Nitrogen Chemical Shifts Research Articles

Four-Component Relativistic Calculations of NMR Shielding Constants of the Transition Metal Complexes—Part 2: Nitrogen-Coordinated Complexes of Cobalt

Both four-component relativistic and nonrelativistic computations within the GIAO-DFT(PBE0) formalism have been carried out for 15N and 59Co NMR shielding constants and chemical shifts of a number of the nitrogen-coordinated complexes of cobalt. It was found that the total values of the calculated nitrogen chemical shifts of considered cobalt complexes span over a range of more than 580 ppm, varying from -452 to +136 ppm. At that, the relativistic corrections to nitrogen shielding constants and chemical shifts were demonstrated to be substantial, changing accordingly from ca. -19 to +74 ppm and from -68 to +25 ppm. Solvent effects on 15N shielding constants and chemical shifts were shown to have contributions no less important than the relativistic effects, namely from -35 to +63 ppm and from -74 to +23 ppm, respectively. Cobalt shielding constants and chemical shifts were found to vary in the ranges of, accordingly, -20,157 to -11,373 ppm and from +3781 to +13,811. The relativistic effects are of major importance in the cobalt shielding constants, resulting in about 4% for the shielding-type contributions, while solvent corrections to cobalt shielding constants appeared to be of less significance, providing corrections of about 1.4% to the gas phase values.

New pecS-n (n = 1, 2) basis sets for quantum chemical calculations of the NMR chemical shifts of H, C, N, and O nuclei.

This paper demonstrates the performance of our previously suggested property-energy consistent method on the example of the generation of effective basis sets, pecS-1 and pecS-2, suited for the calculation of hydrogen, carbon, nitrogen, and oxygen chemical shifts. The new basis sets were successfully approbated in the GIAO-DFT calculations of the chemical shifts of 35 molecules using six different functionals. The pecS-1 basis set demonstrated very good accuracy, which makes this small basis set an effective means for the large-scale computations. At the same time, the pecS-2 basis set also gave very accurate results, thus putting it on a par with the other commensurate basis sets suited for the chemical shifts calculations.

Temperature dependence of NMR chemical shifts: Tracking and statistical analysis.

Isotropic chemical shifts measured by solution nuclear magnetic resonance (NMR) spectroscopy offer extensive insights into protein structure and dynamics. Temperature dependences add a valuable dimension; notably, the temperature dependences of amide proton chemical shifts are valuable probes of hydrogen bonding, temperature‐dependent loss of structure, and exchange between distinct protein conformations. Accordingly, their uses include structural analysis of both folded and disordered proteins, and determination of the effects of mutations, binding, or solution conditions on protein energetics. Fundamentally, these temperature dependences result from changes in the local magnetic environments of nuclei, but correlations with global thermodynamic parameters measured via calorimetric methods have been observed. Although the temperature dependences of amide proton and nitrogen chemical shifts are often well approximated by a linear model, deviations from linearity are also observed and may be interpreted as evidence of fast exchange between distinct conformational states. Here, we describe computational methods, accessible via the Shift‐T web server, including an automated tracking algorithm that propagates initial (single temperature) 1H—15N cross peak assignments to spectra collected over a range of temperatures. Amide proton and nitrogen temperature coefficients (slopes determined by fitting chemical shift vs. temperature data to a linear model) are subsequently calculated. Also included are methods for the detection of systematic, statistically significant deviation from linearity (curvature) in the temperature dependences of amide proton chemical shifts. The use and utility of these methods are illustrated by example, and the Shift‐T web server is freely available at http://meieringlab.uwaterloo.ca/shiftt.

11B NMR Chemical Shift Predictions via Density Functional Theory and Gauge-Including Atomic Orbital Approach: Applications to Structural Elucidations of Boron-Containing Molecules.

11B nuclear magnetic resonance (NMR) spectroscopy is a useful tool for studies of boron-containing compounds in terms of structural analysis and reaction kinetics monitoring. A computational protocol, which is aimed at an accurate prediction of 11B NMR chemical shifts via linear regression, was proposed based on the density functional theory and the gauge-including atomic orbital approach. Similar to the procedure used for carbon, hydrogen, and nitrogen chemical shift predictions, a database of boron-containing molecules was first compiled. Scaling factors for the linear regression between calculated isotropic shielding constants and experimental chemical shifts were then fitted using eight different levels of theory with both the solvation model based on density and conductor-like polarizable continuum model solvent models. The best method with the two solvent models yields a root-mean-square deviation of about 3.40 and 3.37 ppm, respectively. To explore the capabilities and potential limitations of the developed protocols, classical boron–hydrogen compounds and molecules with representative boron bonding environments were chosen as test cases, and the consistency between experimental values and theoretical predictions was demonstrated.

