Heteronuclear Correlation Experiments Research Articles

Structural and dynamic studies of proteins by high-resolution solid-state NMR

Abstract Solid-state NMR (SSNMR) spectroscopy provides unique possibilities for the structural investigation of insoluble or non-crystalline molecules (e.g., membrane proteins) at the atomic level. Recent efforts aim at solving the complete structures of biological macromolecules using high-resolution magic-angle spinning NMR. Structurally homogenous samples of [ 13 C, 15 N]-labeled proteins are used in this type of studies. Sequential correlation of resonances, detection of tertiary inter-atomic contacts and characterization of torsion angles can be achieved using multidimensional homo- and heteronuclear correlation experiments. This review discusses the recent progress made in resonances assignments, structure and dynamics determination, as well as the detection of protein interaction partners by SSNMR methods. To cite this article: A. Bockmann, C. R. Chimie 9 (2006) .

More insight in the structure of silicophosphate gels by 31P–29Si CP MAS multidimensional experiments and 1H–31P–29Si triple resonance experiments

Solid-state 29Si, 31P MAS and CP MAS NMR and X-ray powder diffraction have been used to study phosphosilicate gels of molar composition (TEOS/H3PO4 = 1) treated under various temperature conditions. It was found that the gel treated at 100 °C for 30 h had a structure consisting of a silica matrix including isolated molecules of phosphorous acid. In contrast, cross condensation (leading to the formation of Si–O–P bonds) was observed when the gel was treated at 136 °C for 6 days. Evidence for the formation of Si–O–P bonds was provided first by XRD, which showed the formation of the crystalline phosphosilicate phases Si5O(PO4)6 and Si(HPO4)2·H2O, and then by the use of two-dimensional (2D) NMR techniques: 31P–29Si heteronuclear correlation (HETCOR) experiments, based on cross polarization transfer. Very few data concerning this type of experiments are available in the literature: therefore, CP dynamics was carefully studied under MAS. To cite this article: C. Coelho et al., C. R. Chimie 9 (2006). Des analyses en RMN 29Si, 31P MAS à l'état solide ainsi qu'en diffraction des rayons X sur poudre ont été utilisées pour étudier des gels de phosphosilicates obtenus à partir de mélanges équimolaires de TEOS/H3PO4 et traités sous différentes conditions de température. Dans le cas d'un gel séché à 100 °C pendant 30 h, le spectre RMN 31P MAS montre un signal caractéristique de l'acide phosphorique piégé dans un réseau de silice amorphe. En revanche, le gel traité à 136 °C pendant six jours présente un réarrangement structural différent (phases cristallines Si5O(PO4)6 et Si(HPO4)2·H2O). L'utilisation de techniques RMN 2D (expériences 31P–29Si HECTOR) basées sur le transfert de polarisation croisée a permis de corréler les différents sites 31P et 29Si. Pour citer cet article : C. Coelho et al., C. R. Chimie 9 (2006).

Three new sesquiterpene hemiacetals from Achillea vermicularis

Three new sesquiterpene hemiacetals, tentatively named as achilleanone (1), vermiculone (2) and vermicularone (3) have been isolated from Achillea vermicularis, along with three other known compounds β-amyrin, oleonolic acid and β-sitosterol. The structure elucidation of new compounds was based primarily on two-dimensional (2D) NMR techniques including Nuclear Overhauser Effect/Enhancement (NOE), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlation (HMBC) and nuclear overhauser effect spectroscopy (NOESY) experiments. Compounds 1, 2 and 3 have displayed inhibitory potential against lipoxygenase enzyme in a concentration-dependent fashion with promising IC50 values.

Characterization of Carbon Monoxide Centered Triads in Poly[ethylene‐co‐(butyl acrylate)‐co‐(13C‐carbon monoxide)] by Isotopic Labeling and Two‐Dimensional NMR

AbstractSummary: Poly[ethylene‐co‐(butyl acrylate)‐co‐(carbon monoxide)] (polyEBC) samples, prepared from 13C‐labeled carbon monoxide, were characterized using two dimensional (2D) pulsed field gradient (PFG) 750 MHz NMR spectroscopy. To elucidate the complex mixture of structures present in this terpolymer, 2D 1H/13C‐heteronuclear multiple bond correlation (HMBC) experiments were conducted by selectively exciting the enhanced peaks resulting from 13C‐labeling. High resolution 2D NMR combined with 13C‐labeling of the polymer greatly simplifies the 2D NMR spectra, selectively enhances the weak peaks from low occurrence C‐centered triad structures and aids in their resonance assignments. magnified image

