High-Temperature Pulsed-Field-Gradient Multidimensional NMR of Polymers

The application of pulsed-field-gradient (PFG) techniques has been particularly important in providing the ability to detect 2D and 3D NMR cross peaks from minor structural components in synthetic organic polymers. The lack of mobility in a large percentage of polymers leads to rapid T2 relaxation which prevents the use of pulse sequences, such as the HMBC experiment, that operate based on coherence transfer via small, long-range J couplings. High-temperature NMR increases molecular motion with corresponding line narrowing (e.g., polyethylenes are typically analyzed at 120 degrees C). However, until now, the requirement for high temperature has precluded the use of PFG methods. Here we present data from a new probe which is capable of performing high-temperature PFG coherence selection experiments at temperatures typical of those used in many polymer analyses. We illustrate the performance of this probe with PFG-HMBC spectra of a copolymer from ethylene/1-hexene/1-butene at 120 degrees C.     

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.     

Velocity distributions remotely measured with a single-sided NMR sensor

The pulsed field gradient nuclear magnetic resonance (PFG NMR) method has proved to be a powerful non-invasive technique to measure molecular displacement in various systems. It has been largely implemented with conventional NMR magnets where the volume for housing the flow setup is restricted. In this work we present the first approach to measure velocity distributions ex situ implementing a pulsed field gradient sequence on a single-sided NMR sensor. The open geometry of these sensors provides access to NMR measurements of a large number of applications previously excluded by the geometry of conventional closed magnets. Both, the distortions to the displacement encoding observed when implementing a PFG sequence in the presence of strongly inhomogeneous B0 and B1 fields, and the performance of the modifications proposed to eliminate these distortions are shown by means of numerical simulations. An alternating stimulated spin-echo PFG sequence implemented to remotely measure velocity distributions was combined with a multi-echo acquisition scheme to significantly increase the sensitivity of the method. The technique was implemented to measure the velocity propagator in a fluid undergoing laminar flow and good agreement with the theoretical result is observed.     

Characterization of molecular motion in the solid state by carbon-13 spin-lattice relaxation times

Relaxation calculations for rapidly spinning samples show that spin-lattice relaxation time (T(1Z)) anisotropy varies with the angle between the rotor spinning axis and the external field. When the rate of molecular motion is in the extreme narrowing limit, the measurement of T(1Z) anisotropies for two different values of the spinning angle allows the determination of two linear combinations of the three static spectral densities, J(0)(0), J(1)(0), and J(2)(0). These functions are sensitive to molecular geometry and the rate and trajectory of motion. The utility of these linear combinations in the investigation of molecular dynamics in solids has been demonstrated with natural abundance (13)C NMR experiments on ferrocene. In an isolated (13)C-(1,2)H group, the dipole-dipole interaction has the same orientational dependence as the quadrupole interaction. Thus, the spectral densities that are responsible for dipolar relaxation of (13)C are the same as those responsible for deuteron quadrupolar relaxation. For ferrocene-d(10), deuteron T(1Z) and T(1Q) anisotropies and the relaxation time of the (13)C magic angle spinning peak provide sufficient information to determine the orientation dependence of all three individual spectral densities.     

Peptide internal motions on nanosecond time scale derived from direct fitting of (13)C and (15)N NMR spectral density functions

NMR relaxation-derived spectral densities provide information on molecular and internal motions occurring on the picosecond to nanosecond time scales. Using (13)C and (15)N NMR relaxation parameters [T(1), T(2), and NOE] acquired at four Larmor frequencies (for (13)C: 62.5, 125, 150, and 200 MHz), spectral densities J(0), J(omega(C)), J(omega(H)), J(omega(H) + omega(C)), J(omega(H) - omega(C)), J(omega(N)), J(omega(H) + omega(N)), and J(omega(H) - omega(N)) were derived as a function of frequency for (15)NH, (13)C(alpha)H, and (13)C(beta)H(3) groups of an alanine residue in an alpha-helix-forming peptide. This extensive relaxation data set has allowed derivation of highly defined (13)C and (15)N spectral density maps. Using Monte Carlo minimization, these maps were fit to a spectral density function of three Lorentzian terms having six motional parameters: tau(0), tau(1), tau(2), c(0), c(1), and c(2), where tau(0), tau(1) and tau(2) are correlation times for overall tumbling and for slower and faster internal motions, and c(0), c(1), and c(2) are their weighting coefficients. Analysis of the high-frequency portion of these maps was particularly informative, especially when deriving motional parameters of the side-chain methyl group for which the order parameter is very small and overall tumbling motions do not dominate the spectral density function. Overall correlation times, tau(0), are found to be in nanosecond range, consistent with values determined using the Lipari-Szabo model-free approach. Internal motional correlation times range from picoseconds for methyl group rotation to nanoseconds for backbone N-H, C(alpha)-H, and C(alpha)-C(beta) bond motions. General application of this approach will allow greater insight into the internal motions in peptides and proteins.     

