Selective Excitation with Asymmetric Adiabatic Pulses for NMR Spectroscopy

Existing selective pulses are mainly constructed in the forms of classically shaped pulses, such as the Gaussian pulses, or generated by using numerical optimization methods. However, all of these pulses are highly sensitive to radiofrequency (RF) intensity variation, which means their performance is highly dependent on the accuracy and stability of the RF intensity. Even a slight RF intensity deviation can cause severe degradation in the excitation profile. To solve this problem, we propose a method for narrow selective excitation by sequential application of a pair of phase‐opposite asymmetric adiabatic pulses, all within two scans. By retaining the adiabatic character, the new method is highly robust to RF intensity variation. Moreover, it has flexible excitation bandwidth, ranging from line‐selective to narrow‐band‐selective pulses. The method is tested both in numerical simulations and solution‐state NMR experiments.     

PHRONESIS: A One-Shot Approach for Sequential Assignment of Protein Resonances by Ultrafast MAS Solid-State NMR Spectroscopy

Solid-state NMR (ssNMR) spectroscopy has emerged as the method of choice to analyze the structural dynamics of fibrillar, membrane-bound, and crystalline proteins that are recalcitrant to other structural techniques. Recently, ¹H detection under fast magic angle spinning and multiple acquisition ssNMR techniques have propelled the structural analysis of complex biomacromolecules. However, data acquisition and resonance-specific assignments remain a bottleneck for this technique. Here, we present a comprehensive multi-acquisition experiment (PHRONESIS) that simultaneously generates up to ten 3D ¹H-detected ssNMR spectra. PHRONESIS utilizes broadband transfer and selective pulses to drive multiple independent polarization pathways. High selectivity excitation and de-excitation of specific resonances were achieved by high-fidelity selective pulses that were designed using a combination of an evolutionary algorithm and artificial intelligence. We demonstrated the power of this approach with microcrystalline U-¹³C,¹⁵N GB1 protein, reaching 100 % of the resonance assignments using one data set of ten 3D experiments. The strategy outlined in this work opens up new avenues for implementing novel ¹H-detected multi-acquisition ssNMR experiments to speed up and expand the application to larger biomolecular systems.     

Singlet-state exchange NMR spectroscopy for the study of very slow dynamic processes

Singlet states with lifetimes that are longer than spin-lattice relaxation times TS > T1 offer unique opportunities for studying very slow dynamic processes in solution-state NMR. A set of novel experiments can achieve broadband excitation of singlet states in pairs of coupled spins. The most elaborate of these experiments, two-dimensional singlet-state exchange spectroscopy (SS-EXSY), is independent of the offsets of the two spins, their relative chemical shifts, and their scalar couplings. The new methods open the way to study very slow chemical exchange or translational diffusion using mixing times taum = Ts > T1. The lifetimes TS of singlet states of pairs of protons in a partially deuterated saccharide are shown to be longer than the longitudinal proton relaxation times T1 in the same compound by a factor of ca. 37.     

Improvements in lipid suppression for 1H NMR-based metabolomics: Applications to solution-state and HR-MAS NMR in natural and in vivo samples

Proton nuclear magnetic resonance (NMR) spectra of intact biological samples often show strong contributions from lipids, which overlap with signals of interest from small metabolites. Pioneering work by Diserens et al. demonstrated that the relative differences in diffusivity and relaxation of lipids versus small metabolites could be exploited to suppress lipid signals, in high-resolution magic angle spinning (HR-MAS) NMR spectroscopy. In solution-state NMR, suspended samples can exhibit very broad water signals, which are challenging to suppress. Here, improved water suppression is incorporated into the sequence, and the Carr-Purcell-Meiboom-Gill sequence (CPMG) train is replaced with a low-power adiabatic spinlock that reduces heating and spectral artefacts seen with longer CPMG filters. The result is a robust sequence that works well in both HR-MAS as well as static solution-state samples. Applications are also extended to include in vivo organisms. For solution-state NMR, samples containing significant amount of fats such as milk and hemp hearts seeds are used to demonstrate the technique. For HR-MAS, living earthworms (Eisenia fetida) and freshwater shrimp (Hyalella azteca) are used for in vivo applications. Lipid suppression techniques are essential for non-invasive NMR-based analysis of biological samples with a high-lipid content and adds to the suite of experiments advantageous for in vivo environmental metabolomics.     

