Exploring weak ligand-protein interactions by relaxometry of long-lived spin order.

Relaxation times of nuclear spins often serve as a valuable source of information on the dynamics of various biochemical processes. Measuring relaxation as a function of the external magnetic field turned out to be extremely useful for the studies of weak ligand-protein interactions. We demonstrate that observing the relaxation of the long-lived spin order instead of longitudinal magnetization extends the capability of this approach. We studied the field-dependent relaxation of the longitudinal magnetization and the singlet order (SO) of methylene protons in alanine-glycine dipeptide and citrate in the presence of human serum albumin (HSA). As a result, SO relaxation proved to be more sensitive to ligand-protein interaction, providing higher relaxation contrast for various HSA concentrations. To assess the parameters of the binding process in more details, we utilized a simple analytical relaxation model to fit the experimental field dependences for both SO and T1 relaxation. We also tested the validity of our approach in the experiments with trimethylsilylpropanoic acid (TSP) used as a competitor in ligand binding with HSA.

Microgravity-like Crystallization of Paramagnetic Species in Strong Magnetic Fields.

The crystallization of paramagnetic species in a magnetic field gradient under microgravity-like conditions is an area of interest for both fundamental and applied science. In this paper, a setup for the crystallization of paramagnetic species in the magnetic field up to 7 T generated by a superconducting magnet is described. The research includes calculations of the conditions necessary to compensate for the gravitational force for several types of paramagnetic substances using the magnetic field of superconducting magnets (4.7 T, 7 T, 9.4 T, and 16.4 T). Additionally, for the first time, the crystallization of copper sulfate and cobalt sulfate, as well as a mixture of copper sulfate and cobalt sulfate under gravitational force compensation in a superconducting magnet, was performed. This paper experimentally demonstrates the feasibility of growing paramagnetic crystals within the volume of a test tube on the example of copper and cobalt sulfate crystals. A comparison of crystals grown from the solution of a mixture of copper and cobalt sulfates under the same conditions, with and without the presence of a magnetic field, showed changes in both the number and size of crystals.

UiO-66 framework with an encapsulated spin probe: synthesis and exceptional sensitivity to mechanical pressure.

Probes sensitive to mechanical stress are in demand for the analysis of pressure distribution in materials, and the design of pressure sensors based on metal-organic frameworks (MOFs) is highly promising due to their structural tunability. We report a new pressure-sensing material, which is based on the UiO-66 framework with trace amounts of a spin probe (0.03 wt%) encapsulated in cavities. To obtain this material, we developed an approach for encapsulation of stable nitroxide radical TEMPO ((2,2,6,6-tetramethylpiperidin-1-yl)oxyl) into the micropores of UiO-66 during its solvothermal synthesis. Pressure read-out using electron paramagnetic resonance (EPR) spectroscopy allows monitoring the degradation of the defected MOF structure upon pressurization, where full collapse of pores occurs at as low a pressure as 0.13 GPa. The developed methodology can be used in and ex situ and provides sensitive tools for non-destructive mapping of pressure effects in various materials.

Polarizing insensitive nuclei at ultralow magnetic fields using parahydrogen: A facile route to optimize adiabatic magnetic field sweeps.