有机磷化合物的NMR研究——Ⅱ0,0-二烷基膦酸酯的1H、13C和31PNMR谱

本文报道了21个0,0一二烷基膦酸酯类化合物的¹H、¹³C和³¹P NMR参数。研究和讨论了不等价的二烷基¹H、¹³C化学位移和磷碳偶合常数与立体化学的关系。测定了(CH₃CH₂O)₂P(O)CH(CH₂NO₂)(p-OCH₃C₆H₄)的¹³C自旋一晶格弛豫时间T₁,二乙基¹³C T₁间的差别,说明在类似化合物中,含有化学位移各向异性对弛豫的贡献。     

Water absorption kinetics in different wettability conditions studied at pore and sample scales in porous media by NMR with portable single-sided and laboratory imaging devices

NMR relaxation time distributions of water (1)H obtained by a portable single-sided surface device have been compared with MRI internal images obtained with a laboratory imaging apparatus on the same biocalcarenite (Lecce Stone) samples during capillary water uptake. The aim of this work was to check the ability of NMR methods to quantitatively follow the absorption phenomenon under different wettability conditions of the internal pore surfaces. Stone wettability changes were obtained by capillary absorption of a chloroform solution of Paraloid PB72, a hydrophobic acrylic resin frequently used to protect monuments and buildings, through one face of each sample. Both relaxation and imaging data have been found in good quantitative agreement each other and with masses of water determined by weighing the samples. In particular the Washburn model of water capillary rise applied to the imaging data allowed us to quantify the sorptivity in both treated and untreated samples. Combining relaxation and imaging data, a synergetic improvement of our understanding of the water absorption kinetics at both pore and sample scales is obtained. Since relaxation data have been taken over the course of time without interrupting the absorption process, simply by keeping the portable device on the surface opposite to the absorption, the results show that the single-sided NMR technique is a powerful tool for in situ evaluation of water-repellent treatments frequently used for consolidation and/or protection of stone artifacts.     

植物油的1H NMR和13C NMR的测定

油酸和亚油酸,是人体必需的营养成份,具有抗癌,防止动脉硬化,降脂减压等作用,因而它们在治疗一些癌症及减肥降血脂等方面取得了较好的疗效^[1-3]。通常油酸,亚油酸和其他脂肪酸以甘油酯的形式,大量地存在于植物油中。本文用¹H NMR测定了5种植物油各组峰面积积分,按文献[4]扣除法,整理了各组峰的积分值,计算出油脂中每摩尔所含各功能团的个数,不饱和度,酯化度等数据。此外,还记了豆油和茶油的¹³C NMR谱,从这两种谱图相关谱线积分值推算出各功能团与甲基基团的比率与¹H NMR谱所得结果接近。并计算出,上述两种油中所含脂肪酸甘油酯和各种不饱和脂肪酸甘油酯的克分子百分数。对测得CCH₂C基团质子自旋晶格弛豫时间T₁进行分析后,给出一经验公式来予以拟合,计算结果与测定值吻合。这一事实说明,油脂中各种组分克分子百分数的计算是可以信赖的。     

H,N,C-triple resonance NMR of very large systems at 900 MHz

We provide quantitative signal to noise data and feasibility study at 900 MHz for 1H-15N-13C triple resonance backbone assignment pulse sequences obtained from a medium sized 2H, 13C, 15N labeled protein slowed down in glycerol-water solution to mimic relaxation and spectroscopic properties of a much larger protein system with macromolecular tumbling correlation time of 52 and 80 ns, respectively, at 296 and 283 K (corresponding to molecular weights of 130 and 250 kDa). Comparisons of several different schemes for transferring magnetization from proton to nitrogen and back to proton confirms Yang and Kay's 1999 prediction that avoiding the unfavorable relaxation properties of 1H-15N multiple quantum coherence in the TROSY phase cycle of the final 15N-1H transfer before acquisition is crucial for maximal sensitivity from these very large molecular weight systems. We also show results which confirm some predictions regarding the superiority of TROSY at 900 MHz vs. 800 MHz especially as the molecular weights become very large.     

