Proton-proton Coupling Research Articles

Advanced Structural Determination of Diterpene Esters Using Molecular Modeling and NMR Spectroscopy.

Three new jatrophane esters (1-3) were isolated from Euphorbia amygdaloides ssp. semiperfoliata, including an unprecedented macrocyclic jatrophane ester bearing a hemiketal substructure, named jatrohemiketal (3). The chemical structures of compounds 1-3 and their relative configurations were determined by spectroscopic analysis. The absolute configuration of compound 3 was determined unambiguously through an original strategy combining NMR spectroscopy and molecular modeling. Conformational search calculations were performed for the four possible diastereomers 3a-3d differing in their C-6 and C-9 stereocenters, and the lowest energy conformer was used as input structure for geometry optimization. The prediction of NMR parameters ((1)H and (13)C chemical shifts and (1)H-(1)H coupling constants) by density functional theory (DFT) calculations allowed identifying the most plausible diastereomer. Finally, the stereostructure of 3 was solved by comparison of the structural features obtained by molecular modeling for 3a-3d with NMR-derived data (the values of dihedral angles deduced from the vicinal proton-proton coupling constants ((3)JHH) and interproton distances determined by ROESY). The methodology described herein provides an efficient way to solve or confirm structural elucidation of new macrocyclic diterpene esters, in particular when no crystal structure is available.

Solution Structure of Heparin Pentasaccharide: NMR and DFT Analysis.

High-resolution NMR and density functional theory (DFT) calculations have been applied to analysis of heparin pentasaccharide 3D structure in aqueous solution. The fully optimized molecular geometry of two pentasaccharide conformations (differing from each other in the form, one (1)C4 and the other (2)S0, of the sulfated iduronic acid residue) were obtained using the B3LYP/6-311+G(d,p) level of theory in the presence of solvent, the latter included as explicit water molecules. The presented approach enabled insight into variations of the bond lengths, bond angles, and torsion angles, formations of intra- and intermolecular hydrogen bonds, and ionic interactions in the two pentasaccharide conformations. A rather complex hydrogen bond network is formed, including inter-residue and intraresidue bonds between the NH group in the GlcN,3,6S with oxygens linked to C-2 at the IdoA2S residue and the glycosidic O-1 and the neighboring OSO3(-) group linked to C-3 in the same residue. On the other hand, because the first hydration shell is strongly influenced by strong ion-ion and ion-dipole interactions between sodium ions, sulfates, carboxylates, and -OH groups, ionic interactions play an important role in the stabilization of the 3D structure. The DFT-computed three-bond proton-proton coupling constants also showed that best agreement with experiment was obtained with a weighted average of 15:85 ((1)C4/(2)S0) of the sulfated iduronic acid forms indicating that the ratio is even more shifted toward the (2)S0 form than previously supposed. The DFT-computed pentasaccharide conformation differs from the previously published data, with the main changes at the glycosidic linkages, namely, the ψ1 torsion angles and the ϕ3 angle. The comparison of the glycosidic linkage torsion angle values in solution with the antithrombin-pentasaccharide complex also indicates that the pentasaccharide conformation changes upon binding to antithrombin III. The data supports the assumption that the protein selects the more populated (2)S0 conformer of heparin pentasaccharide and, consequently, the binding process of heparin pentasaccharide with antithrombin III is energetically more favorable than formerly expected.

Dynamics-based selective 2D (1)H/(1)H chemical shift correlation spectroscopy under ultrafast MAS conditions.