Parahydrogen induced polarization (PHIP) provides a powerful tool to enhance inherently weak nuclear magnetic resonance signals, particularly in biologically relevant compounds. The initial source of PHIP is the non-equilibrium spin order of parahydrogen, i.e., dihydrogen, where the two protons make up a singlet spin state. Conversion of this spin order into net magnetization of magnetic heteronuclei, e.g., 13C, provides one of the most efficient ways to exploit PHIP. We propose a facile route to increase the performance of PHIP transfer in experiments with adiabatic sweeps of the ultralow magnetic field. To date, this technique yields the highest efficiency of PHIP transfer, yet, it has been mostly utilized with linear field sweeps, which does not consider the underlying spin dynamics, resulting in sub-optimal polarization. This issue was previously addressed by using the "constant" adiabaticity method, which, however, requires extensive calculations for large spin systems. In this work, the field sweep is optimized by utilizing the field dependence of the average 13C polarization. Both the experimental detection and the numerical simulation of this dependence are straightforward, even for complex multi-spin systems. This work provides a comprehensive survey of PHIP transfer dynamics at ultralow fields for two molecular systems that are relevant for PHIP, namely, maleic acid and allyl pyruvate. The proposed optimization allowed us to increase the resulting 13C polarization in 13C-allyl pyruvate from 6.8% with a linear profile to 8.7% with an "optimal" profile. Such facile optimization routines are valuable for adiabatic experiments in complex spin systems undergoing rapid relaxation or chemical exchange.

Parahydrogen-Induced Hyperpolarization of Unsaturated Phosphoric Acid Derivatives.

Parahydrogen-induced nuclear polarization offers a significant increase in the sensitivity of NMR spectroscopy to create new probes for medical diagnostics by magnetic resonance imaging. As precursors of the biocompatible hyperpolarized probes, unsaturated derivatives of phosphoric acid, propargyl and allyl phosphates, are proposed. The polarization transfer to 1H and 31P nuclei of the products of their hydrogenation by parahydrogen under the ALTADENA and PASADENA conditions, and by the PH-ECHO-INEPT+ pulse sequence of NMR spectroscopy, resulted in a very high signal amplification, which is among the largest for parahydrogen-induced nuclear polarization transfer to the 31P nucleus.

15 N SABRE Hyperpolarization of Metronidazole at Natural Isotope Abundance.

Signal Amplification By Reversible Exchange (SABRE) is gaining increased attention as a tool to enhance weak Nuclear Magnetic Resonance (NMR) signals. In SABRE, spin order is transferred from parahydrogen (H2 in its nuclear singlet spin state) to a substrate molecule in a transient Ir-based complex. In recent years, SABRE polarization of biologically active substrates has been demonstrated, notably of metronidazole - an antibiotic and antiprotozoal drug. In this work, we study 15 N SABRE polarization of metronidazole at natural isotope abundance. We are able to demonstrate significant 15 N polarization reaching 15 %, which corresponds to a signal enhancement of 46,000 at 9.4 T for the nitrogen atom with lone electron pair. Additionally, the other two N-atoms can be polarized, although less efficiently. We present a detailed study of the field dependence of polarization and explain the maxima in the field dependence using the concept of coherent polarization transfer at level anti-crossings in the SABRE complex. A study of spin relaxation phenomena presented here enables optimization of the magnetic field for efficient storage of non-thermal polarization.

Hyperpolarization of cis-15 N2 -Azobenzene by Parahydrogen at Ultralow Magnetic Fields*.

The development of nuclear spins hyperpolarization, and the search for molecules that can be efficiently hyperpolarized is an active area in nuclear magnetic resonance. In this work we present a detailed study of SABRE SHEATH (signal amplification by reversible exchange in shield enabled alignment transfer to heteronuclei) experiments on 15 N2 -azobenzene. In SABRE SHEATH experiments the nuclear spins of the target are hyperpolarized through transfer of spin polarization from parahydrogen at ultralow fields during a reversible chemical process. Azobenzene exists in two isomers, trans and cis. We show that all nuclear spins in cis-azobenzene can be efficiently hyperpolarized by SABRE at suitable magnetic fields. Enhancement factors (relative to 9.4 T) reach up to 3000 for 15 N spins and up to 30 for the 1 H spins. We compare two approaches to observe either hyperpolarized magnetization of 15 N/1 H spins, or hyperpolarized singlet order of the 15 N spin pair. The results presented here will be useful for further experiments in which hyperpolarized cis-15 N2 -azobenzene is switched by light to trans-15 N2 -azobenzene for storing the produced hyperpolarization in the long-lived spin state of the 15 N pair of trans-15 N2 -azobenzene.

