High-resolution 2D NMR Spectra Research Articles

High-resolution NMR spectroscopy for measuring complex samples based on chemical-shift-difference selection.

NMR spectroscopy serves as an immensely powerful tool for component assignments and molecular structure elucidations. However, proton NMR spectra are generally trapped with spectral congestion caused by limited frequency differences and complex multiplets. 2D NMR can effectively relieve spectral congestion, but its resolution and acquisition efficiency are restricted by the broad spectral bandwidth. Herein, we introduce an NMR method based on chemical-shift-difference selection by chirp excitation to record high-resolution 2D NMR spectra for extracting coupling correlation networks and multiplet structures, suitable for measurements on complex samples. The performance of the proposed method is illustrated in determining diastereotopic methylene protons, small frequency-difference coupled proton pairs of furanose, pyranose and benzene rings. This study is expected to benefit molecular structure elucidation and composition analysis of complex samples in chemistry, biochemistry and metabonomics.

Mild Acetylation and Solubilization of Ground Whole Plant Cell Walls in EmimAc: A Method for Solution-State NMR in DMSO-d6.

Lignocellulosic biomass is mainly composed of polysaccharides and lignin. The complexity and diversity of the plant cell wall polymers makes it difficult to isolate the components in pure form for characterization. Many current approaches to analyzing the structure of lignocellulose, which involve sequential extraction and characterization of the resulting fractions, are time-consuming and labor-intensive. The present study describes a new and facile system for rationally derivatizing and dissolving coarsely ground plant cell wall materials. Using ionic liquids (EmimAc) and dichloroacetyl chloride as a solvent/reagent produced mildly acetylated whole cell walls without significant degradation. The acetylated products were soluble in DMSO-d6 from which they can be characterized by solution-state two-dimensional nuclear magnetic resonance (2D NMR) spectrometry. A distinct advantage of the procedure is that it realizes the dissolution of whole lignocellulosic materials without requiring harsh ball milling, thereby allowing the acquisition of high-resolution 2D NMR spectra to revealing structural details of the main components (lignin and polysaccharides). The method is therefore beneficial to understanding the composition and structure of biomass aimed at its improved utilization.

Assessment of the higher order structure of Humira®, Remicade®, Avastin®, Rituxan®, Herceptin®, and Enbrel® by 2D-NMR fingerprinting

The advent of monoclonal antibody biosimilar products has stimulated the development of analytical methods that can better characterize an important quality attribute, namely the higher order structure (HOS). Here, we propose a simple approach based on heteronuclear 2D NMR techniques at natural abundance for generating spectral fingerprints of the HOS at high resolution. We show that the proposed method can assess the HOS of six therapeutic products, adalimumab (Humira®), bevacizumab (Avastin®), infliximab (Remicade®), rituximab (Rituxan®), trastuzumab (Herceptin®), and Etanercept (Enbrel®). After treatment with immobilized papain, the purified fragments (Fab and Fc) were analyzed by 2D proton-nitrogen and proton-carbon NMR correlations. All Fab and Fc fragments produced high-resolution 2D-NMR spectra from which assessment of their higher order structure can be performed in the context of comparability studies. In particular, the two different sequences of Fc fragments could be unambiguously distinguished. The results show that it is possible to obtain structurally dependent information at amino acid resolution of these important therapeutic agents.

High-resolution 2D NMR spectra in inhomogeneous fields based on intermolecular multiple-quantum coherences with efficient acquisition schemes

High-resolution 2D NMR spectra in inhomogeneous fields can be achieved by the use of intermolecular multiple-quantum coherences and shearing reconstruction of 3D data. However, the long acquisition time of 3D spectral data is generally unbearable for invivo applications. To overcome this problem, two pulse sequences dubbed as iDH-COSY and iDH-JRES were proposed in this paper. Although 3D acquisition is still required for the new sequences, the high-resolution 2D spectra can be obtained with a relatively short scanning time utilizing the manipulation of indirect evolution period and sparse sampling. The intermolecular multiple-quantum coherence treatment combined with the raising and lowering operators was applied to derive analytical signal expressions for the new sequences. And the experimental observations agree with the theoretical predictions. Our results show that the new sequences possess bright perspective in the applications on invivo localized NMR spectroscopy.