NMR Heteronuclear Correlation between Quadrupolar Nuclei in Solids

We show for the first time that it is possible to acquire high-resolution heteronuclear NMR correlation experiments in solid state between second-order-broadened half integer quadrupolar nuclei (i.e., 27Al and 17O) using the scalar J-coupling. The sensitivity of the experiment is dramatically improved at high fields (gain proportional to the fourth power of the principal field) with a combination of signal enhancement techniques. This turns a challenging experiment into a real tool. We apply this experiment to characterize a calcium aluminate glass in which we prove the presence of tricluster mu3 oxygen sites and describe the signature of their directly bonded aluminum sites. Applications involve a large range of possible pairs of quadrupolar nuclei in different materials, such as glasses, porous or mesoporous framework materials, zeolites, hybrid organic-inorganic, and bioinvolved materials.

Periplasmic Protein HdeA Exhibits Chaperone-like Activity Exclusively within Stomach pH Range by Transforming into Disordered Conformation

The extremely acidic environment of the mammalian stomach, with a pH range usually between 1 and 3, represents a stressful challenge for enteric pathogenic bacteria such as Escherichia coli before they enter into the intestine. The hdeA gene of E. coli was found to be acid inducible and was revealed by genetic studies to be important for the acid survival of the strain. This study was performed in an attempt to characterize the mechanism of the activity of the HdeA protein. Our data provided in this report strongly suggest that HdeA employs a novel strategy to modulate its chaperone activity: it possesses an ordered conformation that is unable to bind denatured substrate proteins under normal physiological conditions (i.e. at neutral pH) and transforms into a globally disordered conformation that is able to bind substrate proteins under stress conditions (i.e. at a pH below 3). Furthermore, our data indicate that HdeA exposes hydrophobic surfaces that appear to be involved in the binding of denatured substrate proteins at extremely low pH values. In light of our observations, models are proposed to explain the action of HdeA in both a physiological and a molecular context.

Poly(vinylidene chloride- co-vinyl acetate): comprehensive microstructure analysis by NMR spectroscopy

The microstructure analysis of poly(vinylidene chloride- co-vinyl acetate) was done using chemical shift modeling and 2D NMR spectroscopy. Chemical shift modeling was applied to analyze the compositional sensitive resonances of quaternary carbon of vinylidene chloride unit. Reactivity ratios determination was done from the diad and triad fractions. To resolve the complex 1H and 13C{ 1H} NMR spectra of copolymers, 2D 1H/ 13C hetero-nuclear single quantum coherence (HSQC), 1H/ 1H total correlation spectroscopy (TOCSY) and hetero-nuclear multiple bond correlation (HMBC) experiments were conducted. The combination of 2D NMR experiments supported by chemical shift modeling enabled to assign the complex and overlapping proton and carbon-13 resonances unambiguously.

Reversion of Human Pgp-Dependent Multidrug Resistance by New Sesquiterpenes from Zinowiewia costaricensis

In an intensive study of South American medicinal plants, herein we report the isolation, structure elucidation and biological activity of fourteen new and five known dihydro-beta-agarofuran sesquiterpenes from the leaves of Zinowiewia costaricensis (1-19). Their structures were determined by means of (1)H and (13)C NMR spectroscopic studies, including homonuclear and heteronuclear correlation experiments. The absolute configurations of the new compounds were determined by CD studies, chemical correlations or biogenetic grounds. All the natural compounds and derivative 20 have been tested on human MDR1-transfected NIH-3T3 cells, to determine their ability to revert the multidrug resistance phenotype due to P-glycoprotein overexpression. Six compounds from this series (1, 8, 11, 12, 13 and 14) showed similar effectiveness to the classical P-glycoprotein modulator verapamil when reversing resistance to daunorubicin, but it is up to sixteen times greater than that of verapamil when reversing resistance to vinblastine. The structure-activity relationships are discussed.