Sequential assignment of NMR spectra of peptides at natural isotopic abundance with zero- and ultra-low-field total correlation spectroscopy (ZULF-TOCSY).

A novel method dubbed ZULF-TOCSY results from the combination of Zero and Ultra-Low Field (ZULF) with high-field, high-resolution NMR, leading to a generalization of the concept of total correlation spectroscopy (TOCSY). ZULF-TOCSY is a new building block for NMR methods, which has the unique property that the polarization is evenly distributed among all NMR-active nuclei such as 1H, 13C, 15N, 31P, etc., provided that they belong to the same coupling network, and provided that their relaxation is not too fast at low fields, as may occur in macromolecules. Here, we show that ZULF-TOCSY correlations can be observed for peptides at natural isotopic abundance, such as the protected hexapeptide Boc-Met-enkephalin. The analysis of ZULF-TOCSY spectra readily allows one to make sequential assignments, thus offering an alternative to established heteronuclear 2D experiments like HMBC. For Boc-Met-enkephalin, we show that ZULF-TOCSY allows one to observe all expected cross-peaks between carbonyl carbons and α-CH protons, while the popular HMBC method provides insufficient information.

Singlet to triplet conversion in molecular hydrogen and its role in parahydrogen induced polarization.

Detailed experimental and comprehensive theoretical analysis of singlet-triplet conversion in molecular hydrogen dissolved in a solution together with organometallic complexes used in experiments with parahydrogen (the H2 molecule in its nuclear singlet spin state) is reported. We demonstrate that this conversion, which gives rise to formation of orthohydrogen (the H2 molecule in its nuclear triplet spin state), is a remarkably efficient process that strongly reduces the resulting NMR (nuclear magnetic resonance) signal enhancement, here of 15N nuclei polarized at high fields using suitable NMR pulse sequences. We make use of a simple improvement of traditional pulse sequences, utilizing a single pulse on the proton channel that gives rise to an additional strong increase of the signal. Furthermore, analysis of the enhancement as a function of the pulse length allows one to estimate the actual population of the spin states of H2. We are also able to demonstrate that the spin conversion process in H2 is strongly affected by the concentration of 15N nuclei. This observation allows us to explain the dependence of the 15N signal enhancement on the abundance of 15N isotopes.

Correlation of high-field and zero- to ultralow-field NMR properties using 2D spectroscopy.

The field of zero- to ultralow-field (ZULF) nuclear magnetic resonance (NMR) is currently experiencing rapid growth, owing to progress in optical magnetometry and attractive features of ZULF-NMR such as low hardware cost and excellent spectral resolution achieved under ZULF conditions. In this work, an approach is proposed and demonstrated for simultaneous acquisition of ZULF-NMR spectra of individual 13C-containing isotopomers of chemical compounds in a complex mixture. The method makes use of fast field cycling such that the spin evolution takes place under ZULF conditions, whereas signal detection is performed in a high-field NMR spectrometer. This method has excellent sensitivity, also allowing easy assignment of ZULF-NMR spectra to specific analytes in the mixture. We demonstrate that the spectral information is the same as that given by ZULF-NMR, which makes the method suitable for creating a library of ZULF-NMR spectra of various compounds and their isotopomers. The results of the field-cycling experiments can be presented in a convenient way as 2D-NMR spectra with the direct dimension giving the high-field 13C-NMR spectrum (carrying the chemical-shift information) and the indirect dimension giving the ZULF-NMR spectrum (containing information about proton-carbon J-couplings). Hence, the method can be seen as a variant of heteronuclear J-resolved spectroscopy, one of the first 2D-NMR techniques.

Nuclear singlet relaxation by chemical exchange.