Dynamics plays important roles in determining the physical, chemical, and functional properties of a variety of chemical and biological materials. However, a material (such as a polymer) generally has mobile and rigid regions in order to have high strength and toughness at the same time. Therefore, it is difficult to measure the role of mobile phase without being affected by the rigid components. Herein, we propose a highly sensitive solid-state NMR approach that utilizes a dipolar-coupling based filter (composed of 12 equally spaced 90° RF pulses) to selectively measure the correlation of (1)H chemical shifts from the mobile regions of a material. It is interesting to find that the rotor-synchronized dipolar filter strength decreases with increasing inter-pulse delay between the 90° pulses, whereas the dipolar filter strength increases with increasing inter-pulse delay under static conditions. In this study, we also demonstrate the unique advantages of proton-detection under ultrafast magic-angle-spinning conditions to enhance the spectral resolution and sensitivity for studies on small molecules as well as multi-phase polymers. Our results further demonstrate the use of finite-pulse radio-frequency driven recoupling pulse sequence to efficiently recouple weak proton-proton dipolar couplings in the dynamic regions of a molecule and to facilitate the fast acquisition of (1)H/(1)H correlation spectrum compared to the traditional 2D NOESY (Nuclear Overhauser effect spectroscopy) experiment. We believe that the proposed approach is beneficial to study mobile components in multi-phase systems, such as block copolymers, polymer blends, nanocomposites, heterogeneous amyloid mixture of oligomers and fibers, and other materials.

Minimalist Relativistic Force Field: Prediction of Proton-Proton Coupling Constants in (1)H NMR Spectra Is Perfected with NBO Hybridization Parameters.

We previously developed a reliable method for multiparametric scaling of Fermi contacts to achieve fast and accurate prediction of proton-proton spin-spin coupling constants (SSCC) in (1)H NMR. We now report that utilization of NBO hybridization coefficients for carbon atoms in the involved C-H bonds allows for a significant simplification of this parametric scheme, requiring only four general types of SSCCs: geminal, vicinal, 1,3-, and long-range constants. The method is optimized for inexpensive B3LYP/6-31G(d) molecular geometries. A new DU8 basis set, based on a training set of 475 experimental spin-spin coupling constants, is developed for hydrogen and common non-hydrogen atoms (Li, B, C, N, O, F, Si, P, S, Cl, Se, Br, I) to calculate Fermi contacts. On a test set of 919 SSCCs from a diverse collection of natural products and complex synthetic molecules the method gave excellent accuracy of 0.29 Hz (rmsd) with the maximum unsigned error not exceeding 1 Hz.

J-edited pure shift NMR for the facile measurement of (n)J(HH) for specific protons.

We report a novel 1D J-edited pure shift NMR experiment (J-PSHIFT) that was constructed from a pseudo 2D experiment for the direct measurement of proton-proton scalar couplings. The experiment gives homonuclear broad-band (1)H-decoupled (1)H NMR spectra, which provide a single peak for chemically distinct protons, and only retain the homonuclear-scalar-coupled doublet pattern at the chemical-shift positions of the protons in the coupled network of a specific proton. This permits the direct and unambiguous measurement of the magnitudes of the couplings. The incorporation of a 1D selective correlation spectroscopy (COSY)/ total correlation spectroscopy (TOCSY) block in lieu of the initial selective pulse, results in the exclusive detection of the correlated spectrum of a specific proton.

NMR characterization of the binding properties and conformation of glycosaminoglycans interacting with interleukin-10.

The cytokine interleukin-10 (IL-10) is an important regulator of the host immune system with both pro- and anti-inflammatory functions. Glycosaminoglycans (GAGs) play a decisive role in the biology of many growth factors, e.g., for receptor binding or protection from proteolytic degradation. GAGs of the extracellular matrix inhibit IL-10 signaling, however, the molecular mechanism is so far unknown. Here, we studied the interaction between GAGs and IL-10 using a combination of nuclear magnetic resonance (NMR) spectroscopy and computer simulations. The binding region of a set of heparin and chondroitin sulfate GAG disaccharides with varying sulfation pattern were determined by saturation transfer difference (STD) NMR spectroscopy. From the initial growth rate of the STD amplification factor binding affinities were determined and KD values in the low millimolar to micromolar range were obtained. We observed the highest binding affinity to IL-10 with fully sulfated heparin; however, a hyaluronan hexasaccharide did not exhibit binding, which suggests that GAG sulfation is necessary for interaction with IL-10. For octasaccharides or longer GAGs, a cooperative binding behavior was observed, which could indicate simultaneous interaction with both dimer subunits of IL-10. Finally, structural information about the bound GAG was exemplarily obtained for a heparin tetrasaccharide fragment (ΔUA,2S-GlcNS,6S-IdoA,2S-GlcNS,6S) using transferred NOESY experiments, proton-proton scalar couplings and molecular dynamics simulations. The overall backbone conformation is only slightly changed in the presence of IL-10 and the conformational equilibrium between (1)C4 chair and (2)So skew-boat structure of the internal iduronic acid residue is preserved.

