Solution-state NMR Research Articles

Designing, physiochemical confirmation, evaluation of biological and in-silico potential of Triorganotin(IV) complexes

FTIR, NMR, CHN and single crystal X-ray crystallography were used to validate a series of three new triorganotin(IV) carboxylate complexes, R3Sn(L) for R = Methyl (1), n-Butyl (2) and Phenyl (3), obtained from LH = 4-[(2,5-dimethoxyphenyl)carbamoyl]butanoic acid. The coupling constant and θC-Sn-C values in solution-state NMR data suggest a 5-coordinated environment around the Sn centre. In the crystal of 1, the carboxylate is bidentate bridging leading to a zigzag chain with the Sn centre having a distorted trigonal-bipyramidal geometry. The compounds were evaluated for their interaction with salmon sperm DNA and found that they interact through an intercalative mode resulting in hypochromism and bathochromic shift as confirmed by the UV–visible spectroscopic and viscometric techniques. The findings of anti-microbial activity performed on five bacterial and two fungus strains demonstrate that some of the compounds exhibit >80% inhibition of certain bacteria and >100% inhibition of certain fungal strains. The compounds were also evaluated for cell viability tested on human embryonic kidney cell (HEC-239) and human red blood cells (RBC). The anti-cancer potential of the compounds was assessed using cis-platin as a standard against human malignant glioma U87 (MG-U87) cell lines, and 1 was shown to be the most potent (IC50: 148.979 µM) at a 50 µM dose. The DPPH anti-oxidant activity results revealed a 91% maximum scavenging activity for 1. The compounds follow the principles of drug-likeness and have good bioavailability potential, according to an in silico analysis conducted using the SwissADME webserver.

A denoising method for multidimensional magnetic resonance spectroscopy and imaging based on compressed sensing

Both in spectroscopy and imaging, t1-noise arising from instabilities such as temperature alterations, field-related frequency drifts, electronic and sample-spinning instabilities, or motions in in vivo experiments, affects many 2D Magnetic Resonance experiments. This work introduces a post-processing method that aims to attenuate t1-noise, by suitably averaging multiple signals/representations that have been reconstructed from the sampled data. The ensuing Compressed Sensing Multiplicative (CoSeM) denoising starts from a fully sampled 2D MR data set, discards random indirect-domain points, and makes up for these missing, masked data, by a compressed sensing reconstruction of the now incompletely sampled 2D data set. This procedure is repeated for multiple renditions of the masked data –some of which will have been more strongly affected by t1-noise than others. This leads to a large set of 2D NMR spectra/images compatible with the collected data; CoSeM chooses out of these those renditions that reduce the noise according to a suitable criterion, and then sums up their spectra/images leading to a reduction in t1-noise. The performance of the method was assessed in synthetic data, as well as in numerous different experiments: 2D solid and solution state NMR, 2D localized MRS of live brains, and 2D abdominal MRI. Throughout all these data, CoSeM processing evidenced 2–3 fold increases in SNR, without introducing biases, false peaks, or spectral/image blurring. CoSeM also retains a quantitative linearity in the information –allowing, for instance, reliable T1 inversion-recovery MRI mapping experiments.

An investigation into the coordination chemistry of tripodal “click” triazole ligands with Mn, Ni, Co and Zn ions

The steric influences of the triazole side chains in two tripodal chelating ligands tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) and tris[(1-phenyl-1H-1,2,3-triazol-4-yl)methyl]amine (TPTA) are explored through structural studies of four new mononuclear d-block metal complexes. The manganese(II) complex [Mn(TPTA)2](ClO4)2·H2O 1 includes two TPTA ligands each coordinated in a tridentate fashion to give an unusual trigonal prismatic coordination geometry with two non-coordinated pendant triazole groups which engage in weak hydrogen bonding interactions. [Ni(TBTA)(OH2)Cl]Cl·3H2O·MeCN 2 contains an octahedral nickel(II) centre bound by a tetradentate TBTA ligand along with aqua and chloride ligands, where the significant degree of lattice solvation leads to an extensive hydrogen bonding network linking complexes through hexa-aqua water clusters. The mononuclear copper(II) complex [Cu(TPTA)Cl2]·MeCN 3 contains no classical hydrogen bond donors but instead intermolecular aggregation takes place through chelating CH···Cl hydrogen bonds involving the acidic triazole CH groups which leads to close association of adjacent complexes. The zinc complex [Zn(TPTA)Cl]2[ZnCl4]·MeOH 4, in which cationic [Zn(TPTA)Cl]+ species are accompanied by tetrachlorozincate anions, exhibits a tris-triazole chelating coordination geometry for the TPTA ligand and associates through tetrameric π···π stacking interactions despite the positive charge present on each species. Structural analysis of these complexes, supported by solution-state mass spectrometry, NMR spectroscopic and magnetic susceptibility measurements, where applicable, provides new insights into the breadth of coordination geometries and intermolecular packing modes available to this important class of chelating ligand.