The population imbalance between nuclear singlet states and triplet states of strongly coupled spin-1/2 pairs, also known as nuclear singlet order, is well protected against several common relaxation mechanisms. We study the nuclear singlet relaxation of 13C pairs in aqueous solutions of 1,2-13C2 squarate over a range of pH values. The 13C singlet order is accessed by introducing 18O nuclei in order to break the chemical equivalence. The squarate dianion is in chemical equilibrium with hydrogen-squarate (SqH-) and squaric acid (SqH2) characterized by the dissociation constants pK1 = 1.5 and pK2 = 3.4. Surprisingly, we observe a striking increase in the singlet decay time constants TS when the pH of the solution exceeds ∼10, which is far above the acid-base equilibrium points. We derive general rate expressions for chemical-exchange-induced nuclear singlet relaxation and provide a qualitative explanation of the TS behavior of the squarate dianion. We identify a kinetic contribution to the singlet relaxation rate constant, which explicitly depends on kinetic rate constants. Qualitative agreement is achieved between the theory and the experimental data. This study shows that infrequent chemical events may have a strong effect on the relaxation of nuclear singlet order.

Simultaneous 15 N polarization of several biocompatible substrates in ethanol-water mixtures by signal amplification by reversible exchange (SABRE) method.

Signal amplification by reversible exchange (SABRE) is a popular method for generating strong signal enhancements in nuclear magnetic resonance (NMR). In SABRE experiments, the source of polarization is provided by the nonthermal spin order of parahydrogen (pH2 , the H2 molecule in its nuclear singlet spin state). Polarization formation requires that both pH2 and a substrate molecule bind to an Ir-based complex where polarization transfer occurs. Subsequently, the complex dissociates and free polarized substrate molecules are formed. In this work, we present approaches towards biocompatible SABRE, meaning that several small biomolecules are simultaneously polarized by using the SABRE method in water-ethanol solutions at room temperature. We are able to demonstrate significant 15 N-NMR signal enhancements in water-ethanol solutions for biomolecules like nicotinamide, metronidazole, adenosine-5'-monophosphate, and 4-methylimidazole and found that the first three substrates are polarized at the same level as a well-known pyridine. We show that simultaneous polarization of several molecules is indeed feasible when the reactions are carried out at an ultralow field of about 400-500 nT. The achieved enhancements are between 100-fold and 15,000-fold. The resulting 15 N polarization (maximal value about 4% achieved for metronidazole and pyridine at 45°C) strongly depends on the sample temperature, pH2 bubbling pressure, and pH2 flow. One more parameter, which is important for optimizing the enhancement, is the solvent pH. Hence, this study presents a step in developing biocompatible SABRE polarization and gives a clue on how such SABRE experiments should be optimized to achieve the highest NMR signal enhancement.

Validation of Structural Grounds for Anomalous Molecular Mobility in Ionic Liquid Glasses.

Ionic liquid (IL) glasses have recently drawn much interest as unusual media with unique physicochemical properties. In particular, anomalous suppression of molecular mobility in imidazolium IL glasses vs. increasing temperature was evidenced by pulse Electron Paramagnetic Resonance (EPR) spectroscopy. Although such behavior has been proven to originate from dynamics of alkyl chains of IL cations, the role of electron spin relaxation induced by surrounding protons still remains unclear. In this work we synthesized two deuterated imidazolium-based ILs to reduce electron-nuclear couplings between radical probe and alkyl chains of IL, and investigated molecular mobility in these glasses. The obtained trends were found closely similar for deuterated and protonated analogs, thus excluding the relaxation-induced artifacts and reliably demonstrating structural grounds of the observed anomalies in heterogeneous IL glasses.

Spin dynamics in experiments on orthodeuterium induced polarization (ODIP).

