Sensitivity enhancement, assignment, and distance measurement in 13C solid-state NMR spectroscopy for paramagnetic systems under fast magic angle spinning

Despite success of previous studies, high-resolution solid-state NMR (SSNMR) of paramagnetic systems has been still largely unexplored because of limited sensitivity/resolution and difficulty in assignment due to large paramagnetic shifts. Recently, we demonstrated that an approach using very-fast magic angle spinning (VFMAS; spinning speed 20kHz) enhances resolution/sensitivity in (13)C SSNMR for paramagnetic complexes [Y. Ishii, S. Chimon, N.P. Wickramasinghe, A new approach in 1D and 2D (13)C high resolution solid-state NMR spectroscopy of paramagnetic organometallic complexes by very fast magic-angle spinning, J. Am. Chem. Soc. 125 (2003) 3438-3439]. In this study, we present a new strategy for sensitivity enhancement, signal assignment, and distance measurement in (13)C SSNMR under VFMAS for unlabeled paramagnetic complexes using recoupling-based polarization transfer. As a robust alternative of cross-polarization (CP), rapid application of recoupling-based polarization transfer under VFMAS is proposed. In the present approach, a dipolar-based analog of INEPT (dipolar INEPT) methods is used for polarization transfer and a (13)C signal is observed under VFMAS without (1)H decoupling. The resulting low duty factor permits rapid signal accumulation without probe arcing at recycle times ( approximately 3 ms/scan) matched to short (1)H T(1) values of small paramagnetic systems ( approximately 1 ms). Experiments on Cu(dl-Ala)(2) showed that the fast repetition approach under VFMAS provided sensitivity enhancement by a factor of 8-66 for a given sample, compared with the (13)C MAS spectrum under moderate MAS at 5kHz. The applicability of this approach was also demonstrated for a more challenging system, Mn(acac)(3), for which (13)C and (1)H paramagnetic shift dispersions reach 1500 and 700 ppm, respectively. It was shown that effective-evolution-time dependence of transferred signals in dipolar INEPT permitted one to distinguish (13)CH, (13)CH(2), (13)CH(3), (13)CO2- groups in 1D experiments for Cu(DL-Ala)(2) and Cu(Gly)(2). Applications of this technique to 2D (13)C/(1)H correlation NMR under VFMAS yielded reliable assignments of (1)H resonances as well as (13)C resonances for Cu(DL-Ala)(2) and Mn(acac)(3). Quantitative analysis of cross-peak intensities in 2D (13)C/(1)H correlation NMR spectra of Cu(DL-Ala)(2) provided distance information between non-bonded (13)C-(1)H pairs in the paramagnetic system.     

一维NOE NMR 技术用于蒂巴因衍生物的立体结构研究

基于高效镇痛剂双氢埃托啡游离碱,对蒂巴因衍生物的立体化学结构的NMR 测定方法进行了研究．结果表明,采用多频率位移选择激发一维NOESY(1D NOESY)和二维NMR(2D NMR)相结合的方法,可有效解决该类化合物¹H 和¹³C NMR 谱复杂重叠的信号归属困难的问题．同时在阐明溶液构象的基础上,提出采用一维NOE(1D NOE)测定C-7和差向异构体的新方法．该方法简单、有效、不受样品浓度影响和杂质信号干扰,结果可靠,不需要标准品对照等优点．该结果有助于此类化合物的立体结构与生物活性关系的研究．     

吉非替尼谱学性质计算研究

吉非替尼是第一个被批准上市用于治疗晚期非小细胞肺癌(NSCLC)的药物．该文采用5种密度泛函理论(DFT)方法B3LYP,BHandHLYP,M06-2X,CAM-B3LYP和LC-wPBE在6-311++G**水平上对吉非替尼分子的红外、紫外可见光谱及核磁共振谱进行了计算,并通过比较计算值和实验值得到最佳的计算条件．研究结果表明,CAM-B3LYP和M06-2X是最佳的用于描述吉非替尼分子红外光谱的方法;B3LYP//GIAO(Gauge-Including Atomic Orbital)方法预测得到的吉非替尼在(CH₃)₂SO中的¹H NMR与实验值最为接近,用于预测¹³C NMR的最佳方法是B3LYP//CSGT(Circularty Sgmmetrical Gabor Transform)．     

Characterization of microporous aluminophosphate IST-1 using (1)H Lee-Goldburg techniques