Improved methods for1H-3H heteronuclear shift correlation

A better understanding of the structure of complex 3H-labeled molecules can be obtained by complete assignment of their 1H and 3H solution-state NMR spectra. The assignment process is aided by the detection of heteronuclear chemical shift correlations between 1H and 3H nuclei. Heteronuclear correlation (HETCOR) experiments previously applied to this task exhibit several drawbacks caused by the nature of both the pulse sequences and 1H-3H spin systems. The range of J-couplings involved in 1H-3H coupling networks make it challenging to perform correlation experiments using methods that rely on coherences created during free precession periods and interrupted by transfer pulses. Two alternative HETCOR experiments are demonstrated for 1H-3H systems in the present work and are shown to have advantages over earlier methods. The first experiment is known as hetero-TOCSY and correlates heteronuclear chemical shifts using J-cross polarization. This experiment achieves both homonuclear and heteronuclear mixing and connects the chemical shifts of all 1H and 3H nuclei in a coupling network. A second HETCOR experiment uses the heteronuclear Overhauser effect to obtain through-space correlations between nearby nuclei. The 1H-3H HETCOR experiments are phase sensitive and typically contain more correlations than other methods, which is beneficial for assignment purposes, while being sensitive enough to be applicable to routine analytical samples. The experiments were used to analyze 3H incorporation in sub-milligram quantities of 3H-labeled pharmaceutical derivatives with complex labeling schemes.

Synthesis and NMR Spectroscopic Characterization of Some Fluoro-2H-1-Benzopyran Derivatives

The synthesis of some new fluoro-2H-1-benzopyran derivatives utilizing a reaction between titanium phenolates and β-phenylcinnamaldehydes in toluene is reported. These compounds were characterized by NMR and UV/visible spectroscopy as well as mass spectrometry. In solution all the compounds are photochromic. Complete assignment of the 1H and 13C resonances was achieved by concerted application of homonuclear (gs-COSY), proton-detected (C, H) one-bond (gs-HMQC), and long-range (gs-HMBC) heteronuclear two-dimensional chemical shift correlation experiments using a 500 MHz NMR spectrometer equipped with a cryoplatform and a 5 mm cryoprobe. The mass spectra of the different compounds were characterized by intense molecular and high fragment ions. The introduction of an atom of fluorine as a molecular probe is of interest in determining the mechanistic aspects of the photochemical process.

Structural analysis of the O-antigen polysaccharide from Escherichia coli O152

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O152 has been determined. Component analysis together with 1H, 13C and 31P NMR spectroscopy were used to elucidate the structure. Inter-residue correlations were determined by 1H, 31P COSY, 1H, 1H NOESY and 1H, 13C heteronuclear multiple-bond correlation experiments. The PS is composed of pentasaccharide repeating units with the following structure: ▪ The structure is similar to that of the O-antigen polysaccharide from E. coli O173. The cross-reactivity between E. coli O152 and E. coli O3 may be explained by structural similarities in the branching region of their O-antigen polysaccharides.

Testosterone 1 beta-hydroxylation by human cytochrome P450 3A4.

Human cytochrome P450 3A4 forms a series of minor testosterone hydroxylation products in addition to 6 beta-hydroxytestosterone, the major product. One of these, formed at the next highest rate after the 6 beta- and 2 beta-hydroxy products, was identified as 1 beta-hydroxytestosterone. This product was characterized from a mixture of testosterone oxidation products using an HPLC-solid phase extraction-cryoprobe NMR/time-of-flight mass spectrometry system, with an estimated total of approximately 6 microg of this product. Mass spectrometry established the formula as C(19)H(29)O(3) (MH(+) 305.2080). The 1-position of the added hydroxyl group was established by correlated spectroscopy and heteronuclear spin quantum correlation experiments, and the beta-stereochemistry of the added hydroxyl group was assigned with a nuclear Overhauser correlated spectroscopy experiment (1 alpha-H). Of several human P450s examined, only P450 3A4 formed this product. The product was also formed in human liver microsomes.

Solid-state NMR characterization of pyrene-cuticular matter interactions.