Towards an Accurate Prediction of Nitrogen Chemical Shifts by Density Functional Theory and Gauge‐Including Atomic Orbital

AbstractAn efficient, yet accurate, computational protocol for predicting nitrogen nuclear magnetic resonance (NMR) chemical shifts based on density functional theory and the gauge‐including atomic orbital approach is proposed. A database of small and relatively rigid compounds containing nitrogen atoms is compiled. Scaling factors for the linear correlation between experimental 15N chemical shifts and calculated isotropic shielding constants are systematically investigated with seven different levels of theory in both chloroform and dimethyl sulfoxide, two commonly used solvents for NMR experiments. The best method yields a root‐mean‐square deviation of about 5.30 and 7.00 ppm in CHCl3 and dimethyl sulfoxide (DMSO), respectively. Moreover, another set of scaling factors for –NH2 chemical shifts is also proposed based on a separate database with three levels of theory. Furthermore, it is encouraging that a reasonable transferability for the linear correlation is found between these two solvents. This finding will enable broader applications of the developed empirical scaling factors to other commonly used solvents in NMR experiments. The consistency between theoretical predictions and experimental results for structural elucidations is illustrated for selected examples including regioisomers, tautomers, oxidation states, and protonated structures.

Modeling (15)N NMR chemical shift changes in protein backbone with pressure.

Nitrogen chemical shift is a useful parameter for determining the backbone three-dimensional structure of proteins. Empirical models for fast calculation of N chemical shift are improving their reliability, but there are subtle effects that cannot be easily interpreted. Among these, the effects of slight changes in hydrogen bonds, both intramolecular and with water molecules in the solvent, are particularly difficult to predict. On the other hand, these hydrogen bonds are sensitive to changes in protein environment. In this work, the change of N chemical shift with pressure for backbone segments in the protein ubiquitin is correlated with the change in the population of hydrogen bonds involving the backbone amide group. The different extent of interaction of protein backbone with the water molecules in the solvent is put in evidence.

1H–15N HMBC spectra of C18-diterpenoid alkaloids

Abstract The normally underused 1 H– 15 N HMBC spectra of thirteen C 18 -diterpenoid alkaloids have been determined for the first time. As a result, the significant effects of the substituents of nitrogen atoms, the conformations of A ring, and protonation, on the nitrogen-15 chemical shifts are demonstrated.

Solvent effects on the nitrogen NMR chemical shifts in 1-methylazoles – a theoretical study

We have investigated solvent effect on the nitrogen chemical shifts in a series of 1-methylazoles.

Characterizing RNA Excited States Using NMR Relaxation Dispersion.

Changes in RNA secondary structure play fundamental roles in the cellular functions of a growing number of noncoding RNAs. This chapter describes NMR-based approaches for characterizing microsecond-to-millisecond changes in RNA secondary structure that are directed toward short-lived and low-populated species often referred to as "excited states." Compared to larger scale changes in RNA secondary structure, transitions toward excited states do not require assistance from chaperones, are often orders of magnitude faster, and are localized to a small number of nearby base pairs in and around noncanonical motifs. Here, we describe a procedure for characterizing RNA excited states using off-resonance R1ρ NMR relaxation dispersion utilizing low-to-high spin-lock fields (25-3000 Hz). R1ρ NMR relaxation dispersion experiments are used to measure carbon and nitrogen chemical shifts in base and sugar moieties of the excited state. The chemical shift data are then interpreted with the aid of secondary structure prediction to infer potential excited states that feature alternative secondary structures. Candidate structures are then tested by using mutations, single-atom substitutions, or by changing physiochemical conditions, such as pH and temperature, to either stabilize or destabilize the candidate excited state. The resulting chemical shifts of the mutants or under different physiochemical conditions are then compared to those of the ground and excited states. Application is illustrated with a focus on the transactivation response element from the human immune deficiency virus type 1, which exists in dynamic equilibrium with at least two distinct excited states.