Localizing Fatty Acid Binding Sites on Human Serum Albumin by 2D-NMR

The most abundant protein in plasma, human serum albumin (HSA), is the principal carrier for endogenous lipophilic compounds and lipophilic drugs. Non-esterified long chain fatty acids (FA) are its primary physiological ligand, with multiple binding sites of varying affinities. Detailed structural understandings of HSA-ligand interactions are vital during the development of drugs. HSA ligand interactions have been studied by various biophysical methods: x-ray crystallography, fluorescence spectroscopy, and nuclear magnetic resonance (NMR). Numerous crystal structures of HSA, with and without drugs and/or FAs, have been published. Compared to crystallography, our solution state NMR studies are focused on the more physiologically relevant state. Previously, we identified three high affinity and several low affinity FA binding sites on HSA by 1D-NMR at low resolution. Now, we are correlating crystallographic data with high-resolution 2D-NMR spectra of 13C-methyl-labeled oleic acid (OA). Our 2D-HSQC spectra show the 13C-methyl group of OA bound to HSA. The unique microenvironments that each methyl group experiences in the binding pockets gives rise to different NMR signals of varying intensities. These correspond to the different affinities that HSA has for OA, and our 2D-NMR spectra clearly differentiate the three highest affinity-binding sites from six lower affinity-binding sites. We have identified Sudlow's Drug Sites by utilizing the known FA competitors and Sudlow's site binders: warfarin and diazepam. By expanding this drug competition strategy with ibuprofen and diflunisal, we have identified additional FA binding sites. The highest affinity FA binding sites do not have known competitors and these sites are identified with the use of site-directed mutagenesis of HSA. In summary, we are reporting an approach for studying the interactions of FAs with HSA and FA-competitive drugs in a site-specific manner through the use of state-of-the-art 2D-NMR techniques.

NMR solution structure of the N3' --> P5' phosphoramidate duplex d(CGCGAATTCGCG)2 by the iterative relaxation matrix approach.

High-resolution 2D NMR spectra of the duplex CGCGAATTCGCG with deoxyribose sugars but with the normal phosphodiester linker replaced by an N3' --> P5' phosphoramidate (NP) group have been used to establish a solution structure for the duplex. Distance, angle, and base pair hydrogen-bonding constraints were used to refine the structure by use of the iterative relaxation matrix approach (IRMA). The phosphoramidate NH proton signal could be observed in DMSO at low temperature but not in H2O and D2O. For this reason, the structure was refined with the -NH in each of the two possible low-energy configurations. The structure with the nitrogen lone pair located between the nonbridging oxygen atoms of the 5'-phosphate group consistently had the lowest energy and RMSD values, consistent with an X-ray analysis of the same duplex [Tereshko, V., Gryaznov, S. , and Egli, M. (1998) J. Am. Chem. Soc. 120, 269-283]. In the refined structure, the sugars are in the C3'-endo conformation with the change from the normal C2'-endo conformation of deoxyribose apparently being driven by the gauche effect and the change in electronegativity from the 3'O to the 3'NH group. In agreement with preliminary studies [Ding, D., Gryaznov, S. M., Lloyd, D. H., Chandrasekaran, S., Yao, S., Ratmeyer, L., Pan, Y., and Wilson, W. D. (1996) Nucleic Acids Res. 24, 354-360], the backbone conformation in the NP duplex is very close to classical A-form values. Comparison of phosphodiester and phosphoramidate structures suggests that their backbones have global conformations that are primarily a function of the low-energy state of the sugar ring. A somewhat more complex situation arises when base pair conformation is analyzed with many of the base pairs having a conformation between those of classical A- and B-form helices. The effects of the 2' substituent are obviously important in specifying the final conformation of the stacked bases in either an A-form or B-form helix. It is clear, however, that conversion of the normal phosphodiester of DNA into a phosphoramidate linkage yields a nucleic acid that behaves much more like RNA than DNA, and it has been shown that NP sequences can bind to RNA-directed proteins [Rigl, C. T., Lloyd, D. H., Tsou, D. S., Gryaznov, S. M., and Wilson, W. D. (1997) Biochemistry 36, 650-659].