Heteronuclear Cross-polarization Research Articles

Analytical Evaluation of Low-Field 31P NMR Spectroscopy for Lipid Analysis.

We investigate the potential of 31P NMR with simple, maintenance-free benchtop spectrometers to probe phospholipids in complex mixtures. 31P NMR-based lipidomics has become an important topic in a wide range of applications in food- and health-sciences, and the continuous improvements of compact, maintenance- and cryogen-free instruments opens new opportunities for NMR routine analyses. A prior milestone is the evaluation of the analytical performance provided by 31P NMR at low magnetic field. To address this, we assess the ability of state-of-the-art benchtop NMR spectrometers to detect, identify, and quantify several types of phospholipids in mixtures. Relying on heteronuclear cross-polarization experiments, phospholipids can be detected in 2 h with a limit of detection of 0.5 mM at 1 T and 0.2 mM at 2 T, while the headgroups of phosphatidylcholine (PC), phosphatidyl-ethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidyl-glycerol (PG) can be unambiguously assigned based on 2D 1H-31P total correlated spectroscopy (TOCSY) spectra. Furthermore, two quantitative methods to obtain absolute concentrations are proposed and discussed, and the performance is evaluated regarding precision and accuracy.

Hybridizing cross-polarization with NOE or refocused-INEPT enhances the sensitivity of MAS NMR spectroscopy

Heteronuclear cross polarization (CP) has been commonly used to enhance the sensitivity of dilute low-γ nuclei in almost all solid-state NMR experiments. However, CP relies on heteronuclear dipolar couplings, and therefore the magnetization transfer efficiency becomes inefficient when the dipolar couplings are weak, as is often the case for mobile components in solids. Here, we demonstrate methods that combine CP with heteronuclear Overhauser effect (referred to as CP-NOE) or with refocused-INEPT (referred to as CP-RINEPT) to overcome the efficiency limitation of CP and enhance the signal-to-noise ratio (S/N) for mobile components. Our experimental results reveal that, compared to the conventional CP, significant S/N ratio enhancement can be achieved for resonances originating from mobile components, whereas the resonance signals associated with rigid groups are not significantly affected due to their long spin–lattice relaxation times. In fact, the S/N enhancement factor is also dependent on the temperature, CP contact time as well as on the system under investigation. Furthermore, we also demonstrate that CP-RINEPT experiment can be successfully employed to independently detect mobile and rigid signals in a single experiment without affecting the data collection time. However, the resolution of CP spectrum obtained from the CP-RINEPT experiment could be slightly compromised by the mandatory use of continuous wave (CW) decoupling during the acquisition of signals from rigid components. In addition, CP-RINEPT experiment can be used for spectral editing utilizing the difference in dynamics of different regions of a molecule and/or different components present in the sample, and could also be useful for the assignment of resonances from mobile components in poorly resolved spectra. Therefore, we believe that the proposed approaches are beneficial for the structural characterization of multiphase and heterogeneous systems, and could be used as a building block in multidimensional solid-state NMR experiments.

Performance Evaluation of Quantitative Adiabatic 13C NMR Pulse Sequences for Site-Specific Isotopic Measurements

(2)H/(1)H and (13)C/(12)C site-specific isotope ratios determined by NMR spectroscopy may be used to discriminate pharmaceutically active ingredients based on the synthetic process used in production. Extending the Site-specific Natural Isotope Fractionation NMR (SNIF-NMR) method to (13)C is highly beneficial for complex organic molecules when measurements of (2)H/(1)H ratios lead to poorly defined molecular fingerprints. The current NMR methodology to determine (13)C/(12)C site-specific isotope ratios suffers from poor sensitivity and long experimental times. In this work, several NMR pulse sequences based on polarization transfer were evaluated and optimized to measure precise quantitative (13)C NMR spectra within a short time. Adiabatic 180 degrees (1)H and (13)C pulses were incorporated into distortionless enhancement by polarization transfer (DEPT) and refocused insensitive nuclei enhanced by polarization transfer (INEPT) to minimize the influence of 180 degrees pulse imperfections and of off-resonance effects on the precision of the measured (13)C peak areas. The adiabatic DEPT sequence was applied to draw up a precise site-specific (13)C isotope profile of ibuprofen. A modified heteronuclear cross-polarization (HCP) experiment featuring (1)H and (13)C spin-locks with adiabatic 180 degrees pulses is also introduced. This sequence enables efficient magnetization transfer across a wide (13)C frequency range although not enough for an application in quantitative (13)C isotopic analysis.