A comprehensive description of the spin dynamics underlying the formation of Ortho-Deuterium Induced Polarization (ODIP) is presented. ODIP can serve as a tool for enhancing Nuclear Magnetic Resonance (NMR) signals of 2H nuclei, being important probes of molecular structure and dynamics. To produce ODIP, in the first step, the D2 gas is brought to thermal equilibrium at low temperature, here 30 K, so that the ortho-component, corresponding to the total spin of the 2H nuclei equal to 0 and 2, is enriched, here to 92%. In the second step, the orthodeuterium molecule is attached to a substrate molecule using a suitable hydrogenation catalyst such that the symmetry of the two 2H nuclei is broken. As a result, the non-thermal spin order of orthodeuterium is converted into enhancement of observable NMR signals. In this work, we perform a theoretical study of ODIP and calculate the shape of ODIP spectra and their dependence on the magnetization flip angle. These results are compared with experiments performed for a number of substrates; good agreement between experimental and calculated ODIP spectra is found. We also discuss the performance of NMR techniques for converting anti-phase ODIP spectral patterns into in-phase patterns, which are more suitable for signal detection and for transferring ODIP to heteronuclei, here to 13C spins. Experimental procedures reported here allowed us to reach signal enhancement factors of more than 1000 for 2H nuclei in the liquid phase. These results are useful for extending the scope of spin hyperpolarization to the widely used 2H nuclei.

Algorithmic cooling of nuclear spins using long-lived singlet order.

Algorithmic cooling methods manipulate an open quantum system in order to lower its temperature below that of the environment. We achieve significant cooling of an ensemble of nuclear spin-pair systems by exploiting the long-lived nuclear singlet state, which is an antisymmetric quantum superposition of the "up" and "down" Zeeman states. The effect is demonstrated by nuclear magnetic resonance experiments on a molecular system containing a coupled pair of near-equivalent 13C nuclei. The populations of the system are subjected to a repeating sequence of cyclic permutations separated by relaxation intervals. The long-lived nuclear singlet order is pumped well beyond the unitary limit. The pumped singlet order is converted into nuclear magnetization which is enhanced by 21% relative to its thermal equilibrium value.

Oxidative damage to epigenetically methylated sites affects DNA stability, dynamics and enzymatic demethylation.

DNA damage can affect various regulatory elements of the genome, with the consequences for DNA structure, dynamics, and interaction with proteins remaining largely unexplored. We used solution NMR spectroscopy, restrained and free molecular dynamics to obtain the structures and investigate dominant motions for a set of DNA duplexes containing CpG sites permuted with combinations of 5-methylcytosine (mC), the primary epigenetic base, and 8-oxoguanine (oxoG), an abundant DNA lesion. Guanine oxidation significantly changed the motion in both hemimethylated and fully methylated DNA, increased base pair breathing, induced BI→BII transition in the backbone 3' to the oxoG and reduced the variability of shift and tilt helical parameters. UV melting experiments corroborated the NMR and molecular dynamics results, showing significant destabilization of all methylated contexts by oxoG. Notably, some dynamic and thermodynamic effects were not additive in the fully methylated oxidized CpG, indicating that the introduced modifications interact with each other. Finally, we show that the presence of oxoG biases the recognition of methylated CpG dinucleotides by ROS1, a plant enzyme involved in epigenetic DNA demethylation, in favor of the oxidized DNA strand. Thus, the conformational and dynamic effects of spurious DNA oxidation in the regulatory CpG dinucleotide can have far-reaching biological consequences.

Ultrafast Single-Scan 2D†NMR Spectroscopic Detection of a PHIP-Hyperpolarized Protease Inhibitor.

Two-dimensional NMR spectroscopy is one of the most important spectroscopic tools for the investigation of biological macromolecules. However, due to the low sensitivity of NMR spectroscopy, it takes usually from several minutes to many hours to record such spectra. Here, the possibility of detecting a bioactive derivative of the sunflower trypsin inhibitor-1 (SFTI-1), a tetradecapeptide, by combining parahydrogen-induced polarization (PHIP) and ultrafast 2D NMR spectroscopy is shown. The PHIP activity of the inhibitor was achieved by labeling with O-propargyl-l-tyrosine. In 1D PHIP experiments a signal enhancement of a factor of approximately 1200 compared to standard NMR was found. This enhancement permits measurement of 2D NMR correlation spectra of low-concentrated SFTI-1 in less than 10 seconds, employing ultrafast single-scan 2D NMR detection. As experimental examples PHIP-assisted ultrafast single-scan TOCSY spectra of SFTI-1 are shown.