One- and two-dimensional nuclear magnetic resonance (NMR) experiments were performed on Agave americana cutan and tomato cutin to examine the interactions between a hydrophobic pollutant, pyrene, and cuticular material. Variable-temperature NMR experiments show that cutan, an acid- and base-resistant cuticular biopolymer, undergoes the characteristic melting behavior of "polyethylene-like" crystallites, while the tomato cutin does not. The melting point of A. americana cutan was found to be approximately 360 K, which is consistent with the thickness of the polyethylene crystallites of 30-40 methylene units. Sorption models predict that the sorption behavior of hydrophobic pollutants should depend on the phase of the cuticular material. 13C NMR experiments on labeled pyrene were performed. The 13C T1 of pyrene decreases significantly from that of crystalline pyrene upon sorption to both tomato fruit cutin and A. americana cutan, indicating that the pyrene is mobile upon sorption. Magic angle spinning experiments at low spinning frequencies (2-4 kHz) provided the chemical shift anisotropy (CSA) parameters delta, the anisotropy, and eta, the asymmetry parameter, for crystalline and sorbed pyrene. For crystalline pyrene, two types of crystallographically distinctive pyrenes were observed. The first had delta = -97.4+/-0.5 ppm and eta = 0.934+/-0.006, while the second had delta = -98.1+/-0.5 ppm and eta = 0.823+/-0.008. After sorption to cutan, these CSA parameters were found to be delta = -78.9+/-5.3 ppm and eta < 0.70 independent of the length of time since completion of the sorption procedure. In tomato cutin, the CSA parameters were found to be dependent upon the time since completion of the sorption procedure. One and one-half months after sorption, delta was found to have a value of -30.4 ppm < delta < 0.0 ppm and eta was undeterminable, while after 22 months these values become delta = -80.0 +/-3.3 ppm and eta< 0.42. These changes in the CSA parameters demonstrate that upon sorption of pyrene to cutan, the pyrene undergoes anisotropic motion, while in cutin pyrene initially can tumble isotropically, but after 22 months this motion also becomes anisotropic. 2D heteronuclear correlation experiments indicate that pyrene is in close proximity to aliphatic cuticular materials after sorption. This work is directly relevant toward understanding the physical and chemical mechanisms of pollutant sorption to soil organic matter and, thus, help develop improved sorption models and pollution remediation techniques.

19F and 13C NMR Signal Assignment and Analysis in a Perfluorinated Ionomer (Nafion) by Two-Dimensional Solid-State NMR

The 19F and 13C NMR resonances of the perfluorinated ionomer, Nafion, are assigned to their corresponding chemical groups using two-dimensional (2D) 13C−19F heteronuclear correlation and 19F-exchange NMR experiments under 28 or 30 kHz magic-angle spinning, combined with peak area and relaxation time information. On the basis of these new experimental data, we revise the assignment of more than half of the resolved 19F NMR peaks. In particular, the backbone CF group is shown to resonate at −138 ppm, the side-group CF at −144 ppm, and the SCF2 group, which can be selected by a T2 filter, at −117 ppm. The OCF2 groups resonate slightly downfield from the CF3 fluorines. Deconvolution of the 19F and 13C spectra based on cross sections from the 2D spectra provides the peak widths and positions of all side group and several backbone sites. The inhomogeneous broadening observed in both 13C and 19F NMR spectra for the sites near the backbone CF group reveals static disorder near the branch point, which contrasts with the high conformational order of the rest of the backbone and the mobility of the ends of the side group.

NMR analysis of surfaces and interfaces in 2-nm CdSe.

Solid-state NMR analysis on wurtzite 2-nm hexadecylamine-capped CdSe nanocrystals (CdSe-HDA) provides evidence of discrete nanoparticle reconstruction within the Se sublattice of the nanomaterial. The cadmium and selenium atoms are probed with (1)H-(113)Cd and (1)H-(77)Se cross-polarization magic angle spinning (MAS) experiments, which demonstrate five ordered selenium sites in the nanoparticle that can be assigned to contributions arising from different surface sites and a selenium site one layer down from the surface. Intriguingly, in these materials both HDA and thiophenol are observed to selectively bind to specific sites on the nanoparticle surface. 2D heteronuclear chemical shift correlation (HETCOR) experiments provide evidence for thiophenol selectively binding at surface vacancies. Analysis of the NMR provides a model of a 2-nm CdSe-HDA molecular surface.