Low-power cross polarization in fast magic-angle spinning NMR experiments

We describe a low-power approach for heteronuclear cross-polarization (CP) at high magic-angle spinning (MAS) frequencies. It is based on second-order CP at the n = 0 Hartmann–Hahn condition. The mechanism for the polarization transfer in the low-power CP experiment relies on second-order cross terms between homonuclear and heteronuclear dipolar couplings. At a MAS frequency of 65 kHz, rf-field amplitudes below 10 kHz are sufficient to efficiently transfer polarization from 1H to 13C. The low-rf field requirements of this approach make it well suited for the investigation of proteins and other temperature-sensitive samples without the risk of sample heating and degradation.

Efficient cross polarization with simultaneous adiabatic frequency sweep on the source and target channels

In this work, we propose a new and efficient heteronuclear cross polarization scheme, in which adiabatic frequency sweeps from far off-resonance toward on-resonance are applied simultaneously on both the source and target spins. This technique, which we call as Simultaneous ADIabatic Spin-locking Cross Polarization (SADIS CP), is capable of efficiently locking both the source and target spins with moderate power even in the presence of large spectral distribution and fast relaxation. It is shown that by keeping the time-dependent Hartmann–Hahn mismatch minimal throughout the mixing period, polarization transfer can be accelerated. Experiments are demonstrated in a powder sample of l-alanine.

Spin state selective Hadamard encoding during transfer periods using multiple selective CW-HCP

Hadamard spectroscopy today represents an alternative to conventional Fourier transform spectroscopy. The selective inversion of several narrow frequency bands is typically achieved by taylored inversion pulses in place of t 1-evolution periods. However, band-selective inversion can also be achieved during coherence transfer steps, thereby shortening the period during which the magnetization is in the transverse plane. Using CW heteronuclear cross polarization (CW-HCP) as an example for highly selective coherence transfer, the implementation of Hadamard encoding within a transfer step is presented. Transfer characteristics, the preparation of multiple frequency selective CW-HCP and the possibility of acquiring spin state selective spectra are discussed in detail. The theoretical results are verified on two examples involving a cyclic pentapeptide and ubiquitin.

Solution-State NMR Experiments Based on Heteronuclear Cross-Polarization

Heteronuclear coherence transfer in liquid-state NMR applications has been traditionally performed using pulse-interrupted delay schemes such as INEPT-type pulse trains. So far, the alternative use of heteronuclear cross- polarization (HCP) has only been limited to a few cases involving exclusively in-phase to in-phase transfers. In this revi- sion work a theoretical description on the effect and the characteristic anisotropic features of HCP is introduced in terms of product operator formalism. A very intuitive and simple graphical black-box approach based on a pictorial non- classical vector representation that only consider the available input/output magnetization is also presented to understand the general transformations that are undergoing under such rather complex HCP processes. The appropriate manipulation of magnetization components during the HCP block offers novel concepts in pulse sequence design. In this way, new liq- uid-state multidimensional NMR methods incorporating HCP-driven processes have recently been developed and success- fully applied for both small-to-medium-sized molecules and large labeled bio-molecules, as will be discussed in this re- view.

IFSERF, an isotope-filtered SERF experiment for the precise measurement of proton–proton coupling constants between chemically equivalent protons

An isotope-filtered selective refocusing (IFSERF) experiment is presented for the sensitive and precise measurement of the proton–proton coupling constant between chemically equivalent protons. The 2D NMR method combines an initial doubly selective isotope filter based on heteronuclear cross-polarization followed by a selective J-resolved block. The coupling topologies obtained from several 2D variants of the IFSERF experiment are described for the simultaneous measurement of both proton–proton and proton–carbon coupling constants in the involved AA′XX′ spin system. Application on the determination of the relative configuration of double bonds in symmetrical molecules is illustrated.