Conformational Preferences of N,N-Dimethylsuccinamate as a Function of Alkali and Alkaline Earth Metal Salts: Experimental Studies in DMSO and Water As Determined by 1H NMR Spectroscopy

The fraction of gauche conformers of N,N-dimethylsuccinamic acid (1) and its Li+, Na+, K+, Mg2+, Ca2+, and N(Bu)4+ salts were estimated in DMSO and D2O solution by comparing the experimental vicinal proton–proton couplings determined by 1H NMR spectroscopy with those calculated using the Haasnoot, de Leeuw, and Altona (HLA) equation. In DMSO, the gauche preferences were found to increase with decreasing Ahrens ionic radius of the metal counterion. The same trend was not seen in D2O, where the gauche fraction for all of the metallic salts were estimated to be approximately statistical or less. This highlights the importance of metal chelation on the conformation of organic molecules in polar aprotic media, which has implications for protein folding.

Glycosaminoglycan monosaccharide blocks analysis by quantum mechanics, molecular dynamics, and nuclear magnetic resonance.

Glycosaminoglycans (GAGs) play an important role in many biological processes in the extracellular matrix. In a theoretical approach, structures of monosaccharide building blocks of natural GAGs and their sulfated derivatives were optimized by a B3LYP6311ppdd//B3LYP/6-31+G(d) method. The dependence of the observed conformational properties on the applied methodology is described. NMR chemical shifts and proton-proton spin-spin coupling constants were calculated using the GIAO approach and analyzed in terms of the method's accuracy and sensitivity towards the influence of sulfation, O1-methylation, conformations of sugar ring, and ω dihedral angle. The net sulfation of the monosaccharides was found to be correlated with the 1H chemical shifts in the methyl group of the N-acetylated saccharides both theoretically and experimentally. The ω dihedral angle conformation populations of free monosaccharides and monosaccharide blocks within polymeric GAG molecules were calculated by a molecular dynamics approach using the GLYCAM06 force field and compared with the available NMR and quantum mechanical data. Qualitative trends for the impact of sulfation and ring conformation on the chemical shifts and proton-proton spin-spin coupling constants were obtained and discussed in terms of the potential and limitations of the computational methodology used to be complementary to NMR experiments and to assist in experimental data assignment.

BASHD‐J‐resolved‐HMBC, an efficient method for measuring proton–proton and heteronuclear long‐range coupling constants

Natural products often possess various spin systems consisting of a methine group directly bonded to a methyl group (e.g. -CHa-CHb(CH3)-CHc-). The methine proton Hb splits into a broadened multiplet by coupling with several vicinal protons, rendering analysis difficult of (n)JC-H with respect to Hb in the J-resolved HMBC-1. In purpose of the reliable and easy measurements of (n)JC-H and (n)JH-H in the aforesaid spin system, we have developed a new technique, named BASHD-J-resolved-HMBC. This method incorporates band selective homo decoupled pulse and J-scaling pulse into HMBC. In this method, high resolution cross peaks can be observed along the F1 axis by J-scaling pulse, and band selective homo decoupled pulse simplified multiplet signals. Determinations of (n)JC-H and (n)JH-H of multiplet signals can easily be performed using the proposed pulse sequence.

