Two-dimensional Heteronuclear Correlation Research Articles

Crystal state and solution conformation of the B blood group trisaccharide alpha- l-Fucp-(12)-[alpha- d-Galp]-(13)]-beta- d-Galp-OCH3

The crystal structure of the human B blood group related trisaccharide alpha-L-Fucp-(1-->2)-[alpha-D-Galp]-(1-->3)-beta-D-Galp-OCH3 (1) has been determined. The solution structure of 1 was studied by two-dimensional NMR techniques at 600 MHz in D2O solution and the conformational properties were analyzed in terms of the torsional angles phiH and psiH, derived from 3JCH coupling constants, and 10 inter-residue proton-proton distances. 3JCH could be accurately measured by a recently introduced two-dimensional heteronuclear correlation experiment (EXSIDE). The nuclear Overhauser enhancement-derived distances and the calculated torsion angles were compared with the same information available from the crystal structure. The agreement is excellent, indicating that the trisaccharide adopts a restricted conformation in solution, which was also predicted by the Hard Sphere Exo-Anomeric forcefield. The data of 1 are complemented by NMR studies of the closely related alpha-L-Fucp-(1-->2)-[6-deoxy-alpha-D-Galp]-(1-->3)-beta-D-Galp O-(CH2 )7CH3 trisaccharide (2).

Determination of Coupling Signs 1J(119Sn, 15N ) and 2J(119Sn, 117Sn) in Trimethylstannylamines

The l5N-labelled trimethylstannylamines 1 - 3 [(Me3Sn)3N, (Me3Sn)2NPh, (Me3Sn)2NBCgHi4 (BCgHi4 = 9-borabicyclo[3.3.0]nonyn and the non-labelled 4, Me3Sn-N(BC8Hi4)2 , were prepared and studied by 1H, 1C, 15N and 119Sn NMR. The 15N ultrahigh resolution NMR spectra of 1 revealed otherwise unobserved parameters such as 2J(15N ,Sn ,13C ) and the isotope induced chemical shift 2Δ12/13C(15N). 119Sn NMR spectra of 1, recorded under similar conditions, also show new parameters such as 3J(119Sn,N,Sn,13C) which are not resolved in the non-labelled derivative. By using various types o f two-dimensional heteronuclear shift correlations, absolute coupling signs of 1J(119Sn,15N) (all < 0) in 1 - 3 were determined. By the same techniques it proved possible to confirm the negative sign o f :7(Sn,Sn) (-195.4 Hz) in 1. In contrast, the coupling constants 2J(Sn,Sn) for 2 (+71.7) and 3 (+62.0) possess a positive sign. This sign inversion, observed here for the first time for apparently similar compounds, demonstrates the enormous influence of substituents on the nature of the lone pair of electrons at the nitrogen atom. It also shows that these experiments for sign determinations of coupling constants are necessary in order to interpret these data correctly.

Structural Details of Aqueous Attack on a Phosphate Glass by 1H/31P Cross-Polarization NMR

One- and two-dimensional heteronuclear correlation solid-state nuclear magnetic resonance experiments allow a direct study of the chemistry of water incorporation into a simple phosphate glass network. One main pathway for water incorporation into phosphate glass networks is through the formation of P−OH groups, and previous 1H magic-angle-spinning NMR investigations of proton environments in silicate and phosphate glasses suggest that hydroxyl groups are involved in multiple hydrogen-bonding motifs with backbone units within the glass networks. The presence of multiple proton environments presents more complex structural issues for aqueous incorporation into glass systems, which have yet to be explained unambiguously. The spectral editing capabilities of two-dimensional cross-polarization NMR identify specific subunits in modified sodium phosphate glasses, and strong evidence is presented for the formation of intramolecular as well as weaker intermolecular hydrogen bonds. Using a combination of one- and ...

Cation sites in mixed-alkali phosphate glasses

Two lithium/sodium mixed-alkali phosphate glasses were investigated with solid-state two-dimensional heteronuclear correlation NMR. The systems represent two distinct families of mixed-alkali glasses, as they contain a fixed sodium-to-lithium ratio of 1:3, but different overall concentrations of cations. In both glasses, direct NMR evidence is found for the presence of multiple sodium cation environments using frequency-selective editing of two-dimensional correlation spectra. Frequency-specific subspectra were obtained by projection of 23Na resonances correlated to distinct phosphate tetrahedral environments. Dynamic-angle-spinning (DAS) NMR provides for high-resolution subspectra of quadrupolar 23Na nuclei allowing spectral separation of multiple distinct sodium environments. Identification of distinct cation sites is important for understanding the physical origin of the `mixed-alkali effect', where non-linear behavior of physical properties is observed in glasses as a function of relative cation populations.

Two-dimensional 1H and 15N NMR titration studies of hisactophilin

We have used two-dimensional 1H-15N heteronuclear single quantum correlation spectroscopy to measure the pH dependence of backbone amide group chemical shifts in the actin binding protein hisactophilin over the pH range 5.7-11.1. Most of the resonances can be analyzed using a simple equation involving a single apparent ionization constant, pKapp. The majority of resonances in the protein titrate with pKapp values of 5.6-7.4. The results can be rationalized in terms of titration of many histidine residues in hisactophilin. The titration data provide direct experimental support for the proposed models of the atomic basis of actin and membrane binding by hisactophilin.Key words: hisactophilin, histidine, ionization constants, nuclear magnetic resonance, NMR.