Measurement of coupling constants in symmetrical spin systems using a full multiple-step cross-polarization-driven NMR pulse scheme

New NMR pulse schemes completely driven under homonuclear and heteronuclear cross-polarization conditions are proposed for the study and the measurement of coupling constants in symmetrical molecules in solution. The appropriate superimposition of independent magnetization components can afford several spin-selective multiplet patterns that are suitable for the determination of the magnitude and the sign of proton-proton and proton-carbon coupling constants with optimum sensitivity levels. A detailed product operator formalism analysis for the proposed doubly selective 1D and nonselective 2D HCP-TOCSY versions is provided and experimental verification for the configurational analysis of symmetric olefinic systems having chemical equivalence is demonstrated.

Simultaneous recording of spin-state-selective NMR spectra for different InS spin systems.

Heteronuclear magnetization transfer occurring during heteronuclear cross-polarization mixing processes in liquid-state NMR experiments can be easily monitored as a function of the involved in-phase, antiphase, and multiple-quantum magnetization components. The theoretical background on the simultaneous detection of E.COSY-type, TROSY-type, or spin-edited multiplet patterns for different IS and I(2)S spin systems in the same solution-state NMR spectrum is described. The proposed pulse scheme preserves high sensitivity levels and shows good tolerance to the presence of undesired cross-talk artifacts for both NH and NH(2) multiplicities providing an interesting NMR tool for biomolecular applications.

Spin-state-selective excitation in gradient-selected heteronuclear cross-polarization NMR experiments

Several heteronuclear coherence transfer mechanisms involved in proton-detected heteronuclear J-cross-polarization (HCP) NMR experiments have been theoretically derived and experimentally verified in isotropic solution. It is shown that in-phase and/or anti-phase heteronuclear coherence transfer can take place separately or simultaneously during the HCP process as a function of the relative phase between the HCP mixing sequence and the corresponding input magnetization. As the more important consequence, clean coherence-order and spin-state selective (S 3) excitation with maximum sensitivity can be achieved from gradient-enhanced HCP experiments by proper co-addition/subtraction of in-phase and anti-phase magnetizations, offering an attractive alternative to widely used HSQC-type experiments.

Selective cross-polarization in solution state nuclear magnetic resonance of scalar coupled spin 12 and quadrupolar nuclei

Semiselective heteronuclear cross-polarization for achieving coherence transfer between a spin I=12 scalar coupled to a spin S⩾12 in isotropic solution is considered. The expansion of the density operator as products of irreducible tensor operators provides a compact formalism for describing cross-polarization involving scalar coupled quadrupolar nuclei. An analytical description of cross-polarization is presented in the limit of strong radio-frequency (rf) fields, with respect to the scalar-coupling constant. Numerical simulations show that reducing the rf field amplitudes does not have a detrimental effect on the efficiency of the transfer provided they are comparable to or greater than the scalar-coupling constant. The use of weak rf fields largely circumvents the reduced efficacy due to Hartmann–Hahn mismatch. Applications of the method for selective observation of scalar-coupled quadrupolar nuclei are considered and experimental results are presented for a mixture of beryllium fluoride complexes.

Simulations of NMR pulse sequences during equilibrium and non-equilibrium chemical exchange.

The McConnell equations combine the differential equations for a simple two-state chemical exchange process with the Bloch differential equations for a classical description of the behavior of nuclear spins in a magnetic field. This equation system provides a useful starting point for the analysis of slow, intermediate and fast chemical exchange studied using a variety of NMR experiments. The McConnell equations are in the mathematical form of an inhomogeneous system of first-order differential equations. Here we rewrite the McConnell equations in a homogeneous form in order to facilitate fast and simple numerical calculation of the solution to the equation system. The McConnell equations can only treat equilibrium chemical exchange. We therefore also present a homogeneous equation system that can handle both equilibrium and non-equilibrium chemical processes correctly, as long as the kinetics is of first-order. Finally, the same method of rewriting the inhomogeneous form of the McConnell equations into a homogeneous form is applied to a quantum mechanical treatment of a spin system in chemical exchange. In order to illustrate the homogeneous McConnell equations, we have simulated pulse sequences useful for measuring exchange rates in slow, intermediate and fast chemical exchange processes. A stopped-flow NMR experiment was simulated using the equations for non-equilibrium chemical exchange. The quantum mechanical treatment was tested by the simulation of a sensitivity enhanced 15N-HSQC with pulsed field gradients during slow chemical exchange and by the simulation of the transfer efficiency of a two-dimensional heteronuclear cross-polarization based experiment as a function of both chemical shift difference and exchange rate constants.

