Two empirical equations have been devised to transform absolute shieldings (GIAO) into chemical shifts. Both correlations are excellent, but that based on M06-2X/aug-cc-pVDZ calculations needs a correction term for chlorine-substituted boron compounds with B–Cl bonds. These equations have been tested against a collection of closoboranes, neutrals, and dianionic.
Simulations are essential in NMR spectroscopy for understanding and quantitatively predicting the outcomes of experiments. Dynamic NMR experiments are influenced by relaxation and chemical exchange phenomena, which complicate their interpretation. The quantum mechanical theory behind NMR simulations is well established and enables simulations under various conditions or states of the system. In particular, the representation of the spin system in terms of the density matrix, energy and pulse operators, and relaxation and chemical exchange in Liouville space is straightforward and allows for the routine simulation of dynamic NMR experiments. In fact, several advanced open-source software packages are currently available—such as Spinach, GAMMA (pyGAMMA), Simpson, ChemEx, and NMR TITAN—which users can choose from based on their simulation needs. Many of these software packages are optimized for computational speed and make use of sophisticated bases, such as the irreducible spherical tensor basis. Here, we present simulations using the direct method (spin product basis), as implemented in GAMMA, which we believe will help users better understand the underlying theory and thereby explore the other available software resources more effectively. We chose two experiments—CPMG-RD and CEST—as a case study to understand the conformational changes in proteins or macromolecules occurring in the fast and slow exchange regimes, which are frequently investigated by structural biologists.
A new protocol based on the 3D-hNCAnH experiment for rapid, unambiguous backbone resonance assignment and estimation of structural information by intensity quantification of the peaks is described here. Along F1−F3 plane at F2(13Cαi) of the 3D-hNCAnH spectrum, each i to i + 1 sequential connectivity (i.e., HiNi → Hi + 1Ni + 1) is confirmed by two inter-residue sequential correlation peaks: HiNi + 1 and Hi + 1Ni. This allows unambiguous and direct identification of sequential correlations in HSQC peaks, without the need for extensive searching in different planes of 3D spectra. Further, a protocol utilizing the ratio of the intensities of the diagonal and cross peaks along F3(1H) dimension centered at self F2(13Cαi) and sequential F2(13Cαi-1) chemical shifts taken from F2–F3 plane at F1(15Ni) of the 3D-hNCAnH spectrum for estimating one and two bond N–Cα J-coupling constants, respectively, is described. The reliability of this approach is demonstrated using doubly labelled ubiquitin protein, wherein the coupling constants that are measured by the method described here are compared with previously measured values (BMRB Nos.: 15907 and 16582). The application of the approach to other proteins is demonstrated using doubly labelled human SUMO and Ca2+ bound M-crystallin proteins.
One-dimensional quantitative NMR (qNMR) has been predominantly applied to accurately quantify pharmaceutical compounds. However, its application to complex molecules is limited due to several challenges, such as signal response overlap and spectral complexity in 1H qNMR, prolonged acquisition times for 13C, and the potential absence of relevant nuclei for 19F or 31P NMR. In 1H qNMR, complex overlapping NMR peaks in a spectrum hinder quantification by restricting the selection of integration ranges. Specifically, selecting the correct signal response for integration is a major limitation. For example, while developing 1H qNMR methods, signal responses of the target analyte and impurities could occur in the same chemical shift region, leading to measurement errors. Nowadays, two-dimensional (2D) qNMR is emerging as a viable quantitative technique to overcome such limitations. In this work, the application of the 2D NMR method (1H-13C heteronuclear single-quantum coherence spectroscopy [HSQC]) was established to perform quantitative analysis for candesartan cilexetil, thus allowing a more precise quantitation method for a spectrum with complex signal patterns. Quantifying candesartan cilexetil using a 2D 1H-13C HSQC experiment gives a good correlation between the measured and the actual sample weight. The study confirmed that the purity and sample weight can be measured accurately. The present study concludes that advanced NMR approaches can be utilized for the quantification of complex molecules. These advanced 2D qNMR approaches can be extended to other complex molecules and adopted using low-field benchtop instruments.
Saturation transfer difference (STD) NMR is a robust, versatile technique for detecting small molecules binding to large receptors. In addition to identifying binding molecules in the presence of nonbinding molecules, the STD-NMR technique can be used to determine epitope maps and binding constants. In recent years, this technique has been applied to small molecules interacting with nanoparticles. In this tutorial, we introduce the technique of STD-NMR and how it can be used to gain information about small molecules interacting with nanoparticle surfaces. After describing the principle of the STD-NMR technique, we will explain how to best prepare the sample, set up the experiment, and analyze the resulting data when nanoparticles are involved. We will also present extensions to the STD-NMR technique, alternative approaches for when STD-NMR is not ideal, and future directions for the field.
We reveal contrasting behaviors in molecular motion between the two materials, including the identification of resonance-enhanced dynamic features in elastomers. We present a depth-resolved analysis of molecular dynamics in semicrystalline polytetrafluoroethylene (PTFE) and fully amorphous fluorinated elastomer (SIFEL) films using static-gradient solid-state 19F NMR imaging. By measuring spin–lattice relaxation rates ( ) at multiple frequencies and evaluating the corresponding spectral density functions, we reveal distinct dynamic behaviors between the two materials. PTFE exhibits pronounced depth dependence in , indicating enhanced molecular motion near the surface due to a structurally disordered amorphous layer. In contrast, the fluorinated elastomer shows spatially uniform values, reflecting its homogeneous molecular mobility. Notably, the elastomer's spectral density function contains resonance-like peaks at finite frequencies, suggesting the presence of intrinsic vibrational modes superimposed on stochastic motion. This hybrid dynamic signature, captured through nuclear magnetic resonance (NMR) relaxation, offers a unique fingerprint of the elastomer's viscoelastic behavior. Our results demonstrate that static-gradient NMR imaging can probe subtle spatial variations in polymer dynamics noninvasively and with high sensitivity, enabling direct comparison between crystalline and amorphous systems. The findings provide new insights into nanoscale surface dynamics and contribute to the development of advanced materials with tailored thermomechanical properties.