HORRENDOUS NMR: Establishing correlations in solution-state NMR by reinstating non-secular J-coupling terms

Homonuclear isotropic mixing modules allow J-coupled spins to exchange magnetization even when separated by chemical shift offsets that exceed their couplings. This is exploited in TOtal Correlation SpectroscopY (TOCSY) experiments and its variants, which facilitate these homonuclear polarization exchanges by applying broadband RF pulses. These then establish an effective Hamiltonian in which chemical shift offsets are erased, while J-coupling terms –including flip-flop components– remain active. The polarization that these non-secular terms will transfer among systems of chemically inequivalent sites over the course of a mixing period, are widely used modules in 1D and in multidimensional liquid-state NMR. Homonuclear correlation experiments are also common in solids NMR, particularly among X = 13C or 15N nuclei. Solids NMR experiments are often challenged by high-power RF demands which have led to a family of homonuclear solid-state correlation experiments that avoid pulsing on the nuclei of interest, and focus instead on the 1Hs that are bonded to them. These solid experiments usually reintroduce/strengthen 1H-X dipolar couplings; these, in conjunction with assistance from rotational resonance effects, bring back the truncated X-X dipolar interactions and facilitate the generation of cross peaks. The present study explores whether a similar goal can be achieved for solution-state counterparts, based on the reintroduction of truncated flip-flop terms in the J-coupling Hamiltonian via the pulsing on other, heteronuclear species. A proposal to achieve this is derived, and the resulting HOmonucleaR Recoupling by hEteroNuclear DecOUplingS (HORRENDOUS) approach to provide correlations between like nuclei without pulsing on them, is demonstrated.

Sample volume effects in optical overhauser dynamic nuclear polarization

The optical dynamic nuclear polarization (DNP) method has been proposed as an alternative to microwave pumping as a hyperpolarization method for solution-state NMR studies. Using continuous laser illumination to photogenerate triplet states in the presence of a persistent radical produces chemically-induced dynamic electron polarization (CIDEP) via the radical-triplet pair mechanism (RTPM), with cross-relaxation transferring this to nuclear hyperpolarization via an Overhauser mechanism. Numerical simulations have previously indicated that reducing the sample volume while maintaining a constant optical density can significantly increase the NMR signal enhancement, due to the larger steady-state concentration of triplets obtained. Here we provide the first experimental confirmation of these effects, producing a nearly five-fold increase in the optical DNP enhancement factor just by reducing the sample volume with optimal dye and radical concentrations adjusted for each optical path length. The results are supported with an in depth analysis of volume effects in the numerical model, with which they are in good qualitative agreement. These important observations will impact on the future development of the technique, with particular significance for attempts to apply DNP methods to increase sensitivity for volume-limited biological samples.

Hyperpolarized Solution-State NMR Spectroscopy with Optically Polarized Crystals.

Nuclear spin hyperpolarization provides a promising route to overcome the challenges imposed by the limited sensitivity of nuclear magnetic resonance. Here we demonstrate that dissolution of spin-polarized pentacene-doped naphthalene crystals enables transfer of polarization to target molecules via intermolecular cross-relaxation at room temperature and moderate magnetic fields (1.45 T). This makes it possible to exploit the high spin polarization of optically polarized crystals, while mitigating the challenges of its transfer to external nuclei. With this method, we inject the highly polarized mixture into a benchtop NMR spectrometer and observe the polarization dynamics for target 1H nuclei. Although the spectra are radiation damped due to the high naphthalene magnetization, we describe a procedure to process the data to obtain more conventional NMR spectra and extract the target nuclei polarization. With the entire process occurring on a time scale of 1 min, we observe NMR signals enhanced by factors between -200 and -1730 at 1.45 T for a range of small molecules.