Solid-State NMR Connectivity Studies in Dipolarly Coupled Inorganic Networks: Crystal Structure and Site Assignments for the Lithium Polyphosphide LiP5

The crystal structure of LiP5 has been refined from single-crystal X-ray data to a residual of R=0.020 for 2357 structure factors and 56 variable parameters. The structure as determined in the acentric space group Pna21 by H. G. von Schnering and W. Wichelhaus [Naturwissenschaften 59, 78 (1972)] is confirmed. However, the lithium position has been determined with considerably greater accuracy. The polyanionic network of the phosphorus atoms can also be refined in the corresponding centrosymmetric group Pnam, where two adjacent lithium positions with partial occupancy are found. The phosphorus connectivities within this network are studied by means of advanced solid-state NMR techniques. Crystalline polyphosphides represent a particular challenge for such experiments owing to the presence of strong homonuclear 31P–31P dipole–dipole couplings within the network of phosphorus polyanions. To meet this challenge a powerful strategy has been designed by combining magic-angle spinning with 31P–31P double-quantum-filtered two-dimensional exchange, rotational resonance, and heteronuclear cross-polarization spectroscopies. Based on this information, the NMR results are discussed in connection with the local environments of the phosphorus sites.

Novel selective 1D experiments based on heteronuclearJ cross-polarization

Selective 1H–13C inverse NMR experiments based on heteronuclear cross-polarization (HCP) are presented. Practical details on the implementation of one-bond [1J(C,H)] optimized and pseudo-3D HCP experiments combined with gradient coherence selection are reported. On the other hand, accurate values for long-range proton–carbon coupling constants [nJ(C,H); n > 1] can be measured from ultra-clean in-phase 1D proton multiplets resulting of long-range optimized experiments. Alternatively, such heteronuclear connectivities can be also clearly elucidated using a 1H-selective 13C-detected version in which signals exclusively arise from selective transfer of a selected proton to all long-range coupled carbon nuclei. Copyright © 1999 John Wiley & Sons, Ltd.

Site Connectivities in Amorphous Materials Studied by Double-Resonance NMR of Quadrupolar Nuclei: High-Resolution 11B ↔ 27Al Spectroscopy of Aluminoborate Glasses

The utility of heteronuclear double-resonance NMR methods involving the quadrupolar nuclei 11B and 27Al is demonstrated for probing the intermediate-range order in aluminoborate glasses. Results from both rotational echo double resonance (REDOR) and heteronuclear cross-polarization magic angle spinning (CPMAS) studies are presented and discussed. While both techniques are strongly affected by the presence of large quadrupolar splittings, a working strategy is developed on the basis of which reliable structural interpretations are possible. Using this strategy, the effects of the cation type M on the structures of two basic glass compositions, 25M(2)O-45B2O3-30Al2O3 and 40M(2)O-40B2O3-20Al2O3 (M = Na, Ca, Mg), are discussed. Within each series having the same basic network composition, replacement of the modifier cation Na by Ca or Mg produces a large change in the speciation of framework aluminum (“cation effect”): while in the sodium-containing glass the overwhelming fraction of Al is present as tetrahedral AlO4/2 sites, the calcium- and magnesium-containing glasses also possess larger fractions of five- and six-coordinated aluminum sites. 27Al{11B} REDOR results indicate that all of these aluminum environments interact equally strongly with the dominant BO3/2 sites in both glasses. In addition, the reverse 11B{27Al} REDOR results reveal that the extent of the AlO4/2−BO4/2 interaction in the network is highly dependent on the framework composition and on the M cation type. While sodium aluminoborate glasses reveal strong chemical ordering (avoidance of intertetrahedral linkages), the analogous Mg2+-based glasses tend toward a randomization of connectivities. Finally, two-dimensional heteronuclear correlation spectra obtained via 11B{27Al} CPMAS suggest that AlO5 and AlO6 sites can provide a mechanism for stabilizing tetrahedral BO4/2 units.