The presence of two independent methylamine species in microporous aluminophosphate IST-1 (|(CH(3)NH(2))(4)(CH(3)NH(+)(3))(4)(OH(-))(4)|[Al(12)P(12)O(48)]) has been shown previously by synchrotron powder X-ray diffraction. One of these species, [N(1)-C(1)], links to a six-coordinated framework Al-atom [Al(1)], while the other methylamine [N(2)-C(2)] is protonated and hydrogen-bonded to three O-atoms [O(1), O(2) and O(12)]. We revisit the structure of IST-1 and report the complete assignment of the (1)H NMR spectra by combining X-ray data and high-resolution heteronuclear/homonuclear solid-state NMR techniques based on frequency-switched Lee-Goldburg homonuclear decoupling and (31)P-(31)P homonuclear recoupling. Careful analysis of the 2D (1)H-X homonuclear correlation (X=(1)H) and 2D heteronuclear correlation (X=(13)C, (31)P and (27)Al) spectra allowed the distinction of both methylamine species and the assignment of all (31)P and (13)C resonances. For the first time at a relatively high (9.4 T) magnetic field, symmetric doublet patterns have been observed in the (13)C spectra, caused by the influence of the (14)N second-order quadrupolar interaction.     

Measurement of longitudinal relaxation times in crowded 1H NMR spectra using one- and two-dimensional maximum quantum (MAXY) NMR spectroscopy

Methods for measuring longitudinal relaxation times of protons in heavily overlapped ¹H NMR spectra are introduced and exemplified using a solution of cholesteryl acetate. The methods are based on 1-dimensional and 2-dimensional maximum quantum NMR spectroscopy (MAXY), which makes possible the selective detection of CH, CH₂ and CH₃ ¹H NMR resonances. A modification of the BIRD pulse sequence to achieve selective inversion of protons bonded to either ¹²C or ¹³C is given. The approach should find application in studies of molecular dynamics where isotopic enrichment is not possible and the level of available sample dictates the use of ¹H NMR spectroscopy.     

A C solid-state NMR analysis of vitamin D compounds

¹³C cross-polarization/magic-angle spinning (CP/MAS) solid-state NMR spectroscopy has been employed to analyze four vitamin D compounds, namely vitamin D3 (D3), vitamin D2 (D2), and the precursors ergosterol (Erg) and 7-dehydrocholesterol (7DHC). The ¹³C NMR spectrum of D3 displays a doublet pattern for each of the carbon atoms, while that of Erg contains both singlet and doublet patterns. In the cases of 7DHC and D2, the ¹³C spectra display various multiplet patterns, viz. singlets, doublets, triplets, and quartets. To overcome the signal overlap between the ¹³C resonances of protonated and unprotonated carbons, we have subjected these vitamin D compounds to 1D ¹H-filtered ¹³C CP/MAS and {¹H}/¹³C heteronuclear correlation (Hetcor) NMR experiments. As a result, assisted by solution NMR data, all of the ¹³C resonances have been successfully assigned to the respective carbon atoms of these vitamin D compounds. The ¹³C multiplets are interpreted due to the presence of s-cis and s-trans configurations in the α- and β-molecular conformers, consistent with computer molecular modeling determined by molecular dynamics and energy minimization calculations. To further characterize the ring conformations in D3, we have successfully extracted chemical shift tensor elements for the ¹³C doublets. It is demonstrated that ¹³C solid-state NMR spectroscopy provides a robust and high sensitive means of characterizing molecular conformations in vitamin D compounds.     

假耧斗菜碱的二维核磁共振研究

利用1D和2D NMR及NOE差谱等方法研究了首次从自然界分离到的头对尾型BBI生物碱假耧斗菜碱(Paraquileginine)的化学位移及立体结构,对其:¹H和¹³C NMR谱线进行了全归属。本工作对研究毛莫科植物的化学分类及寻找新的有生物功能的天然药物具有参考价值。     

七叶皂苷钠混合物的波谱学分析

混合物的分析在药物分析中起着重要的作用,而NMR是混合物分析的重要手段之一. 本文利用HPLC对七叶皂苷钠混合物中的主要成分A、B、C、D的相对含量进行了测定, 并应用谱带选择性2D CT-HMBC及多种2D NMR实验方法,对这些主要成分的波谱学特征进行了分析,利用各组分的特征谱峰和选择性2D方法的高分辨率对结构相似的成分的谱峰进行了精确的区分和指认,进而对所有的1H、13C NMR信号进行了归属. 实验结果表明利用选择性2D NMR技术能够有利于混合物的分析和谱峰归属.     