The research team established a quantitative 1H NMR method to determine the relative ethoxy content (EO%) in ethylcellulose using a CDCl3/TFA-d solvent mixture. High-field NMR spectroscopy enabled direct measurement without the use of internal or external calibrants by integrating the methyl proton signals (δ 1.15 ppm) and the methylene/methine proton signals (δ 2.5–5.5 ppm), and using their equivalent 1H numbers and corresponding mass fractions for calculations. The EO% was calculated as: EO% = × 100, where r = (IA methyl 1Hs integral; Iᴃ methylene/methine 1Hs integral). Round robin multi-laboratory validation testing showed that the qNMR results deviated by less than 3% from the certificate of analysis (COA) values (44%–51%). The COA values, derived using the pharmacopeial gas chromatography-flame ionization detection (GC-FID) method, may be affected by factors related to incomplete derivatization and the volatility and instability of the product (iodoethane) and the inert internal reference standard (n-octane). Therefore, the qNMR method with its operational simplicity, reduced chemical hazards, and robust performance presents an alternative for quality control of pharmaceutical-grade ethylcellulose.
Epithelial splicing regulatory protein 2 (ESRP2) plays a pivotal role in alternative splicing regulation, particularly in maintaining epithelial cell identity and suppressing epithelial-to-mesenchymal transition (EMT). Despite its biological significance, the structural basis for its RNA-binding specificity remains poorly understood. In this study, we report the solution structure and RNA-binding properties of the RNA Recognition Motif (RRM3) of human ESRP2 using an integrative approach combining nuclear magnetic resonance (NMR) spectroscopy, ITC, molecular docking, and MD simulations. Our structural analysis revealed that ESRP2-RRM3 adopts a canonical RRM fold (βαββαβ), featuring a positively charged β-sheet surface conducive to RNA interaction. ITC assays demonstrated that RRM3 binds UG-rich RNA sequences with moderate affinity, and NMR titrations identified key interacting residues within the conserved RNP motifs, particularly F522 and R480. RNA docking and MD simulations further corroborated these interactions, revealing π–π stacking and hydrogen bonding at the protein-RNA interface. These findings represent the first atomic-level characterization of ESRP2's interaction with its RNA targets and provide mechanistic insight into how it may guide alternative splicing events in vivo. This work lays the groundwork for understanding the modular RNA recognition by ESRP2's multiple RRMs and its broader role in splicing regulation, development, and cancer suppression.
Acute respiratory distress syndrome (ARDS) is a life-threatening condition often complicated by sepsis, leading to worse clinical outcomes. The role of biomarkers in distinguishing ARDS with and without sepsis remains unclear. This study aimed to evaluate the differences in serum metabolites between the two groups, comparing levels on Day 1 and Day 7 of intensive care unit (ICU) admission, and to assess the variation in outcomes via clinical characteristics. A cohort of n = 151 patients was included, with n = 91 providing serum samples on Day 1 and n = 60 on Day 7. Of the n = 91 patients on Day 1, n = 40 had ARDS with sepsis and n = 51 without sepsis, while on Day 7, n = 24 had ARDS with sepsis and n = 36 without. Serum samples were analyzed using 1H NMR-based metabolomics to identify altered metabolites and perturbed pathways. In contrast, no significant differences were found between the patient groups with and without sepsis on either Day 1 or Day 7. Mortality rates were also similar in both groups, with a 50% survival rate on Day 7. No notable differences in the clinical data were observed. These findings suggest that ARDS, with and without sepsis, exhibits a similar metabolic profile, likely due to shared pathophysiological mechanisms. In light of these similarities, the findings indicate a unified approach to ARDS management that may improve the clinical outcomes across both groups.
Photo-chemically induced dynamic nuclear polarisation (photo-CIDNP) is a nuclear spin-selective magnetic resonance phenomenon that has traditionally been used to mechanistically study chemical reactions involving the (transient) formation of radical molecular species, extract EPR observables of short-lived radicals, probe biomolecular structure and interactions and, less importantly, increase the sensitivity of a nuclear magnetic resonance (NMR) measurement. Recently, the introduction of significant methodological advances as well as the advent of benchtop NMR spectroscopy has rekindled interest in this technique, which—serendipitously discovered more than half a century ago—has, as of late, matured into a powerful, highly sensitive and extremely versatile NMR hyperpolarisation method. In this tutorial, aimed primarily at the nonexpert user, we provide practical information on how to plan, set up and perform one-dimensional 1H and heteronuclear photo-CIDNP NMR experiments using a high-field NMR spectrometer and a continuous-wave (CW) illuminant. In particular, strategies for selecting the appropriate experimental setup are described, including aspects such as light source requirements, introduction of appropriate light coupling methods and photosensitiser selection. In addition, examples of suitable one-dimensional 1H and heteronuclear photo-CIDNP pulse schemes are presented, photo-CIDNP-specific acquisition parameters—including the implementation of sequence commands required to trigger the light source—are explained, and ‘hands-on’ practical advice on photo-CIDNP sample preparation is provided. Finally, special attention as to how to acquire and analyse one-dimensional photo-CIDNP data in a meaningful way is given.
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