(13)C chemical shift and (13)C-(14)N dipolar coupling tensors determined by (13)C rotary resonance solid-state NMR

This work explores the utility of simple rotary resonance experiments for the determination of the magnitude and orientation of (13)C chemical shift tensors relative to one or more (13)C--(14)N internuclear axes from (13)C magic-angle-spinning NMR experiments. The experiment relies on simultaneous recoupling of the anisotropic (13)C chemical shift and (13)C--(14)N dipole--dipole coupling interactions using 2D rotary resonance NMR with RF irradiation on the (13)C spins only. The method is demonstrated by experiments and numerical simulations for the (13)C(alpha) spins in powder samples of L-alanine and glycine with (13)C in natural abundance. To investigate the potential of the experiment for determination of relative/absolute tensor orientations and backbone dihedral angles in peptides, the influence from long-range dipolar coupling to sequential (14)N spins in a peptide chain ((14)N(i)--(13)C(alpha)(i)--(14)N(i+1) and (14)N(i+1)--(13)C'(i)--(14)N(i) three-spin systems) as well as residual quadrupolar-dipolar coupling cross-terms is analyzed numerically.     

Determination of Orientational Anisotropy in Glassy Solids by 2D Dipolar Spectra With Sample Flipping

A method is proposed for the quantitative measurement of orientational anisotropy in glassy solids based on 2D dipolar NMR spectra with sample flipping (dipolar DECODER experiment). Purely dipolar spectra are obtained by chemical shift refocusing by a multiple pulse sequence. The experiment is applied to an investigation of a doubly¹³C-labeled sample of bisphenol-A polycarbonate deformed in a channel-die apparatus. The orientational distribution function is determined by an expansion of the distribution in terms of spherical harmonics up to degree four.     

Correlation of fast and slow chemical shift spinning sideband patterns under fast magic-angle spinning

A new two-dimensional solid-state NMR experiment, which correlates slow and fast chemical shift anisotropy sideband patterns is proposed. The experiment, dubbed ROSES, is performed under fast magic-angle spinning and leads to an isotropic spectrum in the directly detected omega(2) dimension. In the evolution dimension omega(1), the isotropic chemical shift is reduced by a factor S, and spinning sidebands are observed spaced by a scaled effective spinning speed omega(R)/S. These spinning sidebands patterns are not identical to those observed with standard slow magic-angle spinning experiments. Chemical shift anisotropy parameters can be accurately extracted with standard methods from these spinning sideband patterns. The experiment is demonstrated with carbon-13 experiments on powdered samples of a dipeptide and a cyclic undecapeptide, cyclosporin-A.     

高分子化学位移的量化计算与固体NMR实验研究

该文以2种不同立构聚丙烯（iPP和sPP）为讨论对象,首先研究了量化计算方法在预测高分子¹³C 各向同性和各向异性化学位移（CSA）中的应用,然后讨论了近年来发展的测定¹³C CSA粉末线形的2种重要固体NMR实验技术（SUPER和RAI）的特点和实验优化问题. 最后,利用可获得接近无扭曲线形的SUPER技术测定了等规立构聚丙烯样品的¹³C CSA粉末线形,并与量化计算理论结果比较. 结果表明：¹³C 各向同性化学位移及CSA粉末线形的理论计算结果均与固体NMR实验结果有很好的符合,预示通过¹³C CSA量化计算结合固体NMR实验是阐明高分子微观结构的有力工具.     

3hJ coupling between C(alpha) and H(N) across hydrogen bonds in proteins

J couplings between (13)C(alpha) and (1)H(N) across hydrogen bonds in proteins are reported for the first time, and a two- or three-dimensional NMR technique for their measurement is presented. The technique exploits the TROSY effect, i.e., the degree of interference between dipolar and chemical shift anisotropy relaxation mechanisms, for sensitivity enhancement. The 2D or 3D spectra exhibit E.COSY patterns where the splittings in the (13)CO and (1)H(N) dimensions are (1)J((13)C(alpha), (13)CO) and the desired (3h)J((13)C(alpha), (1)H(N)), respectively. A demonstration of the new method is shown for the (15)N,(13)C-labeled protein chymotrypsin inhibitor 2 where 17 (3h)J((13)C(alpha), (1)H(N)) coupling constants ranging from 0 to 1.4 Hz where identified and all of positive sign.     