13C-NMR Assignments for Methyls and Quaternary Carbons of the Limonoid Obacunone

The high field ¹H and ¹³C resonances of obacunone were established by a series of I D and 2D NMR experiments. The ¹³C-NMR signals of the five tertiary methyl groups and all quatenary carbons were unambiguously assigned. The results confirm the stereochemical assignments at all stereogenic centres.     

A solid-state NMR application of the anomeric effect in carbohydrates: galactosamine, glucosamine, and N-acetyl-glucosamine

Simple 2D 13C/15N heteronuclear correlation solid-state NMR spectroscopy was implemented to resolve the 15N resonances of the alpha and beta anomers of three amino monosaccharides: galactosamine (GalN), glucosamine hydrochloride (GlcN), and N-acetyl-glucosamine (GlcNAc) labeled specifically with 13C1/15N spin pairs. Although the 15N resonances could not be distinguished in normal 1D spectra, they were well resolved in 2D double CP/MAS correlation spectra by taking advantage of the 13C spectral resolution. The alpha and beta resonances shifted apart by 3-5 ppm in their 13C chemical shifts, and differed by 1-2 ppm in the extended 15N dimension. Aside from this, the detection of other 13C/15N correlations over short distances was also achieved arising from the C2, C3 and CO carbons present in natural abundance. 2D double CP/MAS chemical shift correlation NMR spectroscopy is a simple and powerful technique to characterize the anomeric effect of amino monosaccharides. Applications of the 2D method reveal well-resolved 15N and 13C chemical shifts might be useful for structural determination on carbohydrates of biological significance, such as glycopeptide or glycolipids.     

Side-chain H and C resonance assignment in protonated/partially deuterated proteins using an improved 3D(13)C-detected HCC-TOCSY

We propose the use of (13)C-detected 3D HCC-TOCSY experiments for assignment of (1)H and (13)C resonances in protonated and partially deuterated proteins. The experiments extend 2D C-13-start and C-13-observe TOCSY type experiments proposed earlier. Introduction of the third (1)H dimension to 2D TOCSY: (i) reduces the peak overlap and (ii) increases the sensitivity per unit time, even for highly deuterated (>85%) protein samples, which makes this improved method an attractive tool for the side-chain H and C assignment of average sized proteins with natural isotope abundance as well as large partially deuterated proteins. The experiments are demonstrated with a 16 kDa (15)N, (13)C-labeled non-deuterated apo-CcmE and a 48 kDa uniformly (15)N, (13)C-labeled and fractionally ( approximately 90%) deuterated dimeric sFkpA. It is predicted that this method should be suitable for the assignment of methyl (13)C and (1)H chemical shifts of methyl protonated, highly deuterated and (13)C-labeled proteins with even higher molecular weight.     

Unambiguous Assignment of the 13C NMR Resonances of the Side-Chain Carbon Atoms of Dipropylglyoxal Bis(Amidinohydrazone) by DEPT and Selective Heteronuclear Proton Decoupling Techniques

The previously unassigned carbon-13 NMR resonances of the side-chain carbon atoms of the enzyme inhibitor dipropylglyoxal bis(amidinohydrazone) (DPGBG) have been unambiguously assigned with the aid of DEPT measurements and experiments involving the selective decoupling of the protons of one of the methylene groups. The chemical shifts of the side-chain carbon atoms of DPGBG decrease in a nearly linear fashion as a function of the position of the atom in the side chain, the terminal methyl groups having the lowest shift value. The carbon-13 shifts are positively correlated with the chemical shifts of the corresponding hydrogen atoms.     

DBU与全氟炔酸酯的迈克尔型加成及其产物的NMR谱学研究

1，8-二氮杂二环[5，4，0]十一碳-7-烯(DBU)与全氟炔酸酯反应生成成环化合物. 通过对成环产物的¹H NMR、¹H-¹H COSY、¹³C NMR和¹H-¹³C COSY的分析，归属了各NMR谱线，对相关化合物进行了结构确证，同时对反应机理进行了推测.     

