Assignment Of Carbon Chemical Shifts Research Articles

NMR Assignment of Methyl Groups in Immobilized Proteins Using Multiple-Bond 13C Homonuclear Transfers, Proton Detection, and Very Fast MAS.

In nuclear magnetic resonance spectroscopy of proteins, methyl protons play a particular role as extremely sensitive reporters on dynamics, allosteric effects, and protein–protein interactions, accessible even in high-molecular-weight systems approaching 1 MDa. The notorious issue of their chemical shift assignment is addressed here by a joint use of solid-state 1H-detected methods at very fast (nearly 100 kHz) magic-angle spinning, partial deuteration, and high-magnetic fields. The suitability of a series of RF schemes is evaluated for the efficient coherence transfer across entire 13C side chains of methyl-containing residues, which is key for establishing connection between methyl and backbone 1H resonances. The performance of ten methods for recoupling of either isotropic 13C–13C scalar or anisotropic dipolar interactions (five variants of TOBSY, FLOPSY, DIPSI, WALTZ, RFDR, and DREAM) is evaluated experimentally at two state-of-the-art magic-angle spinning (55 and 94.5 kHz) and static magnetic field conditions (18.8 and 23.5 T). Model isotopically labeled compounds (alanine and Met-Leu-Phe tripeptide) and ILV-methyl and amide-selectively protonated, and otherwise deuterated chicken α-spectrin SH3 protein are used as convenient reference systems. Spin dynamics simulations in SIMPSON are performed to determine optimal parameters of these RF schemes, up to recently experimentally attained spinning frequencies (200 kHz) and B 0 field strengths (28.2 T). The concept of linearization of 13C side chain by appropriate isotope labeling is revisited and showed to significantly increase sensitivity of methyl-to-backbone correlations. A resolution enhancement provided by 4D spectroscopy with non-uniform (sparse) sampling is demonstrated to remove ambiguities in simultaneous resonance assignment of methyl proton and carbon chemical shifts.

Structure Elucidation of Poly-Faldaprevir: Polymer Backbone Solved Using Solid-State and Solution Nuclear Magnetic Resonance Spectroscopy

A large-scale synthesis of the hepatitis C virus drug Faldaprevir revealed precipitation of an unknown insoluble solid from methanol solutions of the drug substance. The unknown impurity was determined to be a polymer of Faldaprevir based on analytical methods that included size exclusion chromatography in combination with electrospray ionization mass spectrometry, solution nuclear magnetic resonance (NMR), matrix-assisted laser desorption ionization–time of flight, ultracentrifugation, elemental analysis, and sodium quantitation by atom absorption spectroscopy. Structure elucidation of the polymeric backbone was achieved using solid-state NMR cross-polarization/magic angle spinning (CP/MAS), cross polarization-polarization inversion, and heteronuclear correlation (HETCOR) experiments. The polymerization was found to occur at the vinyl cyclopropane via a likely free radical initiation mechanism. Full proton and carbon chemical shift assignments of the polymer were obtained using solution NMR spectroscopy. The polymer structure was corroborated with chemical synthesis of the polymer and solution NMR analysis.

Specific 13C labeling of leucine, valine and isoleucine methyl groups for unambiguous detection of long-range restraints in protein solid-state NMR studies

Here we present an isotopic labeling strategy to easily obtain unambiguous long-range distance restraints in protein solid-state NMR studies. The method is based on the inclusion of two biosynthetic precursors in the bacterial growth medium, α-ketoisovalerate and α-ketobutyrate, leading to the production of leucine, valine and isoleucine residues that are exclusively 13C labeled on methyl groups. The resulting spectral simplification facilitates the collection of distance restraints, the verification of carbon chemical shift assignments and the measurement of methyl group dynamics. This approach is demonstrated on the type-three secretion system needle of Shigella flexneri, where 49 methyl–methyl and methyl–nitrogen distance restraints including 10 unambiguous long-range distance restraints could be collected. By combining this labeling scheme with ultra-fast MAS and proton detection, the assignment of methyl proton chemical shifts was achieved.