Evaluation and design of conformationally stable halogen-substituted beta-hairpins using molecular simulation

Determining the folding stability of cyclic peptides is critical to the rational molecular design of potential therapeutics. Therefore, we expect molecular simulation-based methods to play an increasingly important role towards this goal. In this work, we investigate the ability of molecular simulation to predict the stability of a series of halogen-substituted 10-mer cyclic peptide hairpin mimics designed by the Erdelyi group, who extensively studied their folding properties by solution-state NMR, and found that hydroxyl, O-methyl, methyl, and halogen substitutions in cross-strand residue pairs can significantly modulate folding stability.

The effect of dopamine on model lipid membrane structure

Dopamine is a neurotransmitter released by neurons to send signals to other nerve cells. Previous work on the interaction of dopamine with model lipid bilayers made using solution-state NMR spectroscopy has shown that dopamine interacts with phosphatidylcholine (PC) and phosphatidylserine (PS) lipid headgroups primarily through the aromatic side opposite to the hydroxyl groups [1]. Here we use low-angle x-ray scattering (LAXS), solid-state deuterium NMR, as well as electron paramagnetic resonance (EPR) to determine the effect of dopamine on the physical properties of lipid membranes.

Resonance assignments toward solution-state NMR structure determination of novel box jellyfish protein

The box jellyfish eye lens protein, J2, belongs to the structural family known as crystallins, which form hydrogels to refract light and help contribute to varying levels of sight in different animals, ranging from rough light intensity to detailed images, although exactly how remains unknown. Current structure prediction software indicates that this crystallin has very little similarity to previously characterized proteins. This supports our hypothesis that it has a novel fold; potentially one of very few unique conformations identified in recent decades, which will be validated through structure determination.

Conformational characterization of duplex DNA with solution-state NMR spectroscopy

DNA is the hereditary material for living organisms. Understanding the structural features of DNA is essential to comprehend various biological processes. This review outlines the application of solution-state NMR methods to study the structure and dynamics of duplex DNA. A brief overview of the context of NMR in the earlier days of nucleic acids research is discussed. Sample preparation methods and pertinent NMR measurables are introduced, followed by sequential resonance assignment protocol of DNA duplexes. A summary of results obtained from the application of NMR methodology to compare, contrast, and validate initial crystal crystallographic studies of DNA structure, especially the Drew-Dickerson dodecamer and A-tract sequences, is presented. This is followed by a survey of structural insights obtained in studies involving sequence-specific characteristics and mispairs. Results from relaxations experiments that reveal base pairs opening dynamics and short lived transient states of DNA is summarized. Overall, this review aims to introduce the atomistic characterization of DNA using NMR spectroscopy to a beginner level researcher.

Mechanistic Insights into the Formation of 1-Alkylidene/Arylidene-1,2,4-triazolinium Salts: A Combined NMR/Density Functional Theory Approach.

In a recent report on the synthetic approach to the novel substance class of 1-alkylidene/arylidene-1,2,4-triazolinium salts, a reaction mechanism suggesting a regioselective outcome was proposed. This hypothesis was tested via a combined NMR and density functional theory (DFT) approach. To this end, three experiments with 13C-labeled carbonyl reactants were monitored in situ by solution-state NMR. In one experiment, an intermediate as described in the former mechanistic proposal was observed. However, incorporation of 13C isotope labels into multiple sites of the heterocycle could not be reconciled with the “regioselective mechanism”. It was found that an unproductive reaction pathway can lead to 13C scrambling, along with metathetical carbonyl exchange. According to DFT calculations, the concurring reaction pathways are connected via a thermodynamically controlled cyclic 1,3-oxazetidine intermediate. The obtained insights were applied in a synthetic study including aliphatic ketones and para-substituted benzaldehydes. The mechanistic peculiarities set the potential synthetic scope of the novel reaction type.

Probing the Interactions of Splicing Regulatory Small Molecules and Proteins with U1 snRNP Using NMR Spectroscopy.

Alternative RNA splicing is an essential part of gene expression that not only increases the protein diversity of metazoan but also provides an additional layer of gene expression regulation. The U1 small ribonucleoparticle (U1 snRNP) plays an essential role in seeding spliceosome assembly and its binding on weak 5'-splice sites is regulated by transient interactions with splicing factors. Recent progress in allele specific splicing correction has shown the therapeutic potential offered by small molecule splicing modifiers that specifically promotes the recruitment of U1 snRNP to modulate alternative splicing and gene expression. Here, we described a method to reconstitute U1 snRNP in vitro and to study labile interactions with protein or synthetic splicing factors using solution state NMR spectroscopy. This approach allowed us to validate direct interactions between splicing regulators and U1 snRNP and could also be useful for the screening of small molecules acting on splicing regulation.