Proton decoupled 13C NMR imaging

Proton decoupled 13C images were obtained at 2.1 Tesla. 13C[1H] images showed an increase in sensitivity over nondecoupled 13C images because of the nuclear Overhauser effect and elimination of multiple lines from scalar 13C-1H spin-spin couplings. The improvement in S/N for 13C[1H] images was smaller than expected because of a significant decrease in decoupling efficiency when 13C spin echoes were acquired in a readout gradient. Images of 13C compounds that had a wide range of chemical shifts showed separated and/or overlapping images, which is consistent with chemical shift imaging artifacts seen in 1H images. This work examines the technical constraints of acquiring and the difficulties of interpreting 13C[1H] images.     

Time-shared X(omega(1))-half-filter for improved sensitivity in subspectral editing

Experiments with X-half-filter elements allow the separation of the resonances from protons bound and unbound to a spin X into different subspectra. This Communication presents a modified half-filter element where the filter delay is simultaneously used for chemical shift labeling and scalar coupling evolution in a semi-constant time experiment. The filter element is demonstrated with a (1)H NOESY spectrum of a 28.5-kDa 2:1 complex between the uniformly (13)C-labeled N-terminal domain of Escherichia coli arginine repressor and operator DNA.     

13C NMR lineshapes of acetone adsorbed on silica

¹³C NMR data, obtained as a function of temperature with magic-angle spinning (MAS) and either cross polarization or direct polarization, are reported on acetone and a sample of acetone (an approximately equal mixture with ¹³C labels at C-1 or C-2) adsorbed on dry silica gel. Various contributions to the observed linewidths and T^C₂ values are considered in terms of a previously established model of the acetone/SiO₂ system; in that model, acetone species are in equilibrium between a physisorbed-acetone (non-hydrogen-bonded) state and a state consisting of acetone units that are hydrogen bonded to silanol moieties on the silica surface. Spin dynamics simulations are useful in interpreting the effects of variations of experimental parameters. It is concluded that the main linewidth contributions, which increase at lower temperatures, are: (a) a dispersion of chemical shifts in the hydrogen-bonded state, associated with the inhomogeneous character of the silica surface; (b) the interference between MAS averaging of the chemical shift anisotropy (especially for the carbonyl carbon) and molecular motion and/or chemical exchange; and (c) chemical exchange broadening. Prominence of the last of these contributions is most consistent with data obtained as a function of magnetic field strength, MAS speed, and temperature.     

Homo-nuclear 13C J-decoupling in uniformly 13C-enriched solid proteins

Recently, we reported an analysis of carbon lineshapes in high resolution solid-state NMR spectra of uniformly 13C-enriched amino acids. Application of a 13C J-decoupling protocol during the carbon chemical shift evolution period allowed us to separate the contribution of the second-order dipolar shift from that of the 13C-13C J-coupling interactions to carbon linewidths. In this work, we have extended this approach to microcrystalline proteins. We describe the performance of the J-decoupling sequence applied to remove homo-nuclear 13C J-couplings in the 13C spectra of ubiquitin. Analysis of the J-decoupling efficiency for C(alpha) and carbonyl protein sites showed that a significant gain in resolution can be achieved.     

Through-bond heteronuclear single-quantum correlation spectroscopy in solid-state NMR, and comparison to other through-bond and through-space experiments

A new through-bond carbon-proton correlation technique, the MAS-J-HSQC experiment, is described for solid-state NMR. This new pulse scheme is compared experimentally with the previously proposed MAS-J-HMQC experiment in terms of proton resolution on a model sample of powdered L-alanine. We show that for natural abundance compounds, the MAS-J-HMQC and MAS-J-HSQC experiments give about the same proton resolution, whereas, for (13)C-labeled materials, narrower proton linewidths are obtained with the MAS-J-HSQC experiment. In addition we show that in scalar as well as in dipolar heteronuclear shift correlation experiments, when the proton chemical shift is encoded by the evolution of a single-quantum coherence, the proton resolution can be enhanced by simply adding a 180 degrees carbon pulse in the middle of the t(1) evolution time.     