Conformations of the iduronate ring in short heparin fragments described by time-averaged distance restrained molecular dynamics

The polyconformational behavior of L-iduronic acid (L-IdoA2S) in heparin derivatives has been previously analyzed in terms of intra-ring proton-proton vicinal coupling constants ((3)JHH) through mathematical fit of experimental and theoretical values (Ferro DR, Provasoli A, Ragazzi M, Casu B, Torri G, Bossennec V, Perly B, Sinay P, Petitou M, Choay J. 1990. Conformer Populations of L-Iduronic Acid Residues in Glycosaminoglycan Sequences. Carbohydr Res. 195:157-167; Muñoz-García JC, López-Prados J, Angulo J, Díaz-Contreras I, Reichardt N, de Paz JL, Martín-Lomas M, Nieto PM. 2012. Effect of the substituents of the neighboring ring in the conformational equilibrium of iduronate in heparin-like trisaccharides. Chemistry. 18:16319-16331.). However, this methodology is subjected to the experimental uncertainties of the J-coupling measurements, the force field deviations and the goodness of the least-squares fit. In the present work, we have used time-averaged distance restrained molecular dynamics (tar-MD) to largely reduce these errors, which enables accurate quantification of the population of conformers, or puckers, of the L-IdoA2S residue, in a set of eight heparin-like trisaccharides following the general sequence d-glucosamine (GlcN)-IdoA-GlcN, directly from the time evolution of the puckering coordinates θ and . Thus, by carrying out tar-MD simulations in explicit water, with the exclusive nuclear overhauser enhancement (NOE)-derived distance H2-H5 of the L-IdoA2S (2)SO conformer as the unique imposed constraint, we have been able to accurately and easily determine the different extents to which the iduronate ring populates the polar ((1)C4 chair) and equatorial ((2)SO skew-boat) areas of the puckering sphere depending on the sulfation pattern of the flanking GlcN residues, under low temperature conditions (278 K). The results indicate that 6-O-sulfation at the reducing-end GlcN residue facilitates the (1)C4 to (2)SO transitions of the iduronate ring by augmenting the flexibility of the C2-C3 torsion, driving the conformational equilibrium toward a majority of equatorial conformers.

On the interference of J(HH) modulation in HSQMBC‐IPAP and HMBC‐IPAP experiments

The effects of phase modulation due to homonuclear proton-proton coupling constants in HSQMBC-IPAP and HMBC-IPAP experiments are experimentally evaluated. We show that accurate values of small proton-carbon coupling constants, (n)J(CH), can be extracted even for phase-distorted cross-peaks obtained from a selHSQMBC experiment applied simultaneously on two mutually J-coupled protons. On the other hand, an assessment of the reliability of (n)J(CH) measurement from distorted cross-peaks obtained in broadband IPAP versions of equivalent HMBC and HSQMBC experiments is also presented. Finally, we show that HMBC-COSY experiments could be an excellent complement to HMBC for the measurement of small (n)J(CH) values.

1H‐NMR Fingerprinting of Vaccinium vitis‐idaea Flavonol Glycosides

The fruits of Vaccinium vitis-idaea L. are a valuable source of biologically active flavonoid derivatives. For studies focused on the purification of its quercetin glycosides (QGs) and related glycosides from plants and for the purpose of biological studies, the availability of numeric datasets from computer-assisted ¹H iterative full spin analysis (HiFSA), that is, ¹H-NMR fingerprinting, can replace and assist the repetitive and tedious two-dimensional NMR identification protocol required for both known and new compounds, respectively. To fully interpret the complex ¹H-NMR fingerprints of eight QGs obtained from the berries of V. vitis-idaea and provide complete and unambiguous signal assignments. Vaccinium vitis-idaea QGs were purified in a single run by long-bed gel permeation chromatography and identified by comparison with commercially available compounds using LC-MS combining ion-trap and time-of-flight detection and one- or two-dimensional NMR. The HiFSA analysis yielded full sets of ¹H chemical shifts and proton-proton coupling constants, allowing for field-independent spectral simulation. Signal assignments were achieved for the reference standards and the QGs that dominated in purified fractions. However, even mixtures of two to three QGs could be fitted using the HiFSA approach. In the case of the overlapped sugar resonances, the initial fitting of the ¹H spectra of reference compounds, together with values extracted from the two-dimensional NMR data and literature data, assisted in the process. The HiFSA method revealed for the first time the presence of Q-3-O-β-glucopyranoside and Q-3-O-β-glucuronopyranoside in the berries of V. vitis-idaea, and unambiguously confirmed the structures of Q-3-O-[4″-(3-hydroxy-3-methylglutaroyl)]-α-rhamnopyranoside, Q-3-O-α-rhamnopyranoside, Q-3-O-β-galactopyranoside, Q-3-O-α-arabinofuranoside, Q-3-O-β-xylopyranoside and Q-3-O-α-arabinopyranoside.