Multi-dimensional 1H-13C HETCOR and FSLG-HETCOR NMR study of sphingomyelin bilayers containing cholesterol in the gel and liquid crystalline states

(13)C cross polarization magic angle spinning (CP-MAS) and (1)H MAS NMR spectra were collected on egg sphingomyelin (SM) bilayers containing cholesterol above and below the liquid crystalline phase transition temperature (T(m)). Two-dimensional (2D) dipolar heteronuclear correlation (HETCOR) spectra were obtained on SM bilayers in the liquid crystalline (L(alpha)) state for the first time and display improved resolution and chemical shift dispersion compared to the individual (1)H and (13)C spectra and significantly aid in spectral assignment. In the gel (L(beta)) state, the (1)H dimension suffers from line broadening due to the (1)H-(1)H homonuclear dipolar coupling that is not completely averaged by the combination of lipid mobility and MAS. This line broadening is significantly suppressed by implementing frequency switched Lee-Goldburg (FSLG) homonuclear (1)H decoupling during the evolution period. In the liquid crystalline (L(alpha)) phase, no improvement in line width is observed when FSLG is employed. All of the observed resonances are assignable to cholesterol and SM environments. This study demonstrates the ability to obtain 2D heteronuclear correlation experiments in the gel state for biomembranes, expands on previous SM assignments, and presents a comprehensive (1)H/(13)C NMR assignment of SM bilayers containing cholesterol. Comparisons are made to a previous report on cholesterol chemical shifts in dimyristoylphosphatidylcholine (DMPC) bilayers. A number of similarities and some differences are observed and discussed.     

Sensitivity enhancement in (13)C solid-state NMR of protein microcrystals by use of paramagnetic metal ions for optimizing (1)H T(1) relaxation

We discuss a simple approach to enhance sensitivity for (13)C high-resolution solid-state NMR for proteins in microcrystals by reducing (1)H T(1) relaxation times with paramagnetic relaxation reagents. It was shown that (1)H T(1) values can be reduced from 0.4-0.8s to 60-70 ms for ubiquitin and lysozyme in D(2)O in the presence of 10 mM Cu(II)Na(2)EDTA without substantial degradation of the resolution in (13)C CPMAS spectra. Faster signal accumulation using the shorter (1)H T(1) attained by paramagnetic doping provided sensitivity enhancements of 1.4-2.9 for these proteins, reducing the experimental time for a given signal-to-noise ratio by a factor of 2.0-8.4. This approach presented here is likely to be applicable to various other proteins in order to enhance sensitivity in (13)C high-resolution solid-state NMR spectroscopy.     

Assignment of congested NMR spectra: carbonyl backbone enrichment via the Entner-Doudoroff pathway

In NMR spectra of complex proteins, sparse isotope enrichment can be important, in that the removal of many (13)C-(13)C homonuclear J-couplings can narrow the lines and thereby facilitate the process of spectral assignment and structure elucidation. We present a simple scheme for selective yet extensive isotopic enrichment applicable for production of proteins in organisms utilizing the Entner-Doudoroff (ED) metabolic pathway. An enrichment scheme so derived is demonstrated in the context of a magic-angle spinning solid-state NMR (MAS SSNMR) study of Pf1 bacteriophage, the host of which is Pseudomonas aeruginosa, strain K (PAK), an organism that uses the ED pathway for glucose catabolism. The intact and infectious Pf1 phage in this study was produced by infected PAK cells grown on a minimal medium containing 1-(13)C d-glucose ((13)C in position 1) as the sole carbon source, as well as (15)NH(4)Cl as the only nitrogen source. The 37MDa Pf1 phage consists of about 93% major coat protein, 1% minor coat proteins, and 6% single-stranded, circular DNA. As a consequence of this composition and the enrichment scheme, the resonances in the MAS SSNMR spectra of the Pf1 sample were almost exclusively due to carbonyl carbons in the major coat protein. Moreover, 3D heteronuclear NCOCX correlation experiments also show that the amino acids leucine, serine, glycine, and tyrosine were not isotopically enriched in their carbonyl positions (although most other amino acids were), which is as expected based upon considerations of the ED metabolic pathway. 3D NCOCX NMR data and 2D (15)N-(15)N data provided strong verification of many previous assignments of (15)N amide and (13)C carbonyl shifts in this highly congested spectrum; both the semi-selective enrichment patterns and the narrowed linewidths allowed for greater certainty in the assignments as compared with use of uniformly enriched samples alone.     

一种新的六氢喹啉衍生物的NMR研究

应用¹H NMR,¹³C NMR,DEPT,¹H-¹H COSY,HSQC,HMBC等多种NMR实验测试分析方法,确证了一种新的六氢喹啉衍生物2,7,7-三甲基-4-(4-羟基-3-甲氧基）苯基-5-氧代-1,4,5,6,7,8-六氢喹啉-3-羧酸甲酯的结构,并对它的¹H、¹³C信号进行了归属.     

