Nuclear Magnetic Resonance Protocol Research Articles

Nutation-based longitudinal-sensing protocols for high-field NMR with nitrogen-vacancy centers in diamond

Nitrogen-vacancy (N-V) centers in diamond enable nuclear magnetic resonance (NMR) spectroscopy of samples at the nano- and microscale. However, at typical tesla-scale NMR magnetic field strengths, N-V–NMR protocols become difficult to implement due to the challenge of driving fast N-V pulse sequences sensitive to nuclear Larmor frequencies above a few megahertz. We perform simulations and theoretical analysis of the experimental viability of N-V NMR at tesla-scale magnetic fields using a measurement protocol called DRACAERIS (Double Rewound ACquisition Amplitude Encoded Radio Induced Signal). DRACAERIS detects the longitudinal magnetization of the NMR sample at a much lower driven Rabi frequency, more suitable technically for N-V detection. We discuss how pulse errors, finite pulse lengths, and nuclear spin-spin couplings affect the resulting NMR spectra. We find that DRACAERIS is less susceptible to pulse imperfections and off-resonance effects than previous protocols for longitudinal magnetization detection. We also identify reasonable parameters for experimental implementation. Published by the American Physical Society 2024

A Novel NMR-Based Protocol to Screen Ultralow Molecular Weight Fragments.

Fragment-based lead discovery has emerged as one of the most efficient screening strategies for finding hit molecules in drug discovery. Recently, a novel strategy based on a class of fragments characterized by an ultralow molecular weight (ULMW) has been proposed. These fragments bind to the target with a very low affinity, requiring reliable biophysical methods for detection. The most notable application of ULMW used a set of 81 fragments, named MiniFrags, and screened them by X-ray crystallography. We extended the utilization of this novel class of fragments to another gold standard technique for fragment-based screening: nuclear magnetic resonance (NMR). Here, we present a novel NMR protocol to detect and analyze such weak interactions in a challenging real-world scenario: a flexible target with a flat, water-exposed binding site. We identified a subset of 69 highly water-soluble MiniFrags that were screened against the antiapoptotic protein human Bfl-1.

High-resolution nanoscale NMR for arbitrary magnetic fields

Nitrogen vacancy (NV) centers are a major platform for the detection of nuclear magnetic resonance (NMR) signals at the nanoscale. To overcome the intrinsic electron spin lifetime limit in spectral resolution, a heterodyne detection approach is widely used. However, application of this technique at high magnetic fields is yet an unsolved problem. Here, we introduce a heterodyne detection method utilizing a series of phase coherent electron nuclear double resonance sensing blocks, thus eliminating the numerous Rabi microwave pulses required in the detection. Our detection protocol can be extended to high magnetic fields, allowing chemical shift resolution in NMR experiments. We demonstrate this principle on a weakly coupled 13C nuclear spin in the bath surrounding single NV centers, and compare the results to existing heterodyne protocols. Additionally, we identify the combination of NV-spin-initialization infidelity and strong sensor-target-coupling as linewidth-limiting decoherence source, paving the way towards high-field heterodyne NMR protocols with chemical resolution.

Suberitamides A-C, Aryl Alkaloids from a Pseudosuberites sp. Marine Sponge that Inhibit Cbl-b Ubiquitin Ligase Activity.

Three new aryl alkaloids named suberitamides A–C (1–3), were isolated from an extract of the marine sponge Pseudosuberites sp. collected along the coast of North Carolina. Their planar structures were established by extensive nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. To assign the challenging relative configuration of the saturated five-membered ring in suberitamide A (1), a simple and efficient NMR protocol was applied that is based on the analysis of 2- and 3-bond 1H-13C spin-spin coupling constants using a PIP (pure in-phase) HSQMBC (heteronuclear single quantum multiple bond correlation) IPAP (in-phase and anti-phase) experiment. Suberitamides A (1) and B (2) inhibited Cbl-b, an E3 ubiquitin ligase that is an important modulator of immune cell function, with IC50 values of approximately 11 μM.

