Intermediates during the anionic polymerization of styrene were observed using hyperpolarized NMR. Dissolution dynamic nuclear polarization (DNP) of monomers provides a sufficient signal-to-noise ratio for detection of 13C NMR signals in real time as the reaction progresses. Because of its large chemical shift dispersion, 13C is well-suited to distinguish and characterize the chemical species that arise during the reaction. At the same time, incorporation of hyperpolarized small-molecule monomers is a unique way to generate polymers that exhibit a transient signal enhancement at the active site. This strategy is applicable despite the decay of the hyperpolarization of the polymer due to rapid spin–lattice relaxation. Real-time measurements on polymerization reactions provide both mechanistic and kinetic information without the need for stable isotope labeling of the molecules of interest. These capabilities are orthogonal to currently established methods that separate synthesis and analysis into two steps, making dissolution DNP an attractive method to study polymerization reactions.

A significant challenge in realizing the promise of the dissolution dynamic nuclear polarization technique for signal enhancement in high-resolution NMR lies in the nonrenewability of the hyperpolarized spin state. This property prevents the application of traditional two-dimensional correlation spectroscopy, which relies on regeneration of spin polarization before each successive increment of the indirect dimension. Since correlation spectroscopy is one of the most important approaches for the identification and structural characterization of molecules by NMR, it is important to find easily applicable methods that circumvent this problem. Here, we introduce the application of scaling of heteronuclear couplings by optimal tracking (SHOT) to achieve this goal. SHOT decoupling pulses have been numerically optimized on the basis of optimal control algorithms to obtain chemical shift correlations in C–H groups, either by acquiring a single one-dimensional 13C spectrum with 1H off-resonance decoupling or vice versa. Vanillin, which contains a number of functional groups, was used as a test molecule, allowing the demonstration of SHOT decoupling tailored toward simplified and accurate data analysis. This strategy was demonstrated for two cases: First, a linear response to chemical shift offset in the correlated dimension was optimized. Second, a pulse with alternating linear responses in the correlated dimension was chosen as a goal to increase the sensitivity of the decoupling response to the chemical shift offset. In these measurements, error ranges of ±0.03 ppm for the indirectly determined 1H chemical shifts and of ±0.4 ppm for the indirectly determined 13C chemical shifts were found. In all cases, we show that chemical shift correlations can be obtained from information contained in a single scan, which maximizes the ratio of signal to stochastic noise. Furthermore, a comprehensive discussion of the robustness of the method toward nonideal conditions is included based on experimental and simulated data. Unique features of this technique include the abilities to control the accuracy of chemical shift determination in spectral regions of interest and to acquire such chemical shift correlations rapidly—the latter being of interest for potential application in real-time spectroscopy.

The emergence of the dissolution dynamic nuclear polarization (D-DNP) technique provides an important breakthrough to overcome inherent sensitivity limitations in nuclear magnetic resonance (NMR) experiments. In dissolution DNP, only a small amount of frozen sample is polarized, dissolved, and injected into an NMR spectrometer. Although substantially enhanced NMR signals can be obtained, the single scan nature of this technique a priori impedes the use of correlation experiments, which represent some of the most powerful applications of NMR spectroscopy. Here, an alternative method for multiscan spectroscopy from D-DNP samples utilizing a flow NMR probe is described. Multiple hyperpolarized segments of sample are sequentially injected using a purpose designed device. Hadamard spectroscopy can then be applied for obtaining chemical shift correlation information even from a small number of scans. This capability is demonstrated with a four-scan data set for obtaining the [13C,1H] correlations in the test molecule 1-butanol. Because of the effects of spin–lattice relaxation and concentration gradients in the D-DNP experiment, the subtractive process for Hadamard reconstruction requires an additional step of intensity scaling. For this purpose, a reconstruction procedure was developed that uses entropy maximization and is robust with respect to noise and signal overlap. In a broader sense, the multiscan NMR as described here is amenable to various correlation NMR experiments, and increases the versatility of D-DNP in small-molecule characterization.

Fluorine NMR spectroscopy is widely used for detection of protein–ligand interactions in drug discovery because of the simplicity of fluorine spectra combined with a relatively high likelihood for a drug molecule to include at least one fluorine atom. In general, an important limitation of NMR spectroscopy in drug discovery is its sensitivity, which results in the need for unphysiologically high protein concentrations and large ligand:protein ratios. An enhancement in the 19F signal of several thousand fold by dynamic nuclear polarization allows for the detection of submicromolar concentrations of fluorinated small molecules. Techniques for exploiting this gain in signal to detect ligands in the strong-, intermediate-, and weak-binding regimes are presented. Similar to conventional NMR analysis, dissociation constants are determined. However, the ability to use a low ligand concentration permits the detection of ligands in slow exchange that are not easily amenable to drug screening by traditional NMR methods. The relative speed and additional information gained may make the hyperpolarization-based approach an interesting alternative for use in drug discovery.