High-throughput backbone resonance assignment of small 13C,15N-labeled proteins by a triple-resonance experiment with four sequential connectivity pathways using chemical shift-dependent, apparent 1J(1H,13C): HNCACBcodedHAHB

The proposed three-dimensional triple-resonance experiment HNCACBcodedHAHB correlates sequential 15N, 1H moieties via the chemical shifts of 13Calpha, 13Cbeta, 1Halpha, and 1Hbeta. The four sequential correlation pathways are achieved by the incorporation of the concept of chemical shift-coding [J. Biomol. NMR 25 (2003) 281] to the TROSY-HNCACB experiment. The monitored 1Halpha and 1Hbeta chemical shifts are then coded in the line shape of the cross-peaks of 13Calpha, 13Cbeta along the 13C dimension through an apparent residual scalar coupling, the size of which depends on the attached hydrogen chemical shift. The information of four sequential correlation pathways enables a rapid backbone assignment. The HNCACBcodedHAHB experiment was applied to approximately 85% labeled 13C,15N-labeled amino-terminal fragment of Vaccinia virus DNA topoisomerase I comprising residues 1-77. After one day of measurement on a Bruker Avance 700 MHz spectrometer and 8 h of manual analysis of the spectrum 93% of the backbone assignment was achieved.     

A high-resolution (13)C 3D CSA-CSA-CSA correlation experiment by means of magic angle turning

It is shown in this paper that a previously reported 90 degrees sample flipping (13)C 2D CSA-CSA correlation experiment may be carried out alternatively by employing constant slow sample rotation about the magic angle axis and by synchronizing the read pulse to 13 of the rotor cycle. A high-resolution 3D CSA-CSA-CSA correlation experiment based on the magic angle turning technique is reported in which the conventional 90 degrees 2D CSA-CSA powder pattern for each carbon in a system containing a number of inequivalent carbons may be separated according to the isotropic chemical shift value. The technique is demonstrated on 1,2,3-trimethoxybenzene in which all of the overlapping powder patterns that cannot be segregated by the 2D CSA-CSA experiment are resolved successfully by the 3D CSA-CSA-CSA experiment, including even the two methoxy groups (M(1) and M(3)) whose isotropic shifts, confirmed by high-speed MAS, are separated by only 1 ppm. A difference of 4 ppm in the principal value component (delta(33)) between M(1) and M(3) is readily obtained.     

Proton MRI of (13)C distribution by J and chemical shift editing.

The sensitivity of (13)C NMR imaging can be considerably favored by detecting the (1)H nuclei bound to (13)C nuclei via scalar J-interaction (X-filter). However, the J-editing approaches have difficulty in discriminating between compounds with similar J-constant as, for example, different glucose metabolites. In such cases, it is almost impossible to get J-edited images of a single-compound distribution, since the various molecules are distinguishable only via their chemical shift. In a recent application of J-editing to high-resolution spectroscopy, it has been shown that a more efficient chemical selectivity could be obtained by utilizing the larger chemical shift range of (13)C. This has been made by introducing frequency-selective (13)C pulses that allow a great capability of indirect chemical separation. Here a double-resonance imaging approach is proposed, based on both J-editing and (13)C chemical shift editing, which achieves a powerful chemical selectivity and is able to produce full maps of specific chemical compounds. Results are presented on a multicompartments sample containing solutions of glucose and lactic and glutamic acid in water.     

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.     

Obtaining molecular and structural information from 13C-14N systems with 13C FIREMAT experiments

The effect of dipolar coupling to 14N on 13C FIREMAT (five pi replicated magic angle turning) experiments is investigated. A method is developed for fitting the 13C FIREMAT FID employing the full theory to extract the 13C-14N dipolar and 13C chemical shift tensor information. The analysis requires prior knowledge of the electric field gradient (EFG) tensor at the 14N nucleus. In order to validate the method the analysis is done for the amino acids alpha-glycine, gamma-glycine, l-alanine, l-asparagine, and l-histidine on FIREMAT FIDs recorded at 13C frequencies of 50 and 100 MHz. The dipolar and chemical shift data obtained with this analysis are in very good agreement with the previous single-crystal 13C NMR results and neutron diffraction data on alpha-glycine, l-alanine, and l-asparagine. The values for gamma-glycine and l-histidine obtained with this new method are reported for the first time. The uncertainties in the EFG tensor on the resultant 13C chemical shift and dipolar tensor values are assessed.     