Artificial intelligence enhanced two-dimensional nanoscale nuclear magnetic resonance spectroscopy

Two-dimensional nuclear magnetic resonance (NMR) is indispensable to molecule structure determination. Nitrogen-vacancy center in diamond has been proposed and developed as an outstanding quantum sensor to realize NMR in nanoscale or even single molecule. However, like conventional multi-dimensional NMR, a more efficient data accumulation and processing method is necessary to realize applicable two-dimensional (2D) nanoscale NMR with a high spatial resolution nitrogen-vacancy sensor. Deep learning is an artificial algorithm, which mimics the network of neurons of human brain, has been demonstrated superb capability in pattern identifying and noise canceling. Here we report a method, combining deep learning and sparse matrix completion, to speed up 2D nanoscale NMR spectroscopy. The signal-to-noise ratio is enhanced by 5.7 ± 1.3 dB in 10% sampling coverage by an artificial intelligence protocol on 2D nanoscale NMR of a single nuclear spin cluster. The artificial intelligence algorithm enhanced 2D nanoscale NMR protocol intrinsically suppresses the observation noise and thus improves sensitivity.

A comparison of high-throughput plasma NMR protocols for comparative untargeted metabolomics

IntroductionWhen analyzing the human plasma metabolome with Nuclear Magnetic Resonance (NMR) spectroscopy, the Carr–Purcell–Meiboom–Gill (CPMG) experiment is commonly employed for large studies. However, this process can lead to compromised statistical analyses due to residual macromolecule signals. In addition, the utilization of Trimethylsilylpropanoic acid (TSP) as an internal standard often leads to quantification issues, and binning, as a spectral summarization step, can result in features not clearly assignable to metabolites.ObjectivesOur aim was to establish a new complete protocol for large plasma cohorts collected with the purpose of describing the comparative metabolic profile of groups of samples.MethodsWe compared the conventional CPMG approach to a novel procedure that involves diffusion NMR, using the Longitudinal Eddy-Current Delay (LED) experiment, maleic acid (MA) as the quantification reference and peak picking for spectral reduction. This comparison was carried out using the ultrafiltration method as a gold standard in a simple sample classification experiment, with Partial Least Squares–Discriminant Analysis (PLS-DA) and the resulting metabolic signatures for multivariate data analysis. In addition, the quantification capabilities of the method were evaluated.ResultsWe found that the LED method applied was able to detect more metabolites than CPMG and suppress macromolecule signals more efficiently. The complete protocol was able to yield PLS-DA models with enhanced classification accuracy as well as a more reliable set of important features than the conventional CPMG approach. Assessment of the quantitative capabilities of the method resulted in good linearity, recovery and agreement with an established amino acid assay for the majority of the metabolites tested. Regarding repeatability, ~ 85% of all peaks had an adequately low coefficient of variation (< 30%) in replicate samples.ConclusionOverall, our comparison yielded a high-throughput untargeted plasma NMR protocol for optimized data acquisition and processing that is expected to be a valuable contribution in the field of metabolic biomarker discovery.

Endogenous dynamic nuclear polarization NMR of hydride-terminated silicon nanoparticles

Silicon nanoparticles (SiNPs) are intriguing materials and their properties fascinate the broader scientific community; they are also attractive to the biological and materials science sub-disciplines because of their established biological and environmental compatibility, as well as their far-reaching practical applications. While characterization of the particle nanostructure can be performed using 29Si solid-state nuclear magnetic resonance (NMR) spectroscopy, poor sensitivity due to low Boltzmann population and long acquisition times hinder in-depth studies of these potentially game-changing materials. In this study, we compare two dynamic nuclear polarization (DNP) NMR protocols to boost 29Si sensitivity in hydride-terminated SiNPs. First, we assess a traditional indirect DNP approach, where a nitroxide biradical (AMUPol or bCTbk) is incorporated into a glassing agent and transferred through protons (e− → 1H → 29Si) to enhance the silicon. In this mode, electron paramagnetic resonance (EPR) spectroscopy demonstrated that the hydride-terminated surface was highly reactive with the exogenous biradicals, thus decomposing the radicals within hours and resulting in an enhancement factor, ε, of 3 (TB = 15 s) for the 64 nm SiNP, revealing the surface components. Secondly, direct DNP NMR methods were used to enhance the silicon without the addition of an exogenous radical (i.e., use of dangling bonds as an endogenous radical source). With radical concentrations <1 mM, 29Si enhancements were obtained for the series of SiNPs ranging from 3 to 64 nm. The ability to use direct 29Si DNP transfer (e− → 29Si) shows promise for DNP studies of these inorganic nanomaterials (ε = 6 (TB = 79 min) for 64 nm SiNPs) with highly reactive surfaces, showing the sub-surface and core features. These preliminary findings lay a foundation for future endogenous radical development through tailoring the surface chemistry, targeting further sensitivity gains.