Herbicidin Congeners, Undecose Nucleosides from an Organic Extract of Streptomyces sp. L-9-10

Four new undecose nucleosides (herbicidin congeners), three known herbicidins, and 9-(β-d-arabinofuranosyl)hypoxanthine (Ara-H) were isolated from the organic extract of a fermentation culture of Streptomyces sp. L-9-10 using proton NMR-guided fractionation. Their structures were elucidated on the basis of comprehensive 1D and 2D NMR and mass spectrometry analyses. These structures included 2′-O-demethylherbicidin F (1), 9′-deoxy-8′,8′-dihydroxyherbicidin B (2), 9′-deoxy-8′-oxoherbicidin B (2a), and the 8′-epimer of herbicidin B (3). This is the first detailed assignment of proton and carbon chemical shifts for herbicidins A, B, and F. The isolated compounds were evaluated for cancer chemopreventive potential based on inhibition of tumor necrosis factor alpha (TNF-α)-induced nuclear factor-kappa B (NF-κB) activity.

Reichardt’s dye: the NMR story of the solvatochromic betaine dye

Reichardt’s dye, 2,6-diphenyl-4-(2,4,6-triphenyl-1-pyridinio)phenolate (1), has a very large negative solvatochromism in the long wavelength absorption in the UV–vis spectrum when going from nonpolar to polar solvents. This shift provides the basis of the important and widely used ET(30) scale of solvent polarity. While many papers have investigated the properties of this dye, only a few describe the 1H and 13C NMR assignments in any detail. We report herein, our detailed analysis of the proton and carbon chemical shift assignments for this molecule based on 1D and 2D NMR measurements, as well as those of the protonated and methoxy derivatives 2 and 3, respectively. Much to our surprise, some of the critical chemical shift values we observed were significantly different from those previously reported. In addition, we discovered a good correlation not only between the solvent polarity and the chemical shifts of carbons C1 and C4 of Reichardt’s dye (1), but also between the concentration of the dye and these chemical shifts.

Mapping Inhibitor Binding Modes on an Active Cysteine Protease via Nuclear Magnetic Resonance Spectroscopy

Cruzain is a member of the papain/cathepsin L family of cysteine proteases, and the major cysteine protease of the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. We report an autoinduction methodology that provides soluble cruzain in high yields (>30 mg/L in minimal medium). These increased yields provide sufficient quantities of active enzyme for use in nuclear magnetic resonance (NMR)-based ligand mapping. Using circular dichroism and NMR spectroscopy, we also examined the solution-state structural dynamics of the enzyme in complex with a covalently bound vinyl sulfone inhibitor (K777). We report the backbone amide and side chain carbon chemical shift assignments of cruzain in complex with K777. These resonance assignments were used to identify and map residues located in the substrate binding pocket, including the catalytic Cys25 and His162. Selective [(15)N]Cys, [(15)N]His, and [(13)C]Met labeling was performed to quickly assess cruzain-ligand interactions for a set of eight low-molecular weight compounds exhibiting micromolar binding or inhibition. Chemical shift perturbation mapping verified that six of the eight compounds bind to cruzain at the active site. Three different binding modes were delineated for the compounds, namely, covalent, noncovalent, and noninteracting. These results provide examples of how NMR spectroscopy can be used to screen compounds for fast evaluation of enzyme-inhibitor interactions to facilitate lead compound identification and subsequent structural studies.

Determination of 13C Chemical Shift Anisotropy Tensors and Molecular Order of 4-Hexyloxybenzoic Acid

