nuclear polarization) juice" containing TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine N-oxide) and irradiated by a microwave field at a temperature of 1.2 K in a magnetic field of 6.7 T. The line shapes of the spin noise power spectra are sensitive to the variation of the microwave irradiation frequency and change from dip to bump, when the electron Larmor frequency is crossed, which is shown to be in good accordance with theory by simulations. Small but significant deviations from these predictions are observed, which can be related to spin noise and radiation damping phenomena that have been reported in thermally polarized systems. The non-linear dependence of the spin noise integral on nuclear polarization provides a means to monitor hyperpolarization semi-quantitatively without any perturbation of the spin system by radio frequency irradiation.

Hyperpolarized NMR of plant and cancer cell extracts at natural abundance.

Natural abundance (13)C NMR spectra of biological extracts are recorded in a single scan provided that the samples are hyperpolarized by dissolution dynamic nuclear polarization combined with cross polarization. Heteronuclear 2D correlation spectra of hyperpolarized breast cancer cell extracts can also be obtained in a single scan. Hyperpolarized NMR of extracts opens many perspectives for metabolomics.

Challenges in preparing, preserving and detecting para-water in bulk: overcoming proton exchange and other hurdles.

Para-water is an analogue of para-hydrogen, where the two proton spins are in a quantum state that is antisymmetric under permutation, also known as singlet state. The populations of the nuclear spin states in para-water are believed to have long lifetimes just like other Long-Lived States (LLSs). This hypothesis can be verified by measuring the relaxation of an excess or a deficiency of para-water, also known as a "Triplet-Singlet Imbalance" (TSI), i.e., a difference between the average population of the three triplet states T (that are symmetric under permutation) and the population of the singlet state S. In analogy with our recent findings on ethanol and fumarate, we propose to adapt the procedure for Dissolution Dynamic Nuclear Polarization (D-DNP) to prepare such a TSI in frozen water at very low temperatures in the vicinity of 1.2 K. After rapid heating and dissolution using an aprotic solvent, the TSI should be largely preserved. To assess this hypothesis, we studied the lifetime of water as a molecular entity when diluted in various solvents. In neat liquid H2O, proton exchange rates have been characterized by spin-echo experiments on oxygen-17 in natural abundance, with and without proton decoupling. One-dimensional exchange spectroscopy (EXSY) has been used to study proton exchange rates in H2O, HDO and D2O mixtures diluted in various aprotic solvents. In the case of 50 mM H2O in dioxane-d8, the proton exchange lifetime is about 20 s. After dissolving, one can observe this TSI by monitoring intensities in oxygen-17 spectra of H2O (if necessary using isotopically enriched samples) where the AX2 system comprising a "spy" oxygen A and two protons X2 gives rise to binomial multiplets only if the TSI vanishes. Alternatively, fast chemical addition to a suitable substrate (such as an activated aldehyde or ketone) can provide AX2 systems where a carbon-13 acts as a spy nucleus. Proton signals that relax to equilibrium with two distinct time constants can be considered as a hallmark of a TSI. We optimized several experimental procedures designed to preserve and reveal dilute para-water in bulk.

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.

Cross polarization from (1)H to quadrupolar (6)Li nuclei for dissolution DNP.

Cross polarization from protons to quadrupolar (6)Li nuclei is combined with dynamic nuclear polarization of protons at 1.2 K and 6.7 T using TEMPOL as a polarizing agent followed by rapid dissolution. Compared to direct (6)Li DNP without cross-polarization, a higher nuclear spin polarization P((6)Li) can be obtained in a shorter time. A double resonance (1)H-(6)Li probe was designed that is equipped for Longitudinally Detected Electron Spin Resonance.

Dynamic Nuclear Polarization and other magnetic ideas at EPFL.

Although nuclear magnetic resonance (NMR) can provide a wealth of information, it often suffers from a lack of sensitivity. Dynamic Nuclear Polarization (DNP) provides a way to increase the polarization and hence the signal intensities in NMR spectra by transferring the favourable electron spin polarization of paramagnetic centres to the surrounding nuclear spins through appropriate microwave irradiation. In our group at EPFL, two complementary DNP techniques are under investigation: the combination of DNP with magic angle spinning at temperatures near 100 K ('MAS-DNP'), and the combination of DNP at 1.2 K with rapid heating followed by the transfer of the sample to a high-resolution magnet ('dissolution DNP'). Recent applications of MAS-DNP to surfaces, as well as new developments of magnetization transfer of (1)H to (13)C at 1.2 K prior to dissolution will illustrate the work performed in our group. A second part of the paper will give an overview of some 'non-enhanced' activities of our laboratory in liquid- and solid-state NMR.

Blatter-type radicals as polarizing agents for electrochemical overhauser dynamic nuclear polarization.

Overhauser dynamic nuclear polarization (O-DNP) refers to a microwave-assisted process where an unpaired electron's (e.g. a radical) spin polarization is transferred to surrounding nuclei in solution, thus increasing the nuclear magnetic resonance (NMR) signal intensity of a given substance by several orders of magnitude. The presence of the unpaired electrons, which induces relaxation of the resulting hyperpolarized state when the radiation is halted, can be avoided by electrochemically removing the radicals on demand. We report the use of Blatter-type (benzo[e][1,2,4]triazinyl) radicals as polarizing agents, potentially opening the way to highly tunable radicals for electrochemical DNP.