Amphiphilic second-order phase transitions determined through NMR

In this work, nuclear magnetic resonance (NMR) spectroscopy is used to investigate surfactant phase behavior over a broad concentration region. This technique is an adaptation of a previously developed method applied to detect the critical micelle concentration (CMC) of surfactants. In this method, a surfactant concentration is correlated to the normalized intensity, or area, of the NMR signal for each surfactant. In this procedure a linear relationship develops on either side of the CMC, with a distinct change in slope where the primary phase change occurs. The research conducted herein investigates the NMR response at higher surfactant concentration, where the phase change consists of a change in micelle shape or other structural configurations. These secondary phase transitions can be expected from 10 to 90 wt.%, where the CMC measurements are conducted at concentrations <1 wt.%. The solutions were also analyzed with dynamic light scattering (DLS) and a cross-polarizer microscope to confirm suspected detected phase changes. Visible changes in the rheological response were observed and therefore carefully examined. In a large part, the results seen with all methods corresponded with visible differences in the surfactant solutions and detected changes in the NMR protocol.

Quantitative In Situ Enhanced Oil Recovery Monitoring Using Nuclear Magnetic Resonance

Quantitative in situ monitoring of oil recovery from sedimentary rock is demonstrated for the first time using advanced two-dimensional (2D) nuclear magnetic resonance (NMR) correlation measurements on a low field spectrometer. The laboratory-scale NMR system was chosen to provide a common physics of measurement with NMR well-logging tools. The NMR protocols are used to monitor recovery of a heavy Middle East crude oil from high permeability sandstone plugs using a brine (water) flood followed by chemical enhanced oil recovery agents: polymer and alkaline–surfactant–polymer solutions. 2D correlations between relaxation time (T 1, T 2) and apparent self-diffusion coefficient D app are used to obtain simultaneously a volumetric determination of the oil and aqueous fluid-phase saturations present in the porous material. The T 1 − T 2 and D app − T 2 correlations are bulk measurements of the entire rock core-plug; excellent agreement is shown between the measures of remaining oil (from NMR) and recovered oil (from gravimetric assay of the effluent). Furthermore, we introduce the capability to measure spatially resolved T 2 distributions on a low field spectrometer using a rapid frequency-encoded y − T 2 map. A non-uniform distribution of remaining oil is observed due to viscous instabilities in the flowing liquids; the final oil saturation ranges from $${S_{\rm o}^{\rm{(final)}} \approx 0}$$ to 20 % along the direction of flow. These results highlight the quantitative nature of the NMR data obtainable in low field NMR core analysis and also the importance of spatially resolved measurements when studying short core-plugs.

Characterization of humic substances by advanced solid state NMR spectroscopy: Demonstration of a systematic approach

Characterization of humic substances is challenging due to their structural complexity and heterogeneity. Solid state nuclear magnetic resonance (NMR) is regarded as one of the best tools for elucidating structures of humic substances. The primary solid state NMR technique that has been used so far is the routine 13C cross polarization-magic angle spinning (CP-MAS) technique. Although this technique has markedly advanced our understanding of humic substances, the full potential of NMR for characterizing humic substances has yet to be realized. Recent technical developments and applications of advanced solid state NMR have revealed the promise to provide deeper insights into structures of humic substances. In this paper, we summarized and demonstrated the systematic solid state NMR protocol for characterization of humic substances using a humic acid as an example. This protocol included (1) identification of specific functional groups using spectral editing techniques, occasionally assisted by 1H 13C two-dimensional heteronuclear correlation (2D HETCOR) NMR, (2) quantification of specific functional groups based on direct polarization-magic angle spinning (DP-MAS) and DP-MAS with recoupled dipolar dephasing, combined with spectral editing techniques, (3) determination of connectivities and proximities of specific functional groups by 1H 13C 2D HETCOR or 2D HETCOR combined with spectral editing techniques, and (4) examination of domains and heterogeneities by 1H 13C 2D HETCOR with 1H spin diffusion. We used a soil humic acid as an example to demonstrate how this protocol was applied to the characterization of humic substances step by step. Afterwards, based on typical 13C NMR spectra of humic substances we described how we could combine different NMR techniques to identify specific functional groups band by band from downfield to upfield. Finally, we briefly mentioned the potential new NMR techniques that could be developed to enrich the current systematic protocol. This systematic protocol is not only applicable to humic substances but also to other natural organic matter samples.