4-Alkoxy benzoic acids belong to an important class of thermotropic liquid crystals that are structurally simple and often used as starting materials for many novel mesogens. 4-Hexyloxybenzoic acid (HBA) is a homologue of the same series and exhibits an enantiotropic nematic phase. As this molecule could serve as an ideal model compound, high resolution (13)C NMR studies of HBA in solution, solid, and liquid crystalline phases have been undertaken. In the solid state, two-dimensional separation of undistorted powder patterns by effortless recoupling (2D SUPER) experiments have been carried out to estimate the magnitude of the components of the chemical shift anisotropy (CSA) tensor of all the aromatic carbons. These values have been used subsequently for calculating the orientational order parameters in the liquid crystalline phase. The CSA values computed by density functional theory (DFT) calculations showed good agreement with the 2D SUPER values. Additionally, (13)C-(1)H dipolar couplings in the nematic phase have been determined by separated local field (SLF) spectroscopy at various temperatures and were used for computing the order parameters, which compared well with those calculated by using the chemical shifts. It is anticipated that the CSA values determined for HBA would be useful for the assignment of carbon chemical shifts and for the study of order and dynamics of structurally similar novel mesogens in their nematic phases.

A new pentacyclic triterpene isolated from Myroxylon balsamum (syn. Myroxylon peruiferum)

The new pentacyclic triterpene, 11a-methoxy-b-amyrin (1), was isolated from Myroxylon balsamum (L.) Harms (syn. Myroxylon peruiferum L.f.). Its structure was established on the basis of IR, MS, 1H NMR, 13C NMR. 2D NMR experiments were also used to establish the structure and the hydrogen and carbon chemical shift assignments of the new triterpene.

Three-dimensional structure of moenomycin A--a potent inhibitor of penicillin-binding protein 1b.

The first three-dimensional structure of moenomycin A in aqueous solution based on NMR-derived distance constraints and molecular dynamics simulations is presented. The antibiotic moenomycin A was obtained from the FlavomycinR complex by ultrafiltration, chromatography on a DEAE-cellulose ion exchanger and reverse-phase chromatography in 98% purity. In contrast to the previously reported behaviour, the compound gave rise to well-resolved NMR spectra in standard solvents. Using several two-dimensional experiments, a complete assignment of proton and carbon chemical shifts was achieved in (CD3)2SO, CD3OD and H2O/D2O (9:1, pH 7.3). A total of 175 interproton distances were determined from 600-MHz rotating-frame NOE (ROE) spectra and were used as restraints in molecular dynamics calculations. These restraints included 84 ROEs between protons of the moenocinol part leading to a very well-defined structure of the lipid part of the molecule. The relative orientation of the subunits was determined by 66 ROEs among different sugar rings and between the sugar rings and moenocinol. As a result of the molecular dynamics calculation, rings D, E, and F as well as the moenocinol part are very well-defined (average rms deviation over all heavy atoms 0.48 A) whereas rings A, B and C display a higher degree of conformational flexibility which might be an artefact due to the lower number of ROEs in this part of the molecule. A three-dimensional pharmacophore hypothesis comprising functional groups of rings E and F and the carboxyl group of glyceric acid is presented on the basis of the degradation and derivatization studies of Welzel and coworkers [Welzel, P., Kunisch, F., Kruggel, F., Stein, H., Scherkenbeck, J., Hiltmann, A., Duddeck, H., Müller, D., Maggio, J. E., Fehlhaber, H.-W., Seibert, G., van Heijenoort, Y. & van Heijenoort, J. (1987) Moenomycin A: Minimum structural requirements for biological activity, Tetrahedron 43, 585-598; Welzel, P. (1993) Transglycosylase inhibition, in Antibiotics and antiviral compounds--Chemical synthesis and modifications (Krohn, K., Kirst, H. A. & Maag, H., eds) pp. 373-378, VCH Weinheim].

A New Depside Isolated from the Bark of Rauwolfia mattfeldiana

From the hexane extract from stem bark of Rauwolfia mattfeldiana Markgr. (Apocynaceae) a new depside (1, methyl 4’-O-methylarthonioate), along with 1-hydroxy-3,6-dimethoxy-8-methylxanthone (4), lupeol (5), sitosterol (6) and stigmasterol (7) were isolated. The structures of these natural products were elucidated by spectral data analysis. 2D NMR experiments were used to establish the structure and the complete hydrogen and carbon chemical shift assignments of the new depside.