Probing the Functional Interaction Interface of Lipopolysaccharide and Antimicrobial Peptides: A Solution-State NMR Perspective.

Antimicrobial peptides (AMPs) have been a topic of substantial research as the next-generation antibiotics. They have been extensively studied for the selectivity and action against microbial membrane lipids in imparting their targeted functioning. To determine the effectivity of the peptides against the Gram-negative pathogens, it is imperative to elucidate their role in interacting with the lipopolysaccharide moieties. Lipopolysaccharide is a major component of the outer membrane of the Gram-negative bacteria. It serves to protect the bacteria as well as govern the functionality of several antibacterial agents. It can prevent the access of the agents into the inner membrane of the bacteria, thus rendering them inactive. Several techniques have been employed to study the interaction for better designing of peptides; NMR spectroscopy is one of the most widely used techniques in determining the interactive properties of peptides with LPS as it can provide the details in atomistic level. NMR spectroscopy provides information about the structural and conformational changes as well as the dynamics of the interactions.

How Much Entropy Is Contained in NMR Relaxation Parameters?

Solution-state NMR relaxation experiments are the cornerstone to study internal protein dynamics at an atomic resolution on time scales that are faster than the overall rotational tumbling time τR. Since the motions described by NMR relaxation parameters are connected to thermodynamic quantities like conformational entropies, the question arises how much of the total entropy is contained within this tumbling time. Using all-atom molecular dynamics simulations of the T4 lysozyme, we found that entropy buildup is rather fast for the backbone, such that the majority of the entropy is indeed contained in the short-time dynamics. In contrast, the contribution of the slow dynamics of side chains on time scales beyond τR on the side-chain conformational entropy is significant and should be taken into account for the extraction of accurate thermodynamic properties.

Progress toward automated methyl assignments for methyl-TROSY applications

Methyl-TROSY spectroscopy has extended the reach of solution-state NMR to supra-molecular machineries over 100kDa in size. Methyl groups are ideal probes for studying structure, dynamics, and protein-protein interactions in quasi-physiological conditions with atomic resolution. Successful implementation of the methodology requires accurate methyl chemical shift assignment, and the task still poses a significant challenge in the field. In this work, we outline the current state of technology for methyl labeling, data collection, data analysis, and nuclear Overhauser effect (NOE)-based automated methyl assignment approaches. We present MAGIC-Act and MAGIC-View, two Python extensions developed as part of the popular NMRFAM-Sparky package, and MAGIC-Net a standalone structure-based network analysis program. MAGIC-Act conducts statistically driven amino acid typing, Leu/Val pairing guided by 3D HMBC-HMQC, and NOESY cross-peak symmetry checking. MAGIC-Net provides model-based NOE statistics to aid in selection of a methyl labeling scheme. The programs provide a versatile, semi-automated framework for rapid methyl assignment.

Prediction of chemical shift in NMR: A review.

Calculation of solution-state NMR parameters, including chemical shift values and scalar coupling constants, is often a crucial step for unambiguous structure assignment. Data-driven (sometimes called empirical) methods leverage databases of known parameter values to estimate parameters for unknown or novel molecules. This is in contrast to popular ab initio techniques that use detailed quantum computational chemistry calculations to arrive at parameter estimates. Data-driven methods have the potential to be considerably faster than ab inito techniques and have been the subject of renewed interest over the past decade with the rise of high-quality databases of NMR parameters and novel machine learning methods. Here, we review these methods, their strengths and pitfalls, and the databases they are built on.

Discovery of antimicrobial agent targeting tryptophan synthase.