13C核磁共振解析甙类化合物中糖的连接方式

根据葡萄双糖的¹³C化学位移数据,用统计的方法找出糖的构型和连接方式对化学位移影响的经验规律和常数,把它们有条件地推广到甙类化合物中的低聚糖体系中去,推引了一些计算这些低聚糖体系中糖碳化学位移值的经验公式,并用来指定某些甙类化合物中糖碳中的化学位移,得到比较令人满意的结果     

Chemical shift anisotropy amplification

A new NMR experiment which allows a measurement of the chemical shift anisotropy (CSA) tensor under magic angle spinning (MAS) is described. This correlates a fast MAS spectrum in the omega2 dimension with a sideband pattern in omega1 in which the intensities mimic those for a sample spinning at a fraction of the rate omega r/N, and these sidebands result from an amplification by a factor N of the modulation caused by the CSA. Standard methods can be used to extract the principal tensor components from the omega1 sideband patterns, and the nature of the experiment is such that the use of a large number of t1 increments can be avoided without compromising the resolution of different chemical sites. The new experiment is useful for accurately measuring narrow shift anisotropies.     

烷基极化效应与羰基13C化学位移

对羰基化合物中羰基碳的¹³C NMR化学位移与烷基（R）极化效应的内在关系 进行了研究. 结果表明：分子中R的极化效应增加使羰基碳的¹³C化学位移值升 高,其关系可表示为δ=a+b·ΣPEI(R),其中a、b为系数,PEI（R）为R极化效应指数.
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CYCLCROP indirect 13C mapping for long-time and chemical-shift selective diffusion measurements in complex hydrocarbon systems

Cyclic cross-polarization from a proton magnetization to ¹³C and from there back to proton coherences permits the indirect, ¹³C chemical shift selective detection of hydrocarbon compounds in the proton NMR channel. This excitation technique can be combined with elements of one-, two- or three-dimensional magnetic resonance imaging permitting the measurement of time-resolved spatial distributions of hydrocarbon components. Beginning this sort of CYCLCROP mapping experiment with a non-equilibrium distribution of the constituents in the system allows one to study the time evolution of the concentrations of all components that can be identified by characteristic ¹³C resonance lines. As applications, studies of ingress, mixing, gel formation, transport and metabolism in living plants, long-time inter- and self-diffusion in complex hydrocarbon systems are suggested. As a test experiment, the diffusion of methanol in swollen polymethylmethacrylate was examined.     

Application of REDOR subtraction for filtered MAS observation of labeled backbone carbons of membrane-bound fusion peptides

Clean MAS observation of 13C-labeled carbons in membrane-bound HIV-1 and influenza fusion peptides was made by using a rotational-echo double-resonance spectroscopy (REDOR) filter of directly bonded 13C-15N pairs. The clean filtering achieved with the REDOR approach is superior to filtering done with sample difference spectroscopy. In one labeling approach, the peptide had labels at a single 13C carbonyl and its directly bonded 15N. The resulting chemical shift distribution of the filtered signal is used to assess the distribution of local secondary structures at the labeled carbonyl. For the influenza peptide, the Leu-2 carbonyl chemical shift distribution is shown to vary markedly with lipid and detergent composition, as well as peptide:lipid ratio, suggesting that the local peptide structure also has a strong dependence on these factors. Because most carboxylic- and amino-labeled amino acids are commercially available, this REDOR approach should have broad applicability to chemically synthesized peptides as well as bacterially synthesized proteins. In a second labeling approach, the HIV-1 fusion peptide had U-13C, 15N labeling over three sequential residues. When a 1.6 ms REDOR dephasing time is used, only backbone 13C signals are observed. The resulting spectra are used to determine spectral linewidths and to assess feasibility of assignment of uniformly labeled peptide.