Quantitative analysis of complex amino acids and RGD peptides by X‐ray photoelectron spectroscopy (XPS)

The C 1 s, N 1 s, and O 1 s core level binding energies (BEs) of the functional groups in amino acids (glycine, aspartic acid, glutamic acid, arginine, and histidine) with varied side‐chains and cell‐binding RGD‐based peptides have been determined and characterized by X‐ray photoelectron spectroscopy with a monochromatic Al Kα source. The zwitterionic nature of the amino acids in the solid state is unequivocally evident from the N 1 s signals of the protonated amine groups and the C 1 s signature of carboxylate groups. Significant adventitious carbon contamination is evident for all samples but can be quantitatively accounted for. No intrinsic differences in the XP spectra are evident between two polymorphs (α and γ) of glycine, indicating that the crystallographic differences have a minor influence on the core level BEs for this system. The two nitrogen centers in the imidazole group of histidine exhibit an N 1 s BE shift that is in line with previously reported data for theophylline and aqueous imidazole solutions, while the nitrogen and carbon chemical shifts reflect the unusual guanidinium chemical environment in arginine. It is shown that the complex envelopes of C 1 s and O 1 s photoemission spectra for short‐chain peptides can be analyzed quantitatively by reference to the less complex XP spectra of the constituent amino acids, provided the peptides are of high enough purity. The distinctive N 1 s photoemission from the amide linkages provides an indicator of peptide formation even in the presence of common impurities, and variations in the relative intensities of N 1 s were found to be diagnostic for each of the three peptides investigated (RGD, RGDS, and RGDSC). Copyright © 2013 John Wiley & Sons, Ltd.

Structural Basis for Galectin-1-dependent Pre-B Cell Receptor (Pre-BCR) Activation

During B cell differentiation in the bone marrow, the expression and activation of the pre-B cell receptor (pre-BCR) constitute crucial checkpoints for B cell development. Both constitutive and ligand-dependent pre-BCR activation modes have been described. The pre-BCR constitutes an immunoglobulin heavy chain (Igμ) and a surrogate light chain composed of the invariant λ5 and VpreB proteins. We previously showed that galectin-1 (GAL1), produced by bone marrow stromal cells, is a pre-BCR ligand that induces receptor clustering, leading to efficient pre-BII cell proliferation and differentiation. GAL1 interacts with the pre-BCR via the unique region of λ5 (λ5-UR). Here, we investigated the solution structure of a minimal λ5-UR motif that interacts with GAL1. This motif adopts a stable helical conformation that docks onto a GAL1 hydrophobic surface adjacent to its carbohydrate binding site. We identified key hydrophobic residues from the λ5-UR as crucial for the interaction with GAL1 and for pre-BCR clustering. These residues involved in GAL1-induced pre-BCR activation are different from those essential for autonomous receptor activation. Overall, our results indicate that constitutive and ligand-induced pre-BCR activation could occur in a complementary manner.

Direct Sequential Hit Strategy for Unambiguous and Accurate Backbone Assignment of 13C/15N Labeled Proteins

Backbone assignment is the very first and key requirement of any study of protein by NMR. However, the process is generally made difficult by severe amide shift degeneracy commonly exhibited by higher MW (>15 kDa in size) alpha-helical and structurally disordered proteins. Pulse sequences and novel strategies have been reported earlier relying heavily on well dispersed nitrogen chemical shifts. In this context, we present here extension of HNN and HN(C)N based assignment protocol (Panchal et al., J Biomol NMR 20:135–147, 2001) by exploiting carbon chemical shifts to resolve the ambiguities arising because of amide 15N/1H shift degeneracy. The resulted modifications have been termed as hNCAnH and hNcoCAnH, respectively and based on the complementary and mutually exclusive information contained in these two spectra, an efficient strategy -named here as strategy—has been presented for unambiguous and accurate backbone assignment of 13C/15N labeled proteins. The strategy provides directly the coordinates of sequential amide peaks (H i−1, N i−1 and H i+1, N i+1) for a particular amide cross peak (H i , N i ), just after performing the simple manual peak picking step. Thus, the sequential (i to i − 1 or i to i + 1) connectivities between the backbone amide cross peaks (H i , N i ) are established directly without making cumbersome search through various planes of the 3D spectrum as is the case with other presently used approaches for backbone assignment. The strategy have been demonstrated here using an intrinsically unfolded 164-residue protein named DLC1 binding domain of nNOS (residues 134–298).