An advanced NMR protocol for the structural characterization of aluminophosphate glasses

In this work a combination of complementary advanced solid-state nuclear magnetic resonance (NMR) strategies is employed to analyse the network organization in aluminophosphate glasses to an unprecedented level of detailed insight. The combined results from MAS, MQMAS and 31P-{ 27Al}-CP-heteronuclear correlation spectroscopy (HETCOR) NMR experiments allow for a detailed speciation of the different phosphate and aluminate species present in the glass. The interconnection of these local building units to an extended three-dimensional network is explored employing heteronuclear dipolar and scalar NMR approaches to quantify P–O–Al connectivity by 31P{ 27Al}-heteronuclear multiple quantum coherence (HMQC), -rotational echo adiabatic passage double resonance (REAPDOR) and -HETCOR NMR as well as 27Al{ 31P}-rotational echo double resonance (REDOR) NMR experiments, complemented by 31P-2D-J-RESolved MAS NMR experiments to probe P–O–P connectivity utilizing the through bond scalar J-coupling. The combination of the results from the various NMR approaches enables us to not only quantify the phosphate units present in the glass but also to identify their respective structural environments within the three-dimensional network on a medium length scale employing a modified Q notation, Q n m , AlO x , where n denotes the number of connected tetrahedral phosphate, m gives the number of aluminate species connected to a central phosphate unit and x specifies the nature of the bonded aluminate species (i.e. 4, 5 or 6 coordinate aluminium).

The integrity of cisplatin in aqueous and plasma ultrafiltrate media studied by 195Pt and 15N nuclear magnetic resonance.

195Pt and 15N nuclear magnetic resonance (NMR) was used to study the chemical equilibria of cisplatin in water and plasma ultrafiltrate (PUF). Cisplatin was found to be stable for at least 2, but no longer than 5 months in a reconstituted clinical formulation, as determined by 195Pt NMR. In aqueous solution, the cis-PtCl2(NH3)2 195Pt and 15N NMR signal intensities decreased with time and the formation of [PtCl(H2O)(NH3)2]+ at PH values of 3.0, 6.5, 7.5 and 9.5 was observed within 24 h of sample preparation. In addition, [Pt(H2O)2(NH3)2]++ was observed at pH 3.0, and [PtCl(OH)(NH3)2] and [Pt(OH)2(NH3)2] were observed at pHs 7.5 and 9.5. During incubation of PUF with cisplatin for 35 h, 15N NMR signals for at least eight cisplatin derivatives appeared at different times, whereas only four were observed by 195Pt NMR. With our NMR protocols, the detection limit for quantifiable cisplatin derivatives is estimated at 500 microM using 195Pt NMR and < or = 200 microM using 15N NMR. In addition to providing useful information about the chemical stability of cisplatin and derivatives formed in aqueous solution, these magnetic resonance techniques, particularly 15N NMR, can provide useful information about the metabolism of cisplatin in biological regimes.

Solving solution structures of physiologically relevant proteins by NMR spectroscopy.

In the last decade or so, high resolution nuclear magnetic resonance (NMR) spectroscopy has become the technique of choice to study protein solution structure and dynamics at the molecular level. This is due, in part, to much improved instrumentation, but it is principally the consequence of the de­ velopment of two-dimensional NMR methodologies. The latter were first proposed by Jeener in the early 1970s (56) and were vigorously applied to protein structural analysis some ten years later, initially by Wuthrich and his co-workers (134). Since then many groups have further developed NMR methodologies and applied them to the study of structure and dynamics of a wide array of proteins (see, for example, 9, 80, 129, 30, 57 for more recent reviews) and other macromolecules. This chapter briefly summarizes the NMR protocol for the eventual realiza­ tion of the three-dimensional solution structure of physiologically relevant proteins given the restraints of the NMR technique. Because of space limita­ tions, this article is not exhaustive, and many fine studies are not mentioned in detail or at all (for example, 32). We have chosen to concentrate on families of related proteins on which detailed NMR structural analyses have been performed.