Conformational Equilibria of N,N-Dimethylsuccinamic Acid and Its Lithium Salt as a Function of Solvent

The conformational preferences of N,N-dimethylsuccinamic acid and its Li(+) salt were estimated by comparing the respective experimental NMR vicinal proton-proton coupling constants to semiempirical coupling constants for each staggered conformer as derived by the Haasnoot-De Leeuw-Altona method. The strong gauche preferences for the Li(+) salts clearly depended more on the solvents' hydrogen-bond donating strength (α) than on their hydrogen-bond accepting (β) counterpart, where α and β are the corresponding Kamlet-Taft parameters.

Constraints on short-range spin-dependent interactions from scalar spin-spin coupling in deuterated molecular hydrogen.

A comparison between existing nuclear magnetic resonance measurements and calculations of the scalar spin-spin interaction (J coupling) in deuterated molecular hydrogen yields stringent constraints on anomalous spin-dependent potentials between nucleons at the atomic scale (∼ 1 Å). The dimensionless coupling constant g(P)(p)g(P)(N)/4 π associated with the exchange of pseudoscalar (axionlike) bosons between nucleons is constrained to be less than 3.6 × 10(-7) for boson masses in the range of 5 keV, representing improvement by a factor of 100 over previous constraints. The dimensionless coupling constant g(A)(p)g(A)(N)/4 π associated with the exchange of an axial-vector boson between nucleons is constrained to be g(A)(p)g(A)(N)/4 π<1.3 × 10(-19) for bosons of mass ≲ 1000 eV, improving constraints at this distance scale by a factor of 100 for proton-proton couplings and more than 8 orders of magnitude for neutron-proton couplings.

Karplus dependence of spin–spin coupling constants revisited theoretically. Part 1: second-order double perturbation theory

A double perturbation theory (DPT) at the second order level of approximation formalism has been applied to examine the dihedral angle dependence of the Fermi-contact (FC) contribution to nuclear spin-spin coupling constants. The unperturbed wave function of the ground state in DPT was approximated by the Hartree-Fock Slater determinant, while the excited states were treated as the single excited determinants. An analytical expression relating the FC term of vicinal proton-proton spin-spin coupling constants across the aliphatic single carbon-carbon bond to the dihedral angle describing inner rotation around the C-C bond in the ten-electron ten-orbital moiety H-C-C-H has been derived and analyzed. In particular, it has been shown that extrema of (3)J(H,H) are observed at φ = πn, n = 0, ±1, ±2,…, which provides a theoretical background of a well-known semiempirical Karplus equation.

Efficient measurement of the sign and the magnitude of long‐range proton‐carbon coupling constants from a spin‐state‐selective HSQMBC‐COSY experiment

A spin state-selective Heteronuclear Single-Quantum Multiple-Bond Connectivities (HSQMBC-COSY) experiment is proposed to measure the sign and the magnitude of long-range proton-carbon coupling constants ((n)J(CH); n > 1) either for protonated or for non-protonated carbons in small molecules. The simple substitution of the selective 180° (1)H pulse in the original selHSQMBC pulse scheme by a hard one allows the simultaneous evolution of both proton-proton and proton-carbon coupling constants during the refocusing period and enables a final COSY transfer between coupled protons. The successful implementation of the IPAP principle leads to separate mixed-phase α/β cross-peaks from which (n)J(CH) values can be easily measured by analyzing their relative frequency displacements in the detected dimension.