Pressure Dependence of 15N Chemical Shifts in Model Peptides Ac-Gly-Gly-X-Ala-NH2

High pressure NMR spectroscopy has developed into an important tool for studying conformational equilibria of proteins in solution. We have studied the amide proton and nitrogen chemical shifts of the 20 canonical amino acids X in the random-coil model peptide Ac-Gly-Gly-X-Ala-NH2, in a pressure range from 0.1 to 200 MPa, at a proton resonance frequency of 800 MHz. The obtained data allowed the determination of first and second order pressure coefficients with high accuracy at 283 K and pH 6.7. The mean first and second order pressure coefficients <B115N> and <B215N> for nitrogen are 2.91 ppm/GPa and −2.32 ppm/GPa2, respectively. The corresponding values <B11H> and <B21H> for the amide protons are 0.52 ppm/GPa and −0.41 ppm/GPa2. Residual dependent 1J1H15N-coupling constants are shown.

Computational Study and Molecular Orbital Analysis of NMR Shielding, Spin–Spin Coupling, and Electric Field Gradients of Azido Platinum Complexes

(195)Pt, (14)N, and (15)N NMR data for five azido (N(3)(-)) complexes are studied using relativistic density functional theory (DFT). Good agreement with experiment is obtained for Pt and N chemical shifts as well as Pt-N J-coupling constants. Calculated (14)N electric field gradients (EFGs) reflect experimentally observed trends for the line broadening of azido (14)N NMR signals. A localized molecular orbital analysis of the nitrogen EFGs and chemical shifts is performed to explain some interesting trends seen experimentally and in the first-principles calculations: (i) (14)N NMR signals for the Pt-coordinating (N(α)) nuclei in the azido ligands are much broader than for the central (N(β)) or terminal (N(γ)) atoms. The N(β) signals are particularly narrow; (ii) compared to N(γ), the N(α) nuclei are particularly strongly shielded; (iii) N(β) nuclei have much larger chemical shifts than N(α) and N(γ) ; and (iv) The Pt-N(α) J-coupling constants are small in magnitude when considering the formal sp hybridization of N(α). It is found that for N(α) a significant shielding reduction due to formation of the dative N(α)-Pt bond is counterbalanced by an increased shielding from spin-orbit (SO) coupling originating at Pt. Upon coordination, the strongly delocalized π system of free azide localizes somewhat on N(β) and N(γ). This effect, along with rehybridization at N(α) upon bond formation with Pt, is shown to cause a deshielding of N(γ) relative to N(α) and a strong increase of the EFG at N(α). The large 2p character of the azide σ bonds is responsible for the particularly high N(β) chemical shifts. The nitrogen s-character of the Pt-N(α) bond is low, which is the reason for the small J-coupling. Similar bonding situations are likely to be found in azide complexes with other transition metals.

Specificity of 15N NMR chemical shifts to the nature of substituents and tautomerism in substituted pyridine N-oxides

1H, 13C, and 15N NMR chemical shifts have been measured for 2-aminopyridine N-oxide (1), its eleven derivatives (2–10, 13, 14), and 3-Cl and 3-Br substituted 4-nitropyridine N-oxides (11, 12). δ(15N) of pyridine ring nitrogen in 2-acetylaminopyridine N-oxides are 5.9–11.5 ppm deshielded from those in 2-aminopyridine N-oxides. When amino and acetylamino substituents are in 4-position, δ(15N) of ring nitrogen is 21.3 ppm deshielded in the acetylated derivative. The strong resonance interaction between 2-amino and 5-nitro groups reflects in the decrease of amino nitrogen shielding about 5.3–17.9 ppm. Also, 1H and 13C NMR spectral data are in agreement with 15N NMR results reflected as deshielded amino protons and carbons C-2 and C-5. The pyridine nitrogen chemical shift in all amino- and acetylamino derivatives vary between −101.2 and −126.7 ppm, which has been connected with the tautomeric balance in our earlier studies.

Resolving Nitrogen-15 and Proton Chemical Shifts for Mobile Segments of Elastin with Two-dimensional NMR Spectroscopy

In this study, one- and two-dimensional NMR experiments are applied to uniformly (15)N-enriched synthetic elastin, a recombinant human tropoelastin that has been cross-linked to form an elastic hydrogel. Hydrated elastin is characterized by large segments that undergo "liquid-like" motions that limit the efficiency of cross-polarization. The refocused insensitive nuclei enhanced by polarization transfer experiment is used to target these extensive, mobile regions of this protein. Numerous peaks are detected in the backbone amide region of the protein, and their chemical shifts indicate the completely unstructured, "random coil" model for elastin is unlikely. Instead, more evidence is gathered that supports a characteristic ensemble of conformations in this rubber-like protein.