Hyperpolarization of nuclear spins through techniques such as dynamic nuclear polarization (DNP) can greatly increase the signal-to-noise ratio in NMR measurements, thus eliminating the need for signal averaging. This enables the study of many dynamic processes which would otherwise not be amenable to study by NMR spectroscopy. A report of solid- to liquid-state DNP of a short peptide, bacitracin A, as well as of a full-length protein, L23, is presented here. The polypeptides are hyperpolarized at low temperature and dissolved for NMR signal acquisition in the liquid state in mixtures of organic solvent and water. Signal enhancements of 300–2000 are obtained in partially deuterated polypeptide when hyperpolarized on 13C and of 30–180 when hyperpolarized on 1H. A simulated spectrum is used to identify different resonances in the hyperpolarized 13C spectra, and the relation between observed signal enhancement for various groups in the protein and relaxation parameters measured from the hyperpolarized samples is discussed. Thus far, solid- to liquid-state DNP has been used in conjunction with small molecules. The results presented here, however, demonstrate the feasibility of hyperpolarizing larger proteins, with potential applications toward the study of protein folding or macromolecular interactions.

Hyperpolarization of nuclear spins is gaining increasing interest as a tool for improving the signal-to-noise ratio of NMR and MRI. While in principle, hyperpolarized samples are amenable to the same or similar experiments as are used in conventional NMR, the large spin polarization may give rise to unexpected effects. Here, spontaneous emission of signal was observed from proton spin systems, which were hyperpolarized to negative spin temperature by dynamic nuclear polarization (DNP). An unexpected feature of these emissions is that, without any radio-frequency excitation, multiple beats arise that cannot be explained by the Bloch equations with radiation damping. However, we show that a simple modification to these equations, which takes into account an additional supply of hyperpolarized magnetization from a reservoir outside of the active detection region, can phenomenologically describe the observed signal. The observed effect demonstrates that even well-known mechanisms of spin evolution can give rise to unexpected effects when working with hyperpolarized samples, which may need to be addressed through the development of new experimental techniques.Graphical abstractEmission of signals from a spin system hyperpolarized to negative spin temperature after injection into the active region of the NMR coil. Top: Free induction decay. Bottom: Time-frequency analysis.Research highlights► Hyperpolarization of protons to negative spin temperature yields unstable spin state. ► Spontaneous emission of signal cannot be described by radiation damping alone. ► Multiple emission features are explained by modified Bloch equations.

A combined simulation and experimental study was performed to investigate how methanol affects the structure of a model peptide BBA5. BBA5 forms a stable β-hairpin-α-helix structure in aqueous solutions. Molecular dynamics simulations were performed in water and methanol/water solutions using all-atom explicit models. NMR experiments were used to test the calculated results. The combined theoretical and experimental studies suggest that methanol strengthens the interactions between the polar backbone of the peptide and thus enhances the secondary structure formation; at the same time methanol weakens the hydrophobic interactions and results in an expansion of the hydrophobic core and an increase in gyration.

Short, secondary-structure-containing peptides are suitable models for the study of protein folding due to their relative simplicity. Here, we investigate thermal denaturation of the tryptophan zipper peptide, trpzip4, a peptide that forms a β-hairpin in solution. In order to monitor the thermal denaturation of peptides or small proteins, chemical shift values of Hα or HN may be used. However, various factors other than secondary structure can influence chemical shift values, such as side-chain orientation of nearby aromatic residues. Nuclear Overhauser effect (NOE) intensity from backbone interproton cross peaks is an alternative way to study thermal denaturation, as long as various factors that give rise to a change in NOE intensity upon changing the temperature are considered. As a relative indicator for denaturation, we define a cutoff temperature, where half of the initial NOE intensity is lost for each backbone interproton cross peak. For trpzip4, this cutoff temperature is highest for residues in the central part of the structure and lowest for residues near the termini. These observations support the notion that the structure of the trpzip4 peptide is stabilized by a hydrophobic cluster formed by tryptophan residues located in the central region of the β-hairpin.

Emerging techniques for hyperpolarization of nuclear spins, foremost dynamic nuclear polarization (DNP), lend unprecedented sensitivity to nuclear magnetic resonance spectroscopy. Sufficient signal can be obtained from a single scan, and reactions even far from equilibrium can be studied in real-time. When following the progress of a reaction by nuclear magnetic resonance, however, spin relaxation occurs concomitantly with the reaction to alter resonance line intensities. Here, we present a model for accounting for spin-relaxation in such reactions studied by hyperpolarized NMR. The model takes into account auto- and cross-relaxation in dipole−dipole coupled spin systems and is therefore applicable to NMR of hyperpolarized protons, the most abundant NMR-active nuclei. Applied to the Diels−Alder reaction of 1,4-dipheneylbutadiene (DPBD) with 4-phenyl-1,2,4-triazole-3,5-dione (PTD), reaction rates could be obtained accurately and reproducibly. Additional parameters available from the same experiment include relaxation rates of the reaction product, which may yield further information about the molecular properties of the product. The method presented is also compatible with an experiment where a single spin in the reactant is labeled in its spin-state by a selective radio frequency pulse for subsequent tracking through the reaction, allowing the unambiguous identification of its position in the product molecule. In this case, the chemical shift specificity of high-resolution NMR can allow for the simultaneous determination of reaction rates and mechanistic information in one experiment.