3D HCCH(3)-TOCSY for resonance assignment of methyl-containing side chains in (13)C-labeled proteins

Two 3D experiments, (H)CCH(3)-TOCSY and H(C)CH(3)-TOCSY, are proposed for resonance assignment of methyl-containing amino acid side chains. After the initial proton-carbon INEPT step, during which either carbon or proton chemical shift labeling is achieved (t(1)), the magnetization is spread along the amino acid side chains by a carbon spin lock. The chemical shifts of methyl carbons are labeled (t(2)) during the following constant time interval. Finally the magnetization is transferred, in a reversed INEPT step, to methyl protons for detection (t(3)). The proposed experiments are characterized by high digital resolution in the methyl carbon dimension (t(2max) = 28.6 ms), optimum sensitivity due to the use of proton decoupling during the long constant time interval, and an optional removal of CH(2), or CH(2) and CH, resonances from the F(2)F(3) planes. The building blocks used in these experiments can be implemented in a range of heteronuclear experiments focusing on methyl resonances in proteins. The techniques are illustrated using a (15)N, (13)C-labeled E93D mutant of Schizosacharomyces pombe phosphoglycerate mutase (23.7 kDa).     

Determination of multiple ***φ***-torsion angles in proteins by selective and extensive (13)C labeling and two-dimensional solid-state NMR.

We describe an approach to efficiently determine the backbone conformation of solid proteins that utilizes selective and extensive (13)C labeling in conjunction with two-dimensional magic-angle-spinning NMR. The selective (13)C labeling approach aims to reduce line broadening and other multispin complications encountered in solid-state NMR of uniformly labeled proteins while still enhancing the sensitivity of NMR spectra. It is achieved by using specifically labeled glucose or glycerol as the sole carbon source in the protein expression medium. For amino acids synthesized in the linear part of the biosynthetic pathways, [1-(13)C]glucose preferentially labels the ends of the side chains, while [2-(13)C]glycerol labels the C(alpha) of these residues. Amino acids produced from the citric-acid cycle are labeled in a more complex manner. Information on the secondary structure of such a labeled protein was obtained by measuring multiple backbone torsion angles phi; simultaneously, using an isotropic-anisotropic 2D correlation technique, the HNCH experiment. Initial experiments for resonance assignment of a selectively (13)C labeled protein were performed using (15)N-(13)C 2D correlation spectroscopy. From the time dependence of the (15)N-(13)C dipolar coherence transfer, both intraresidue and interresidue connectivities can be observed, thus yielding partial sequential assignment. We demonstrate the selective (13)C labeling and these 2D NMR experiments on a 8.5-kDa model protein, ubiquitin. This isotope-edited NMR approach is expected to facilitate the structure determination of proteins in the solid state.     

High-sensitivity multinuclear NMR spectroscopy of a smectite clay and of clay-intercalated polymer

The nuclear magnetic resonance (NMR) properties of a smectite clay low in paramagnetic ions, and NMR experiments to detect organic material near the silicate surfaces with high sensitivity, have been explored by 1H, 29Si, and 13C NMR. In oven-dried hectorite clay, 1H NMR reveals a sharp signal at 0.35 ppm that narrows significantly with spinning speed. It is assigned to the "inner" OH protons of the silicate layers. In fluorohectorite, where the OH groups are replaced by fluorines, no such 1H peak is observed. The assignment is further confirmed by the efficient cross-polarization observed in two-dimensional (2D) 1H-29Si HETCOR spectra, and by 29Si-detected REDOR experiments with 1H-dephasing in the 29Si dipolar field, which yield a 1H-29Si distance of 2.9 + 0.4 A. In these 1H-29Si experiments, the sensitivity of the 29Si signal is enhanced at least fivefold by refocusing the decay resulting from the inhomogeneous broadening of the single 29Si peak, stretching the 29Si signal out over 80 ms. The small 1H linewidth of this signal at spinning frequencies exceeding 4 kHz is attributed to the large proton-proton distances in the clay. The upfield isotropic chemical shift of the OH groups is explained by their inaccessibility to hydrogen-bonding partners, as a result of their location in hexagonal "cavities" of the clay structure. The well-resolved, easily selectable OH-proton signal and the high-sensitivity 29Si detection open excellent perspectives for NMR studies of composites of clays with organic molecules. Two-dimensional 1H-29Si and 1H-1H chemical-shift correlation experiments enable efficient detection of the 1H spectrum of organic segments near the clay surface. Combined with 1H spin diffusion, the organic segments at up to several nanometers from the clay surfaces can be probed. A 2D 1H-13C correlation experiment yields the 13C spectrum of the organic species near the clay surfaces. A mobility gradient of intercalated poly(ethylene oxide), PEO, segments is proven in 1H-3Si WISE experiments with spin diffusion.