Measuring 1H-1H and 1H-13C RDCs in methyl groups: example of pulse sequences with numerically optimized coherence transfer schemes

The optimization of coherence-transfer pulse-sequence elements (CTEs) is the most challenging step in the construction of heteronuclear correlation NMR experiments achieving sensitivity close to its theoretical maximum (in the absence of relaxation) in the shortest possible experimental time and featuring active suppression of undesired signals. As reported in the present article, this complex optimization problem in a space of high dimensionality turns out to be numerically tractable. Based on the application of molecular dynamics in the space of pulse-sequence variables, a general method is proposed for constructing optimized CTEs capable of transferring an arbitrary (generally non-Hermitian) spin operator encoding the chemical shift of heteronuclear spins to an arbitrary spin operator suitable for signal detection. The CTEs constructed in this way are evaluated against benchmarks provided by the theoretical unitary bound for coherence transfer and the minimal required transfer time (when available). This approach is used to design a set of NMR experiments enabling direct and selective observation of individual (1)H-transitions in (13)C-labeled methyl spin systems close to optimal sensitivity and using a minimal number of spectra. As an illustrative application of the method, optimized CTEs are used to quantitatively measure (1)H-(1)H and (1)H-(13)C residual dipolar couplings (RDCs) in a 17 kDa protein weakly aligned by means of Pf1 phages.     

20(R)和20(S)-人参皂甙Rg_2碳氢NMR信号全指定

20 (R)和 2 0 (S) 人参皂甙 Rg2 属于达玛烷型四环三萜类化合物 .应用 2DNMR技术 :1 H 1 HCOSY、HMQC和HMBC全归属 2 0 (R)和 2 0 (S) 人参皂甙 Rg2 碳和氢质子信号 ,为该类型化合物的结构鉴定提供波谱学依据 .     

Fixed and pulsed gradient diffusion methods in low-field core analysis

We review diffusion-weighted relaxation protocols for two-dimensional diffusion/relaxation time (D, T(2)) distributions and their application to fluid-saturated sedimentary rocks at low fields typical of oil-well logging tools (< or = 2 MHz for 1H). Fixed field gradient (FFG) protocols may be implemented in logging tools and in the laboratory; there, pulsed field gradient (PFG) protocols are also available. In either category, direct or stimulated echoes may be used for the diffusion evolution periods. We compare the results of several variant FFG and PFG protocols obtained on liquids and two contrasting sedimentary rocks. For liquids and rocks of negligible internal gradients (g(int)), results are comparable, as expected, for all the studied protocols. For rocks of strong g(int), protocol-dependent artifacts are seen in the joint (D, T2) distributions, consistent with the effects of the internal fields. For laboratory petrophysics, the PFG methods offer several advantages: (a) significantly improved signal-to-noise ratio and acquisition times for repetitions over many samples; (b) freedom from heteronuclear contamination when fluorinated liquids are used in core holders; and (c) a palette of variants--one comparable with the FFG--for the study of rocks of significant g(int). Given suitable hardware, both PFG and FFG methods can be implemented in the same bench-top apparatus, providing a versatile test bed for application in a petrophysical laboratory.     

Dynamics of the biopolymers in articular cartilage studied by magic angle spinning NMR

To understand the viscoelastic properties of cartilage tissue and for the development of tissue-engineered cartilage, we have studied the physicochemical properties of bovine nasal and pig articular cartilage by¹³C nuclear magnetic resonance (NMR) methods. The major macromolecular components of cartilage can be investigated individually by applying¹³C high-resolution (HR) NMR with scalar decoupling (for the polysaccharide component) and solid-state NMR with dipolar decoupling (for the collagen component). Partially resolved NMR spectra of the cartilage polysaccharides can be obtained by HR¹³C NMR indicating that these polysaccharides are highly mobile. Resonance lines have been assigned to chondroitin sulfate, the most mobile component of cartilage. To characterize time scales of molecular motions, we have measuredT ₁ andT ₂ relaxation times as a function of temperature and analyzed these data by means of a broad distribution of molecular correlation times. Typical correlation times for the large amplitude motions of chondroitin sulfate are of the order of 0.1–10 ns. For the detection and dynamical characterization of the cartilage collagen cross-polarization magic angle spinning (CP MAS) and high-power decoupling are indispensable.¹³C CP MAS spectra of cartilage are dominated by resonances from rigid collagen, while only low-intensity signals from the polysaccharides are observed. The good sensitivity at a magnetic field strength of 17.6 T allows the site-specific investigation of cartilage collagen dynamics by two-dimensional NMR methods. The cartilage collagen is essentially rigid with low-amplitude segmental motions on the fast time scale. Considering the high water content of cartilage and the almost isotropic mobility of the chondroitin sulfate molecules it is remarkable how little this affects the collagen dynamics. The dynamics of cartilage macromolecules is broadly distributed from almost completely rigid to highly mobile, which lends cartilage its mechanical strength and shock-absorbing properties.     