Homonuclear Selective One-Dimensional Gradient NMR Experiments in the Structure Determination of Natural Diterpene Derivatives

Summary.  The solution structure of two natural diterpene derivatives, the secondary metabolites esulatin-A and esulatin-B of Euphorbia esula, was investigated by homonuclear NMR experiments. Since the spectral dispersion of the ¹H NMR spectra at 500 MHz was sufficient to separate several skeletal protons of the title compounds, they were selectively excited with a double pulsed field gradient spin-echo (DPFGSE) sequence using 180°Gaussian pulses sandwiched between sine shaped gradients. With the use of selective excitation, scalar as well as dipolar interactions of the selected spins were monitored through one-dimensional (1D) COSY, TOCSY, and NOESY experiments. The chemical shifts of the coupling partners could be accurately extracted from the 1D COSY and TOCSY spectra recorded with high digital resolution. The selective TOCSY experiment provided an excellent opportunity to identify spins belonging to the same scalarly coupled spin system. The solution state conformation was investigated by selective gradient enhanced NOESY experiments. Proton–proton distances were evaluated from the cross-relaxation rates obtained from a quantitative analysis of the NOESY spectra recorded with different mixing times. The NMR derived distances were compared to the results of solid state X-ray diffraction measurements. Corresponding author. E-mail: pforgo@chem.u-szeged.hu Received November 21, 2001. Accepted (revised) January 9, 2002     

Practical aspects of ROESY experiments for identification of bound waters in the cyclic tetrasaccharide

ROESY pulse sequences are presented and evaluated to identify bound waters in the cyclic tetrasaccharide. The first experiment incorporated the double-pulsed field gradient spin-echo (DPFGSE) for selective water excitation at the initial portion of the pulse sequence. Although long, shaped pulses were used in DPFGSE to achieve the highly selective excitation of water resonance that is very close to resonances of the cyclic tetrasaccharide, the approach was not effective because of the loss of sensitivity. Concomitant use of long delays and moderate length of shaped pulses in the portion of DPFGSE gained more sensitivity. A simple approach incorporating spin-echo with long delays instead of DPFGSE also afforded a sensitive spectrum. Practical aspects of these ROESY experiments are illustrated using the cyclic tetrasaccharide cyclo-{-->6}-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->).     

Selective excitation 1D-NMR experiments for the assignment of the absolute configuration of secondary alcohols

Routine selective excitation experiments, easy to set up on modern NMR spectrometers, allow for the determination of the absolute configuration of chiral secondary alcohols by double derivatization directly in the NMR tube. As a general method, TOCSY1D with selective excitation of the α proton in the MPA esters and with a short mixing time reveals only the nearby protons in the coupling network. Typically, the analysis takes less than 30 min. A longer mixing time, selective excitation of other signals, or NOESY1D experiments can be used for measuring ΔδRS of other protons.     

Adiabatic single scan two-dimensional NMR spectrocopy

New excitation schemes, based on the use adiabatic pulses, for single scan two-dimensional NMR experiments (Frydman et al., Proc. Nat. Acad. Sci. 2002, 99, 15 858-15 862) are introduced. The advantages are discussed. Applications in homo- and heteronuclear experiments are presented.     

NMR quantification of trace components in complex matrices by band‐selective excitation with adiabatic pulses

The use of band‐selective excitation with adiabatic pulses to rapidly obtain NMR spectra of trace components in the presence of strong signals is described, along with qualitative and quantitative examples from food matrices like olive oil and honey. Copyright © 2009 John Wiley & Sons, Ltd.     