Heteronuclear Cross Polarization for Enhanced Sensitivity ofin Vivo13C MR Spectroscopy on a Clinical 1.5 T MR System

The potential of heteronuclear {1H-13C} cross polarization was studied for optimization of the signal-to-noise ratio inin vivo13C MR spectroscopy at the clinical field strength of 1.5 T. Experiments on the human calf showed a significant chemical-shift selective signal enhancement on triglyceride signals of 3.9 by heteronuclear cross polarization, compared to a standard pulse-acquire sequence. Studies on a neonatal piglet brain showed an enhancement by cross polarization of 2.2 for the detection of13C-1-glucose. This enhancement allowed a fourfold improvement in time resolution in dynamic13C MR of13C-1-glucose inflow in piglet brain. Phantom experiments demonstrated the efficiency of this technique for interleaved detection of two spectral regions. Tests with a volume coil showed the feasibility of signal enhancement by cross polarization over a large volume of interest.

Doubly Sensitivity-Enhanced 3D HCCH-TOCSY of13C-Labeled Proteins in H2O Using Heteronuclear Cross Polarization and Pulsed Field Gradients

Doubly Sensitivity-Enhanced 3D HCCH-TOCSY of13C-Labeled Proteins in H2O Using Heteronuclear Cross Polarization and Pulsed Field Gradients

Band-selective hetero- and homonuclear cross-polarization using trains of shaped pulses

The performance of solution cross-polarization using trains of shaped pulses on two channels is investigated by computer simulation and experiment. It is determined that a Waltz modulation pattern of Gaussian pulses of individual flip angles of 225°, issued to two coupled spins simulatneously, yields excellent coherence transfer with good phasing behavior. Simulations and experimental verification were carried out for both heteronuclear cross-polarization between two restricted areas (e.g. (1)H(α)-(13)C(α)) and for homonuclear cross-polarization between two spectral regions (e.g. (13)CO-(13)C(α)). It is shown that shaped cross-polarization behaves as pure heteronuclear cross-polarization when the two radiofrequency (rf) fields are far apart, while it behaves in some aspects analogous to homonuclear cross-polarization when the two rf fields approach each other. The novel coherence-transfer sequence, referred to as 'cosine-modulated shaped Waltz' (CSW), was implemented in a 3D (H)C(CCO)NH experiment using an 18-kDa isotopically labeled protein.

The influence of molecular motion on cross-polarization in cross-linked elastomers

The effect of molecular motion on the heteronuclear cross-polarization rate for the case of the spin-lock procedure was investigated. In applying heteronuclear solid state NMR techniques to mobile elastomer systems the influence of molecular motion cannot be neglected. Starting in the slow motion regime a strong collision model was used for predicting changes of the cross-polarization rate in the dipolar spectral density function of abundant spins. The dipolar correlation time and hence the cross-polarization rate is found to scale with the inverse of the correlation time of the molecular motion. The same behavior is obtained using a second approach valid in the intermediate molecular motion regime. This is based on the effect of the motion on the homonuclear and heteronuclear van-Vleck moments and leads to a linear dependence of the cross-polarization rate on the correlation time of molecular motion. This dependence was verified experimentally by 1H 13C high-resolution cross-polarization measurements on sulfur cross-linked elastomer systems. 13C rotating frame spin-lattice relaxation rate measurements were used to corroborate these data and the approximations used to evaluate the influence of molecular motion on cross-polarization rates. The dependence of these rates on the cross-link density of the elastomer network is analyzed and it is shown that they scale with the cross-link density. The correlation of the 1H- 13C cross-polarization rates with the dynamic storage moduli was demonstrated.