Nuclear Magnetic Resonance Spectroscopy and Mass Spectrometry of Solanidine, Leptinidine, and Acetylleptinidine. Steroidal Alkaloids from Solanum chacoense Bitter

Solanidine, leptinidine, and acetylleptinidine from Solanum chacoense Bitter were structurally characterized by high-resolution MS and high-field 1H-NMR and 13C-NMR. 1H-NMR and 13C-NMR chemical shifts were assigned for solanidine and acetylleptinidine using DEPT, HMBC, and HMQC. Complete carbon chemical shift assignments are presented for all three alkaloids. The orientation of the C-23 acetoxyl group of acetylleptinidine was determined to be axial. Keywords: NMR-MS spectroscopy; solanidine; leptinidine; acetylleptinidine

Nuclear magnetic resonance solution structure of the growth factor receptor-bound protein 2 Src homology 2 domain.

A family of NMR solution structures of the growth factor receptor-bound protein 2 (Grb2) SH2 domain has been determined by heteronuclear multidimensional NMR. Proton, nitrogen, and carbon chemical shift assignments have been made for the SH2 domain of Grb2. Assignments were made from a combination of homonuclear two-dimensional and 15N- and 13C-edited three-dimensional spectra at pH 6.2 and 298 K. Structure-induced proton and carbon secondary shifts were calculated and used to facilitate the spectral assignment process. NOE, scalar coupling, secondary chemical shift, and amide proton exchange data were used to characterize the secondary structural elements and hydrogen-bonding network in the Grb2 SH2 domain. The three-dimensional structure of the Grb2 SH2 domain was calculated using 1112 restraints obtained from NOE, coupling constant, and amide proton exchange data. The rmsd for the 24 calculated structures to the mean structure of the Grb2 SH2 domain was 0.75 A for backbone and 1.28 A for all heavy atoms. The three-dimensional fold of the Grb2 SH2 domain is similar to that observed for other SH2 domains and consists of two alpha-helical segments and eight beta-strands, six strands that make up two contiguous antiparallel beta-sheets, and two strands that form two short parallel beta-sheets. The structure of the phosphotyrosine binding pocket of Grb2 is similar to that observed for other SH2 domains. The hydrophobic binding pocket of Grb2 is similar to that observed for Src with the exception that tryptophan 121 of Grb2 occupies part of the pY+3 binding pocket. Structural implications for the Grb2 SH2 domain selectivity at the pY+2 and pY+3 sites are discussed.

PH Dependent C6 and C8 13C chemical shift assignment in N-acetyl neuraminic acid

Heteronuclear correlation NMR spectroscopy was employed in the unambigous carbon chemical shift assignment of N-acetyl neuraminic acid at pH 1.0 and 7.0. Our results show that at pH 1.0 the C6 resonance is slightly downfield from that of C8 and at pH 7.0 the C6 and C8 chemical shifts are transposed. These experiments firmly establish carbon chemical shift assignments in N-acetyl neuraminic acid for the first time.

Carbon-13 Chemical Shift Assignment of 3,3-Dihalogenotricyclo[4.2.1.02,4]Nonane Derivatives by Two-Dimensional NMR Techniques

Abstract We describe therein the Carbpn-13 NMR spectra of six dihalogenotricyclo[4.2.1.02.4]nonane derivatives. The complete assignment of carbon chemical shifts has been made using 2D NMR techniques.

Carbon-13 Chemical Shift Assignment of β-Cedrene and Some Derivatives

Abstract Carbon-13 NMR spectral assignments have been obtained for β-cedrene and three derivatives: β-cedren-9-ol, its acetate and β-cedren-9-one, by combination of two-dimensional NMR techniques and shift reagent experiment.

Definitive Carbon-13 Chemical Shift Assignments in 2-Substituted Adamantane Derivatives by Deuterium Isotope Effects

Abstract Deuterium isotope effects on carbon-13 chemical shifts in 5-[2H]-isotopomeres of ten 2-substituted adamantane derivatives were determined and used for complete assignment of their carbon-13 NMR spectra. The carbon-13 NMR spectra of ten 2-substituted adamantane derivatives have been assignated by consideration of deuterium isotope effects. Some four- and five-bond downfield deuterium effects on certain chemical shifts have been measured and attributed to remote hyperconjugative interactions.