Improved methods for 1H-3H heteronuclear shift correlation

A better understanding of the structure of complex 3H-labeled molecules can be obtained by complete assignment of their 1H and 3H solution-state NMR spectra. The assignment process is aided by the detection of heteronuclear chemical shift correlations between 1H and 3H nuclei. Heteronuclear correlation (HETCOR) experiments previously applied to this task exhibit several drawbacks caused by the nature of both the pulse sequences and 1H-3H spin systems. The range of J-couplings involved in 1H-3H coupling networks make it challenging to perform correlation experiments using methods that rely on coherences created during free precession periods and interrupted by transfer pulses. Two alternative HETCOR experiments are demonstrated for 1H-3H systems in the present work and are shown to have advantages over earlier methods. The first experiment is known as hetero-TOCSY and correlates heteronuclear chemical shifts using J-cross polarization. This experiment achieves both homonuclear and heteronuclear mixing and connects the chemical shifts of all 1H and 3H nuclei in a coupling network. A second HETCOR experiment uses the heteronuclear Overhauser effect to obtain through-space correlations between nearby nuclei. The 1H-3H HETCOR experiments are phase sensitive and typically contain more correlations than other methods, which is beneficial for assignment purposes, while being sensitive enough to be applicable to routine analytical samples. The experiments were used to analyze 3H incorporation in sub-milligram quantities of 3H-labeled pharmaceutical derivatives with complex labeling schemes.     

Broadband RF‐amplitude‐dependent flip angle pulses with linear phase slope

Pulse sequences in NMR spectroscopy sometimes require the application of pulses with effective flip angles different from 90° and 180°. Previously (Magn. Reson. Chem. 2015, 53, 886‐893), offset‐compensated broadband excitation pulses with RF‐amplitude‐dependent effective flip angles (RADFA) were introduced that are applicable in such cases. However, especially RF‐amplitude‐restricted RADFA pulses turned out to perform not as good as desired in terms of achievable bandwidths. Here, a class of RF‐amplitude‐restricted RADFA pulses with linear phase slope is introduced that allows excitation over much larger bandwidths with better performance. In this theoretical work, the basic principle of the pulse class is explained, their physical limits explored, and their properties, also compared with other pulse classes, discussed in detail. Copyright © 2017 John Wiley & Sons, Ltd.     

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.     

Modulation-aided signal enhancement in the magic angle spinning NMR of spin-5/2 nuclei

We report signal enhancement schemes using fast amplitude modulated pulses for the one-dimensional (1D) nuclear magnetic resonance (NMR) of spin-5/2 nuclei under magic-angle spinning. Signal enhancement by a factor of around 2.5 is observed when amplitude modulated pulses precede selective excitation of the central transition. This enhancement is a result of the redistribution of energy level populations through partial saturation of the satellite transitions. Results are shown for ²⁷Al and ¹⁷O. The gain in signal intensity is very useful for the observation of weak signals from low abundance quadrupolar nuclei. The scheme works for wide ranges of quadrupole interactions and rf powers.     

Improved NMR methods for the direct 13C-satellite-selective excitation in overlapped 1H-NMR spectra

Improved pulsed-field gradient echo methods are presented and discussed for the direct selective excitation of the (13)C-satellite lines in overcrowded (1)H NMR spectra of small molecules. Sensitivity enhancements in (13)C spin-state selection can be achieved by combining multiple-proton-frequency excitation and Hadamard phase encoding. Several satellite-selective (SATSEL) NMR experiments are proposed and exemplified by measuring the sign and the magnitude of small, long-range proton-carbon coupling constants for (1)H resonances showing several levels of signal overlapping.     

Elucidating the initial dynamics of electron photodetachment from atoms in liquids using variably-time-delayed resonant multiphoton ionization

We study the photodetachment of electrons from sodium anions in room temperature liquid tetrahydrofuran (THF) using a new type of three-pulse pump-probe spectroscopy. Our experiments use two variably-time-delayed pulses for excitation in what is essentially a resonant 1+1 two-photon ionization: By varying the arrival time of the second excitation pulse, we can directly observe how solvent motions stabilize and trap the excited electron prior to electron detachment. Moreover, by varying the arrival times of the ionization (excitation) and probe pulses, we also can determine the fate of the photoionized electrons and the distance they are ejected from their parent Na atoms. We find that as solvent reorganization proceeds, the second excitation pulse becomes less effective at achieving photoionization, and that the solvent motions that stabilize the excited electron following the first excitation pulse occur over a time of approximately 450 fs. We also find that there is no spectroscopic evidence for significant solvent relaxation after detachment of the electron is complete. In combination with the results of previous experiments and molecular dynamics simulations, the data provide new insight into the role of the solvent in solution-phase electron detachment and charge-transfer-to-solvent reactions.     