Diterpenoids from Calceolaria inamoena

Four 9- epi- ent-labdanes were isolated from the aerial parts of Calceolaria inamoena. Their structures, 2β-hydroxy-9- epi-ent-labda8(17)-13( E)dien-15-oic acid, 2β-hydroxy-9- epi-ent-labda8(17)-13( Z)dien-15-oic acid, 2β-hydroxy-9- epi-ent-labda8(17)-13( E)dien-15-al and 2β-hydroxy-9- epi-ent-labda-8(17)-13( Z)dien-15-al, were established by spectroscopic methods including by analysis of 2 dimensional heteronuclear correlation experiments 1H/ 13C (normal and long range), and NOE and gs-sel-1D-NOESY and TOCSY of their methyl ester or acetyl derivatives.

Poly(n-butyl acrylate-co-carbon monoxide-co-ethylene) Characterization by High-Temperature Two-Dimensional NMR at 750 MHz

Poly(n-butyl acrylate-co-carbon monoxide-co-ethylene) samples with varying monomer compositions and degrees of branching (g‘) were characterized using 750 MHz two-dimensional (2D) pulsed-field gradient (PFG) NMR spectroscopy. The NMR experiments were performed in solution at 100 °C to ensure sample homogeneity and sufficient mobility of the ethylene-based terpolymer. Since the 1H and 13C NMR spectra of the terpolymer samples were complex, 2D-1H/13C-heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond correlation (HMBC) experiments were conducted. The distinctive resonances of the ketone and ester carbonyl groups facilitated spectral assignments, such that chain-end resonances were apparent and the effects of short-chain-branching reactions and monomer sequence distributions were readily observed.

A comprehensive discussion of HSQC and HMQC pulse sequences

AbstractMuch of the progress in NMR applications over the last decade has stemmed from the development of proton‐detected, heteronuclear multidimensional spectroscopy. The basic building blocks of the multitude of double and triple resonance experiments available today are the heteronuclear multiple quantum correlation (HMQC) and heteronuclear single quantum correlation (HSQC) experiments. The goal of this article is to provide a comprehensive, pedagogical description of these two pulse sequences, with the aid of product operator formalism. Building from basic principles, constant‐time experiments, sensitivity‐enhanced techniques and the role of pulsed‐field gradients for coherence transfer pathway selection are explained in detail. This article is intended to be used as a primer for students and researchers seeking a firm theoretical understanding of the fundamental tools of modern NMR pulse sequences. © 2004 Wiley Periodicals, Inc. Concepts Magn Reson 20A: 1–23, 2004

Characterization of n-butyl acrylate centered triads in poly(n-butyl acrylate-co-carbon monoxide-co-ethylene) by isotopic labeling and two dimensional NMR.

Poly(n-butylacrylate-co-carbon monoxide-co-ethylene) (polyEBC) samples prepared from 13C-labeled monomer, n-butyl acrylate, were characterized using two dimensional (2D) pulsed field gradient (PFG) 750 MHz NMR spectroscopy. To elucidate the complex structure of the terpolymer, 2D-1H/13C-heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) experiments were conducted by selectively exciting the enhanced resonances in the spectra of two polymer samples, one polymer resulting from synthesis with 1-13C-n-butylacrylate monomer and a second polymer obtained from a synthesis with 2-13C-n-butylacrylate monomer. High-resolution 2D-NMR combined with 13C-labeling of the polymer greatly simplifies the 2D-NMR spectra, selectively enhances the weak peaks from low occurrence B-centered triad structures, and aids in their resonance assignments. In all experiments, the sample temperature was 120 degrees C, to ensure a homogeneous solution and sufficient molecular mobility. Supplementary material (1D 13C NMR spectra of the 13C-labeled and unlabeled polymers) is available in the online version of this article at http://dx.doi.org/100.1007/s00216-003-2402-3.

Insecticidal sesquiterpene pyridine alkaloids from Maytenus chiapensis.

The new sesquiterpene pyridine alkaloids chiapenines ES-I (1), ES-II (2), ES-III (3), and ES-IV (4), in addition to the known alkaloids wilfordine (5), alatamine (6), wilforidine (7), alatusinine (8), euonine (9), euonymine (10), ebenifoline E-I (11), forrestine (12), mayteine (13), and 4-hydroxy-7-epi-chuchuhuanine E-V (14), were isolated from the leaves of Maytenus chiapensis. Their structures were elucidated by 1D and 2D NMR spectroscopy, including homonuclear and heteronuclear correlation (COSY, ROESY, HSQC, and HMBC) experiments. Wilfordine, alatusinine, and euonine exhibited strong antifeedant activity against Spodoptera littoralis.