Reprint of “Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H–1H spin–spin coupling constants in proteins”

Two-dimensional correlated spectroscopy (COSY) is used for measurements of proton-proton spin-spin coupling constants in protein 1H NMR spectra. High digital resolution along the frequency axis ω2 is achieved by placing the carrier frequency in the center of the spectrum, using quadrature detection in both dimensions and presenting the spectrum in the phase sensitive mode. Compared to other techniques for studies of spin-spin coupling constants, COSY provides greatly improved spectral resolution. This is illustrated by experiments with H2O solutions of the small globular protein BUSI IIA (bull seminal inhibitor IIA).

Performance of the SCC-DFTB Model for Description of Five-Membered Ring Carbohydrate Conformations: Comparison to Force Fields, High-Level Electronic Structure Methods, and Experiment.

The solution conformations of biologically important mono- and di-α-d-arabinofuranosides were investigated using the dispersion-corrected self-consistent charge density functional tight binding (SCC-DFTB) and the AMBER/GLYCAM models. Simulations were performed using both long dynamics and umbrella sampling simulations. Angular distributions about the exocyclic C-C bonds and puckering distributions of the rings obtained from the SCC-DFTB model were quite different from those obtained with the AMBER/GLYCAM approach. The joint probability distribution of rotamer and ring puckering parameters reveals further discrepancies, while both methods predict weak correlation between exocyclic torsions and ring puckering. To assess the reliability of the simulations, ensemble-averaged vicinal proton-proton coupling constants ((3)JH,H) were calculated and compared directly to experimental NMR coupling constants. It is found that the (3)JH,H values obtained from the AMBER/GLYCAM simulations agree with experiment, while those obtained from the SCC-DFTB method, in most cases, differ from experimental (3)JH,H values. Potential energy surfaces (PES) along the exocyclic torsion obtained from ab initio and DFT calculations differ significantly from those obtained with the SCC-DFTB method. This study establishes that a high-quality all-atom force field would be more suitable than one of the superior semiempirical methods, SCC-DFTB, for investigation of rotamer populations and ring puckering in floppy ring systems such as furanosides.

Indirect non-electrostatic mechanism of the proton-proton interaction and proton-lattice coupling in KH2PO4 ferroelectrics

It has been shown that the mechanism previously proposed for the effective interaction of protons in MH2 AO4 ferroelectrics, such as KH2PO4 (KDP), is applicable to the description of the proton-lattice coupling. The expression derived for the total Hamiltonian of the coupled proton-lattice system, along with the single-mode approximation, also allows the consideration of several modes involved in the ferroelectric phase transition. The use of the proposed approach is illustrated by the example of the numerical evaluation of the effect of the proton-lattice coupling on the critical temperature T c of the ferroelectric phase transition in KDP and deuterated KDP.

C-HETSERF: distinction of cis/trans-isomers and measurement of long range couplings between chemically equivalent nuclei in polycyclic aromatic hydrocarbons

The scalar couplings between chemically equivalent protons in symmetric molecules are not reflected in the NMR spectrum. The present study reports the utility of our previously reported two dimensional spin selective correlation experiment to measure the short and long range homo- and hetero-nuclear scalar couplings in such systems. In weakly coupled spin systems, the experiment also yields the relative signs of the couplings. The relative configurations of double bonded symmetrically disubstituted isomers could be determined from the measured long range vicinal proton-proton couplings (3JHH), owing to the fact that these couplings are consistently larger for trans isomers. The study also reveals that long range 2JCH cannot be utilized as an exclusive parameter for the identification of symmetrically disubstituted cis/trans isomers. The application of the methodology for the determination of couplings between the chemically equivalent protons in polycyclic aromatic hydrocarbons and in bigger molecules such as porphyrin, has also been demonstrated. The study, carried out on large number of molecules, shows the generality and wide applicability of the C-HETSERF experiment.