Porous functionalized polymers enable generating and transporting hyperpolarized mixtures of metabolites.

Hyperpolarization by dissolution dynamic nuclear polarization (dDNP) has enabled promising applications in spectroscopy and imaging, but remains poorly widespread due to experimental complexity. Broad democratization of dDNP could be realized by remote preparation and distribution of hyperpolarized samples from dedicated facilities. Here we show the synthesis of hyperpolarizing polymers (HYPOPs) that can generate radical- and contaminant-free hyperpolarized samples within minutes with lifetimes exceeding hours in the solid state. HYPOPs feature tunable macroporous porosity, with porous volumes up to 80% and concentration of nitroxide radicals grafted in the bulk matrix up to 285 µmol g-1. Analytes can be efficiently impregnated as aqueous/alcoholic solutions and hyperpolarized up to P(13C) = 25% within 8 min, through the combination of 1H spin diffusion and 1H → 13C cross polarization. Solutions of 13C-analytes of biological interest hyperpolarized in HYPOPs display a very long solid-state 13C relaxation times of 5.7 h at 3.8 K, thus prefiguring transportation over long distances.

Hyperpolarization of nitrogen-15 nuclei by cross polarization and dissolution dynamic nuclear polarization.

Dynamic Nuclear Polarization (DNP) is often achieved by the direct transfer of polarization from electrons to nuclei such as 13C, induced by microwave saturation of the wings of narrow EPR lines of radicals like trityl. In the indirect approach on the other hand, DNP is used to transfer the polarization from the electrons of radicals such as nitroxides that have broad EPR lines to nuclear spins I = 1H, followed by cross-polarization (CP) from I = 1H to S = 13C or other nuclei with low gyromagnetic ratios. This approach is particularly attractive for S = 15N, since direct DNP yields modest polarizations P(15N) < 4% with build-up times that can be as long as τDNP(15N) > 2 h. In this paper, we show that CP from 1H to 15N at 1.2 K can yield P(15N) = 25% with τCP-DNP(15N) = 10-15 min. After rapid dissolution and transfer to a solution-state NMR spectrometer, a polarization P(15N) = 20% was observed at 300 K. The longitudinal relaxation times in solution can be as long as T1(15N) > 800 s in favorable cases.

Transportable hyperpolarized metabolites.

Nuclear spin hyperpolarization of 13C-labelled metabolites by dissolution dynamic nuclear polarization can enhance the NMR signals of metabolites by several orders of magnitude, which has enabled in vivo metabolic imaging by MRI. However, because of the short lifetime of the hyperpolarized magnetization (typically <1 min), the polarization process must be carried out close to the point of use. Here we introduce a concept that markedly extends hyperpolarization lifetimes and enables the transportation of hyperpolarized metabolites. The hyperpolarized sample can thus be removed from the polarizer and stored or transported for use at remote MRI or NMR sites. We show that hyperpolarization in alanine and glycine survives 16 h storage and transport, maintaining overall polarization enhancements of up to three orders of magnitude.

Hyperpolarization of Frozen Hydrocarbon Gases by Dynamic Nuclear Polarization at 1.2 K.

We report a simple and general method for the hyperpolarization of condensed gases by dynamic nuclear polarization (DNP). The gases are adsorbed in the pores of structured mesoporous silica matrices known as HYPSOs (HYper Polarizing SOlids) that have paramagnetic polarizing agents covalently bound to the surface of the mesopores. DNP is performed at low temperatures and moderate magnetic fields (T = 1.2 K and B0 = 6.7 T). Frequency-modulated microwave irradiation is applied close to the electron spin resonance frequency (f = 188.3 GHz), and the electron spin polarization of the polarizing agents of HYPSO is transferred to the nuclear spins of the frozen gas. A proton polarization as high as P((1)H) = 70% can be obtained, which can be subsequently transferred to (13)C in natural abundance by cross-polarization, yielding up to P((13)C) = 27% for ethylene.

Measuring absolute spin polarization in dissolution-DNP by Spin PolarimetrY Magnetic Resonance (SPY-MR).

Dynamic nuclear polarization at 1.2 K and 6.7 T allows one to achieve spin temperatures on the order of a few millikelvin, so that the high-temperature approximation (ΔE

Hyperpolarized Water to Study Protein-Ligand Interactions.

The affinity between a chosen target protein and small molecules is a key aspect of drug discovery. Screening by popular NMR methods such as Water-LOGSY suffers from low sensitivity and from false positives caused by aggregated or denatured proteins. This work demonstrates that the sensitivity of Water-LOGSY can be greatly boosted by injecting hyperpolarized water into solutions of proteins and ligands. Ligand binding can be detected in a few seconds, whereas about 30 min is usually required without hyperpolarization. Hyperpolarized water also enhances proton signals of proteins at concentrations below 20 µM so that one can verify in a few seconds whether the proteins remain intact or have been denatured.