Proton Relaxometry of Long-Lived Spin Order.

A study of long-lived spin order in chlorothiophene carboxylates at both high and low magnetic fields is presented. Careful sample preparation (removal of dissolved oxygen in solution, chelating of paramagnetic impurities, reduction of convection) allows one to obtain very long-lived singlet order of the two coupled protons in chlorothiophene derivatives, having lifetimes of about 130 s in D2 O and 240 s in deuterated methanol, which are much longer than the T1 -relaxation times (18 and 30 s, respectively, at a field B 0 =9.4 T). In protonated solvents the relaxation times become shorter, but the lifetime is still substantially longer than T 1 . In addition, long-lived coherences are shown to have lifetimes as long as 30 s. Thiophene derivatives can be used as molecular tags to study slow transport, slow dynamics and slow chemical processes, as has been shown in recent years.

Assessment of heteronuclear long-lived states at ultralow magnetic fields.

A study of long-lived spin states in hetero-nuclear spin systems is presented. Since long-lived states are efficiently sustained only when the spins are "strongly coupled", this study necessitates going to "ultralow" magnetic fields, which are much lower than the Earth's field. To do so, we utilize a fast field-cycling device, which rapidly shuttles the sample between an NMR (Nuclear Magnetic Resonance) magnet and a magnetic shield with a very low field inside. While the spin evolution is taking place at an ultralow field, detection is performed at the high field of an NMR spectrometer. We report hetero-nuclear long-lived order in two spin and four-spin systems, given by the CH and CH3 groups of methyl propiolate, and present a detailed analysis of the spectral manifestation of such long-lived states.

Excitation of singlet-triplet coherences in pairs of nearly-equivalent spins.

We present approaches for an efficient excitation of singlet-triplet coherences in pairs of nearly-equivalent spins. Standard Nuclear Magnetic Resonance (NMR) pulse sequences do not excite these coherences at all or with very low efficiency. The single quantum singlet-triplet coherences, here termed the outer singlet-triplet coherences, correspond to lines of low intensity in the NMR spectrum of a strongly-coupled spin pair (they are sometimes referred to as "forbidden transitions"), whereas the zero-quantum coherences, here termed the inner singlet-triplet coherences, do not have a direct spectral manifestation. In the present study, we investigated singlet-triplet coherences in a pair of nearly-equivalent carbon spins of the 13C-isotopomer of a specially designed naphthalene derivative with optimized relaxation properties. We propose and compare several techniques to drive the singlet-triplet coherence in strongly coupled spin pairs. First, we study different methods for efficient excitation of the outer singlet-triplet coherences. The achieved conversion efficiency of magnetization to the coherences of interest is close to the theoretically allowed maximum. Second, we propose methods to convert the outer coherences into the inner singlet-triplet coherence. The inner singlet-triplet coherence is insensitive to field inhomogeneity and can be long-lived. By probing this coherence, we perform a very precise measurement of the spin-spin J-couplings. A remarkable property of this coherence is that it can be preserved even in absence of a spin-locking radiofrequency field. Consequently, it is possible to shuttle the sample between different magnetic fields preserving the coherence. This allows one to study the field dependence of the relaxation time, TIST, of the inner singlet-triplet coherence by performing field-cycling experiments. We observed dramatic changes of the ratio TIST/T1 from about 1 (in strong fields) up to 2.4 (in weak fields), which is the evidence of a significant influence of the chemical shift anisotropy on relaxation. We have detected a remarkably long lifetime of the inner singlet-triplet coherence of about 200 s at the magnetic field of 5 mT.