A convenient method for the measurements of transverse relaxation rates in homonuclear scalar coupled spin systems

A new solution-state NMR method is proposed to determine apparent transverse NMR relaxation rates in both weakly and strongly scalar coupled two-spin systems.     

The Flexibility of SIMPSON and SIMMOL for Numerical Simulations in Solid-and Liquid-State NMR Spectroscopy

 Addressing the need for numerical simulations in the design and interpretation of advanced solid- and liquid-state NMR experiments, we present a number of novel features for numerical simulations based on the SIMPSON and SIMMOL open source software packages. Major attention is devoted to the flexibility of these Tcl-interfaced programs for numerical simulation of NMR experiments being complicated by demands for efficient powder averaging, large spin systems, and multiple-pulse rf irradiation. These features are exemplified by fast simulation of second-order quadrupolar powder patterns using crystallite interpolation, analysis of rotary resonance triple-quantum excitation for quadrupolar nuclei, iterative fitting of MQ-MAS spectra by combination of SIMPSON and MINUIT, simulation of multiple-dimensional PISEMA-type correlation experiments for macroscopically oriented membrane proteins, simulation of Hartman-Hahn polarization transfers in liquid-state NMR, and visualization of the spin evolution under complex composite broad-band excitation pulses.     

Solid-state NMR spectroscopy applied to a chimeric potassium channel in lipid bilayers

We show that solid-state NMR can be used to investigate the structure and dynamics of a chimeric potassium channel, KcsA-Kv1.3, in lipid bilayers. Sequential resonance assignments were obtained using a combination of (15)N- (13)C and (13)C- (13)C correlation experiments conducted on fully labeled and reverse-labeled as well as C-terminally truncated samples. Comparison of our results with those from X-ray crystallography and solution-state NMR in micelles on the closely related KcsA K (+) channel provides insight into the mechanism of ion channel selectivity and underlines the important role of the lipid environment for membrane protein structure and function.     

利用选择性激发核磁共振对混合物中的两种化合物进行结构解析

利用现代NMR的1D、2D技术对一个混合物进行了分析。结果表明：混合物由两种化合物组成。为了同时准确确定两种化合物的结构，本工作应用了1D-TOCSY技术，利用该技术选择性强的特点来补充常规的1D、2DHMR实验所提供的分子结构的信息。在没有进行预分离的条件下，顺利地完成了样品中两种化合物的核磁信号归属，并最终确定了它们的结构。     

Longitudinal Relaxation Enhancement in 1H NMR Spectroscopy of Tissue Metabolites via Spectrally Selective Excitation

Nuclear magnetic resonance spectroscopy is governed by longitudinal (T₁) relaxation. For protein and nucleic acid experiments in solutions, it is well established that apparent T₁ values can be enhanced by selective excitation of targeted resonances. The present study explores such longitudinal relaxation enhancement (LRE) effects for molecules residing in biological tissues. The longitudinal relaxation recovery of tissue resonances positioned both down‐ and upfield of the water peak were measured by spectrally selective excitation/refocusing pulses, and compared with conventional water‐suppressed, broadband‐excited counterparts at 9.4 T. Marked LRE effects with up to threefold reductions in apparent T₁ values were observed as expected for resonances in the 6–9 ppm region; remarkably, statistically significant LRE effects were also found for several non‐exchanging metabolite resonances in the 1–4 ppm region, encompassing 30–50 % decreases in apparent T₁ values. These LRE effects suggest a novel means of increasing the sensitivity of tissue‐oriented experiments, and open new vistas to investigate the nature of interactions among metabolites, water and macromolecules at a molecular level.