Due to its ability to enhance the signal of a single NMR scan by several orders of magnitude, solid-to-liquid state dynamic nuclear polarization (DNP) appears well suited for the analysis of minimal amounts of compounds, as well as for the study of rapid chemical reactions. A key requirement in enabling the application of DNP-NMR to typical small-molecule substances encountered in chemistry and biochemistry is the ability to obtain high-resolution spectra, while at the same time minimizing the loss of polarization due to spin relaxation between the separate steps of DNP polarization and NMR measurement. Here, we present data demonstrating the capability of measuring DNP enhanced NMR spectra of compounds with comparably short relaxation times, with only minimal line broadening attributable to the sample transfer process. We discuss the performance characteristics of a sample injection apparatus specifically designed to provide high-resolution DNP-NMR spectra of small molecule compounds.

Nuclear Magnetic Resonance (NMR) is an important spectroscopic tool for the identification and structural characterization of molecules in chemistry and biochemistry. The most significant limitation of NMR compared to other spectroscopies is its relatively low sensitivity, which thus often requires long measurement times or large amounts of sample. A way of increasing sensitivity of single scan NMR spectra by several orders of magnitude is through hyperpolarization of nuclear spins. Dynamic nuclear polarization allows hyperpolarization of most spins in small molecules encountered in chemistry and biochemistry. NMR spectra of small amounts of samples from natural source, or from chemical synthesis can readily be acquired. Perhaps more interestingly, the availability of the entire hyperpolarized NMR signal in one single scan allows the measurement of transient processes in real time, if applied together with a stopped-flow technique. Through observation of chemical shift, different reactant and product species can be distinguished, and kinetics and mechanisms, for example in enzyme catalyzed reactions, can be elucidated. Real-time hyperpolarization-enhanced NMR is uniquely amenable to correlating atomic positions not only through space, but also over time between reactant and product species. Such correlations carry mechanistic information about a reaction, and can prove reaction pathways. Applications of this technique are emerging in different areas of chemistry concerned with rapid reactions, including not only enzymatic processes, but also chemical catalysis and protein folding.

Due to its ability to enhance the signal of a single NMR scan by several orders of magnitude, solid-to-liquid state dynamic nuclear polarization (DNP) appears well suited for the analysis of minimal amounts of compounds, as well as for the study of rapid chemical reactions. A key requirement in enabling the application of DNP-NMR to typical small-molecule substances encountered in chemistry and biochemistry is the ability to obtain high-resolution spectra, while at the same time minimizing the loss of polarization due to spin relaxation between the separate steps of DNP polarization and NMR measurement. Here, we present data demonstrating the capability of measuring DNP enhanced NMR spectra of compounds with comparably short relaxation times, with only minimal line broadening attributable to the sample transfer process. We discuss the performance characteristics of a sample injection apparatus specifically designed to provide high-resolution DNP-NMR spectra of small molecule compounds.

Nuclear Magnetic Resonance (NMR) is an important spectroscopic tool for the identification and structural characterization of molecules in chemistry and biochemistry. The most significant limitation of NMR compared to other spectroscopies is its relatively low sensitivity, which thus often requires long measurement times or large amounts of sample. A way of increasing sensitivity of single scan NMR spectra by several orders of magnitude is through hyperpolarization of nuclear spins. Dynamic nuclear polarization allows hyperpolarization of most spins in small molecules encountered in chemistry and biochemistry. NMR spectra of small amounts of samples from natural source, or from chemical synthesis can readily be acquired. Perhaps more interestingly, the availability of the entire hyperpolarized NMR signal in one single scan allows the measurement of transient processes in real time, if applied together with a stopped-flow technique. Through observation of chemical shift, different reactant and product species can be distinguished, and kinetics and mechanisms, for example in enzyme catalyzed reactions, can be elucidated. Real-time hyperpolarization-enhanced NMR is uniquely amenable to correlating atomic positions not only through space, but also over time between reactant and product species. Such correlations carry mechanistic information about a reaction, and can prove reaction pathways. Applications of this technique are emerging in different areas of chemistry concerned with rapid reactions, including not only enzymatic processes, but also chemical catalysis and protein folding.