2D CP/MASC Isotropic Chemical Shift Correlation Established byH Spin Diffusion

A new 2D solid-state CP/MAS¹³C NMR exchange experiment for through-space isotropic chemical shift correlation is proposed and demonstrated. Through-space correlation is established via a second cross polarization from¹³C to¹H and subsequent¹H spin diffusion. A third cross polarization results in the final¹³C–¹³C isotropic chemical shift correlation. The¹H spin diffusion time is a variable parameter allowing different mean square magnetization displacements to be probed. Experimental results on mixtures of differently¹³C-labeled alanine and polyethylene indicate that this site-selective 2D technique can be used to characterize domain sizes and proximities over a wide range of length scales (1–200 nm) in solids such as polymers or biological materials.     

氟喹诺酮类抗菌药物与过渡金属离子Cr3+, Mn2+和Fe3+形成配合物配位位置的研究

通过1D ¹H NMR, ¹³C NMR和1H自旋-晶格弛豫时间T₁对氟喹诺酮类抗菌药物与过渡金属离子Cr³⁺, Mn²⁺和Fe³⁺形成配合物的位置进行了研究, 合成了固体配合物对其进行了元素分析. 实验结果表明过渡金属离子Cr³⁺, Mn²⁺和Fe³⁺与氟喹诺酮类抗菌药物形成1∶2的配合物, 并确定了配位位置.     

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.     

A compact high-speed mechanical sample shuttle for field-dependent high-resolution solution NMR

Analysis of NMR relaxation data has provided significant insight on molecular dynamic, leading to a more comprehensive understanding of macromolecular functions. However, traditional methodology allows relaxation measurements performed only at a few fixed high fields, thus severely restricting their potential for extracting more complete dynamic information. Here we report the design and performance of a compact high-speed servo-mechanical shuttle assembly adapted to a commercial 600 MHz high-field superconducting magnet. The assembly is capable of shuttling the sample in a regular NMR tube from the center of the magnet to the top (fringe field ～0.01 T) in 100 ms with no loss of sensitivity other than that due to intrinsic relaxation. The shuttle device can be installed by a single experienced user in 30 min. Excellent 2D-(15)N-HSQC spectra of (u-(13)C, (15)N)-ubiquitin with relaxation at low fields (3.77 T) and detection at 14.1T were obtained to illustrate its utility in R(1) measurements of macromolecules at low fields. Field-dependent (13)C-R(1) data of (3,3,3-d)-alanine at various field strengths were determined and analyzed to assess CSA and (1)H-(13)C dipolar contributions to the carboxyl (13)C-R(1).     

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.     

TRANILAST ^1H^13CNMR谱分析及其结构的鉴定

本文用COSY,CHCOR等二维核磁共振技术对TRANILAST,2[]3-(3.4-二甲基苯基)1-氧代-2-丙烯基]氨基]苯甲到进行了~1H、~(13)C NMR谱数据分析及归属,并结合~(13)C弛豫时间T_1 及变温实验对合成产品进行了结构鉴定.     

2D 1H-31P solid-state NMR studies of the dependence of inter-bilayer water dynamics on lipid headgroup structure and membrane peptides

The dynamics of hydration-water in several phospholipid membranes of different compositions is studied by 2D (1)H-(31)P heteronuclear correlation NMR under magic-angle spinning. By using a (1)H T(2) filter before and a (1)H mixing-time after the evolution period and (31)P detection, inter-bilayer water is selectively detected without resonance overlap from bulk water outside the multilamellar vesicles. Moreover the (1)H T(2) relaxation time of the inter-bilayer water is measured. Lipid membranes with labile protons either in the lipid headgroup or in sterols exhibit water-(31)P correlation peaks while membranes free of exchangeable protons do not, indicating that the mechanism for water-lipid correlation is chemical exchange followed by relayed magnetization transfer to (31)P. In the absence of membrane proteins, the inter-bilayer water (1)H T(2)'s are several tens of milliseconds. Incorporation of charged membrane peptides shortened this inter-bilayer water T(2) significantly. This T(2) reduction is attributed to the peptides' exchangeable protons, molecular motion and intermolecular hydrogen bonding, which affect the water dynamics and the chemically relayed magnetization transfer process.