Antibiotic resistance is a continually growing challenge in the treatment of various bacterial infections worldwide. New drugs and new drug targets are necessary to curb the threat of infectious diseases caused by multidrug-resistant pathogens. The tryptophan biosynthesis pathway is essential for bacterial growth but is absent in higher animals and humans. Drugs that can inhibit the bacterial biosynthesis of tryptophan offer a new class of antibiotics. In this work, we combined a structure-based strategy using in silico docking screening and molecular dynamics (MD) simulations to identify compounds targeting the α subunit of tryptophan synthase with experimental methods involving the whole-cell minimum inhibitory concentration (MIC) test, solution state NMR, and crystallography to confirm the inhibition of L-tryptophan biosynthesis. Screening 1,800 compounds from the National Cancer Institute Diversity Set I against α subunit revealed 28 compounds for experimental validation; four of the 28 hit compounds showed promising activity in MIC testing. We performed solution state NMR experiments to demonstrate that a one successful inhibitor, 3-amino-3-imino-2-phenyldiazenylpropanamide (Compound 1) binds to the α subunit. We also report a crystal structure of Salmonella enterica serotype Typhimurium tryptophan synthase in complex with Compound 1 which revealed a binding site at the αβ interface of the dimeric enzyme. MD simulations were carried out to examine two binding sites for the compound. Our results show that this small molecule inhibitor could be a promising lead for future drug development.

Synthesis, characterization and cytotoxic evaluation of chalcone derivatives

Four chalcones, (E)-3-(3-hydroxyphenyl)-1-phenylprop-2-en-1-one (A), (E)-3-(4-hydroxyphenyl)-1-phenylprop-2-en-1-one (B), (E)-3-(4-hydroxyphenyl)-1-(4-methoxyphenyl)prop‑2-en-1-one (C) and (E)-3-(4-bromophenyl)-1-(4-methoxyphenyl)prop‑2-en-1-one (D), were synthesized and characterized by 1H and 13C solution-state NMR, 13C solid-state NMR and single-crystal X-ray diffraction. The different interactions (hydrogen bonding and π- interactions) observed in the crystal structures of these four molecules were confirmed in the solid-state data as well as in the Hirshfeld surface analysis. The in vitro cytotoxicity of these molecules was also evaluated against human acute lymphoblastic leukemia (CEM), human breast adenocarcinoma (MCF7), human cervical carcinoma (HeLa), human malignant melanoma cell lines (G-361), and normal human skin fibroblasts (BJ). A, B, C and D displayed cytotoxicity in at least two cell lines. A, B, and C were strongly cytotoxic against CEM cells while B and C showed selectivity by not affecting the growth of the normal human skin fibroblasts.

Sensitivity Enhancement by Progressive Saturation of the Proton Reservoir: A Solid-State NMR Analogue of Chemical Exchange Saturation Transfer.

Chemical exchange saturation transfer (CEST) enhances solution-state NMR signals of labile and otherwise invisible chemical sites, by indirectly detecting their signatures as a highly magnified saturation of an abundant resonance—for instance, the 1H resonance of water. Stimulated by this sensitivity magnification, this study presents PROgressive Saturation of the Proton Reservoir (PROSPR), a method for enhancing the NMR sensitivity of dilute heteronuclei in static solids. PROSPR aims at using these heteronuclei to progressively deplete the abundant 1H polarization found in most organic and several inorganic solids, and implements this 1H signal depletion in a manner that reflects the spectral intensities of the heteronuclei as a function of their chemical shifts or quadrupolar offsets. To achieve this, PROSPR uses a looped cross-polarization scheme that repeatedly depletes 1H–1H local dipolar order and then relays this saturation throughout the full 1H reservoir via spin-diffusion processes that act as analogues of chemical exchanges in the CEST experiment. Repeating this cross-polarization/spin-diffusion procedure multiple times results in an effective magnification of each heteronucleus’s response that, when repeated in a frequency-stepped fashion, indirectly maps their NMR spectrum as sizable attenuations of the abundant 1H NMR signal. Experimental PROSPR examples demonstrate that, in this fashion, faithful wideline NMR spectra can be obtained. These 1H-detected heteronuclear NMR spectra can have their sensitivity enhanced by orders of magnitude in comparison to optimized direct-detect experiments targeting unreceptive nuclei at low natural abundance, using modest hardware requirements and conventional NMR equipment at room temperature.

Measuring the Rate of In-vitro Drug Release From Polymeric Nanoparticles by 19F Solution State NMR Spectroscopy.

We report what we believe is the first use of 19F NMR spectroscopy to directly measure in-vitro release (IVR) from polymeric nanoparticles (PNPs). Using 19F NMR we selectively measured IVR of AZD2811 from PNPs. Due to rapid nuclear relaxation in solid-like environments only AZD2811 in solution is detected, and physical separation from the PNPs isn't required. The NMR approach and ultra-centrifugation/UHPLC were shown to be equivalent. The selectivity of 19F NMR means it is readily applied to complex IVR media such as recombinant human serum albumin (rHSA).