Discovery of Marinopyrrole A (Maritoclax) as a Selective Mcl-1 Antagonist that Overcomes ABT-737 Resistance by Binding to and Targeting Mcl-1 for Proteasomal Degradation

The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-X(L) and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-X(L) with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-X(L), and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degradation via the proteasome system, which is associated with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-X(L)-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematologic malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by ∼60- to 2000-fold at 1-2 μM. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degradation.

Probing Transient Hoogsteen Hydrogen Bonds in Canonical Duplex DNA Using NMR Relaxation Dispersion and Single-Atom Substitution

Nucleic acids transiently morph into alternative conformations that can be difficult to characterize at the atomic level by conventional methods because they exist for too little time and in too little abundance. We recently reported evidence for transient Hoogsteen (HG) base pairs in canonical B-DNA based on NMR carbon relaxation dispersion. While the carbon chemical shifts measured for the transient state were consistent with a syn orientation for the purine base, as expected for A(syn)•T(anti) and G(syn)•C(+)(anti) HG base pairing, HG type hydrogen bonding could only be inferred indirectly. Here, we develop two independent approaches for directly probing transient changes in N-H···N hydrogen bonds and apply them to the characterization of transient Hoogsteen type hydrogen bonds in canonical duplex DNA. The first approach takes advantage of the strong dependence of the imino nitrogen chemical shift on hydrogen bonding and involves measurement of R(1ρ) relaxation dispersion for the hydrogen-bond donor imino nitrogens in G and T residues. In the second approach, we assess the consequence of substituting the hydrogen-bond acceptor nitrogen (N7) with a carbon (C7H7) on both carbon and nitrogen relaxation dispersion data. Together, these data allow us to obtain direct evidence for transient Hoogsteen base pairs that are stabilized by N-H···N type hydrogen bonds in canonical duplex DNA. The methods introduced here greatly expand the utility of NMR in the structural characterization of transient states in nucleic acids.

Multinuclear Solid-State Nuclear Magnetic Resonance and Density Functional Theory Characterization of Interaction Tensors in Taurine

A variety of experimental solid-state nuclear magnetic resonance (NMR) techniques has been used to characterize each of the elements in 2-aminoethane sulfonic acid (taurine). A combination of (15)N cross-polarization magic angle spinning (CPMAS), (14)N ultrawideline, and (14)N overtone experiments enabled a determination of the relative orientation of the nitrogen electric field gradient and chemical shift tensors. (17)O spectra recorded from an isotopically enriched taurine sample at multiple magnetic fields allowed the three nonequivalent oxygen sites to be distinguished, and NMR parameters calculated from a neutron diffraction structure using density functional theory allowed the assignment of the (17)O parameters to the correct crystallographic sites. This is the first time that a complete set of (17)O NMR tensors are reported for a sulfonate group. In combination with (1)H and (13)C MAS spectra, as well as a previously reported (33)S NMR study, this provides a very broad set of NMR data for this relatively simple organic molecule, making it a potentially useful structure on which to test DFT calculation methods (particularly for the quadrupolar nuclei (14)N, (17)O, and (33)S) or NMR crystallography approaches.

Rapid calculation of protein chemical shifts using bond polarization theory and its application to protein structure refinement

Although difficult to analyze, NMR chemical shifts provide detailed information on protein structure. We have adapted the semi-empirical bond polarization theory (BPT) to protein chemical shift calculation and chemical shift driven protein structure refinement. A new parameterization for BPT amide nitrogen chemical shift calculation has been derived from MP2 ab initio calculations and successfully evaluated using crystalline tripeptides. We computed the chemical shifts of the small globular protein ubiquitin, demonstrating that BPT calculations can match the results obtained at the DFT level of theory at very low computational cost. In addition to the calculation of chemical shift tensors, BPT allows the calculation of chemical shift gradients and consequently chemical shift driven geometry optimizations. We applied chemical shift driven protein structure refinement to the conformational analysis of a set of Trypanosoma brucei (the causative agent of African sleeping sickness) tryparedoxin peroxidase Px III structures. We found that the interaction of Px III with its reaction partner Tpx seems to be governed by conformational selection rather than by induced fit.