Solution studies of staphylococcal nuclease H124L. 2. Proton, carbon-13, and nitrogen-15 chemical shift assignments for the unligated enzyme and analysis of chemical shift changes that accompany formation of the nuclease-thymidine 3',5'-bisphosphate-calcium ternary complex

Accurate 1H, 15N, and 13C chemical shift assignments were determined for staphylococcal nuclease H124L (in the absence of inhibitor or activator ion). Backbone 1H and 15N assignments, obtained by analysis of three-dimensional 1H-15N HMQC-NOESY data [Wang, J., Mooberry, E.S., Walkenhorst, W.F., & Markley, J. L. (1992) Biochemistry (preceding paper in this issue)], were refined and extended by a combination of homo- and heteronuclear two-dimensional NMR experiments. Staphylococcal nuclease H124L samples used in the homonuclear 1H NMR studies were at natural isotopic abundance or labeled randomly with 2H (to an isotope level of 50%); nuclease H124L samples used for heteronuclear NMR experiments were labeled uniformly with 15N (to an isotope level greater than 95%) or uniformly with 13C (to an isotope level of 26%). Additional nuclease H124L samples were labeled selectively by incorporating single 15N- or 13C-labeled amino acids. The chemical shifts of uncomplexed enzyme were then compared with those determined previously for the nuclease H124L.pdTp.Ca2+ ternary complex [Wang, J., LeMaster, D. M., & Markley, J.L. (1990) Biochemistry 29, 88-101; Wang, J., Hinck, A.P., Loh, S. N., & Markley, J.L. (1990) Biochemistry 29, 102-113; Wang, J., Hinck, A.P., Loh, S.N., & Markley, J.L. (1990) Biochemistry 29, 4242-4253]. The results reveal that the binding of pdTp and Ca2+ induces large shifts in the resonances of several amino acid segments. These chemical shift changes are interpreted in terms of changes in backbone torsion angles that accompany the binding of pdTp and Ca2+; changes at the binding site appear to be transmitted to other regions of the molecule through networks of hydrogen bonds.

Proton and carbon chemical-shift assignments for 6-kestose and neokestose from two-dimensional n.m.r. measurements

The proton and carbon chemical shift assignments of the simplest levan, 6-kestose, [O-β- d-fructofuranosyl-(2→6)-β-fructofuranosyl-(2↔1)-α- d-glucopyranoside], along with another trisaccharide, neokestose, [O-β- d-fructofuranosyl-(2→6)-α- d-glucopyranosyl-(1↔2)-β- d-fructofuranoside], were determined using two-dimensional (2D) homonuclear and heteronuclear n.m.r. methods. The 1H peak of H-1 of the glucose residue was determined by its chemical shift. Using H-H coupling information to this proton, the chemical shifts of most of the proton signals of the glucose moiety were determined. Though the signals from the two fructose residues were very close, a NOESY experiment and long-range CH correlation experiments allowed their complete carbon and proton assignment. This work completes and corrects literature assignments.

Structural and14N EFG Information on Solid Imidazole by13C CP/MAS NMR Data

Abstract Residual dipolar coupling between carbons and 14N nuclei in the 13C CPMAS NMR spectrum of solid imidazole is studied. Calculations of expected splittings with a previously reported equation leads to the complete assignment of the solid state carbon chemical shifts. Additionally, information is provided on the location of 14N electric field gradient axes at the N-H site.

NMR and conformational analysis of ganglioside GD1a

One-dimensional TOCSY and 1D NOESY experiments using Gaussian-shaped pulses as well as 2D NOESY and 2D ROESY measurements were carried out to obtain informations for primary and secondary structural characterizations. These techniques combined with 13 C- 1 H correlation measurements were essential for the complete assignment of hydrogen and carbon chemical shifts. Proton and carbon spin-lattice relaxation time measurements were carried out to probe the dynamic behavior of the hexasaccharide. The interresidual hydrogen distances calculated from the 1D NOESY values were found to be in good accord with the lowest energy model suggested by the HSEA calculations, including those for the glycosidic linkages containing the internal sialic acid residue