Accurate and Precise Measurement of Heteronuclear Long-Range Couplings by a Gradient-Enhanced Two-Dimensional Multiple-Bond Correlation Experiment

We propose a phase-sensitive gradient-enhanced two-dimensional heteronuclear multiple-bond correlation (psge-2D HMBC) experiment for speedy, accurate, and precise measurement of2JCHand3JCH. The experiment does not suppress one-bond correlations. Rather, the value of a desired long-rangeJCHis obtained from the pertinent cross-peak pattern in the HMBC spectrum, using the corresponding1JCHcorrelation pattern as reference. The application of the proposed experiment is illustrated for the trisaccharide raffinose.

1D and 2D solid state NMR investigations of the framework structure of As-synthesized AlPO 4-14

The framework structure of As-synthesized A1PO 4-14 has been investigated with a combination of different one-dimensional 27Al and 31P solid state NMR techniques and 27Al 31P double resonance methods. The results are found to be fully consistent with the assumed structural model. 27Al MAS and DOR experiments at three different magnetic field strengths together with simulations show the presence of two tetrahedral sites, one pentacoordinated and one octahedral aluminum site. The 27Al quadrupolar coupling constants and the 31P isotropic chemical shifts of the tetrahedral sites correlate well with tetrahedral shear-strain parameters and mean P-O-A1 bond angles, respectively. These correlations allow one to assign all of the NMR resonances to specific T-sites in the proposed framework structure. The assignments are then further confirmed by the application of three different two-dimensional heteronuclear correlation methods (i.e., 27Al → 31P TEDOR, CP, and INEPT) which reveal the connectivities between AlO x and PO 4 polyhedra. The two-dimensional INEPT experiment is applied here for the first time in the solid state.

New Multinuclear Experiments on Solid Organotin Fluorides

nBu3SnF and Mes3SnF (Mes = mesityl) are organotin fluorides that have direct Sn–F bonds. The119Sn and19F NMR spectra of these compounds have been measured using a selection of CPMAS probes. Doubly decoupled {1H,19F} as well as singly decoupled {1H}119Sn NMR spectra are shown. Double decoupling gives higher resolution spectra and allows the sample of Mes3SnF to be spun slower to give more spinning-sidebands without overlapping of signals, giving improved analysis. The doubly decoupled spectra are sensitive to the power and offset of the decouplers. The effect of off-resonance decoupling has been observed for119Sn NMR through reduced splittings when the19F decoupler power and offset were varied. Proton-decoupled19F spectra have been measured using a new HF double-resonance probe. In addition, two-dimensional heteronuclear correlation spectra are presented for Mes3SnF, allowing a full assignment for the19F and119Sn chemical shifts of the two sites. Full shielding tensor data are reported for both compounds. Values have been derived for the effective (119Sn,19F) dipolar coupling constants from both119Sn and19F experiments, yielding estimates of coupling anisotropy.

Flavans and A-type proanthocyanidins from Prunus prostrata

A new natural dimeric A-type proanthocyanidin and two new monomeric flavanols have been isolated from Prunus prostrata and identified as ent-epiafzelechin (2 α → O → 7, 4 α → 8) quercetin, 5,7,2′,5′-tetrahydroxy-(2 R,3 R)- flavan-3-ol and, 7-methoxy-(+)-catechin, respectively. The structurally related metabolites ent-epiafzelechin (2 α → O → 7, 4 α → 8) afzelechin, ent-epiafzelechin (2 α → O → 7, 4 α → 8) kaempferol, ent-epiafzelechin (2 α → O → 7, 4 α → 8) epicatechin, ent-epiafzelechin (2 α → O → 7, 4 α → 8) epiafzelechin, ent-epiafzelechin (2 α → O → 7, 4 α → 8) catechin, (+)-catechin and (−)-epicatechin were also isolated. Structure elucidation was achieved by extensive NMR (homo- and hetero-nuclear two-dimensional correlation, NOE difference, and selective INEPT experiments) and CD studies.

Characterization of a buried neutral histidine residue in Bacillus circulans xylanase: NMR assignments, pH titration, and hydrogen exchange.

Bacillus circulans xylanase contains two histidines, one of which (His 156) is solvent exposed, whereas the other (His 149) is buried within its hydrophobic core. His 149 is involved in a network of hydrogen bonds with an internal water and Ser 130, as well as a potential weak aromatic-aromatic interaction with Tyr 105. These three residues, and their network of interactions with the bound water, are conserved in four homologous xylanases. To probe the structural role played by His 149, NMR spectroscopy was used to characterize the histidines in BCX. Complete assignments of the 1H, 13C, and 15N resonances and tautomeric forms of the imidazole rings were obtained from two-dimensional heteronuclear correlation experiments. An unusual spectroscopic feature of BCX is a peak near 12 ppm arising from the nitrogen bonded 1H epsilon 2 of His 149. Due to its solvent inaccessibility and hydrogen bonding to an internal water molecule, the exchange rate of this proton (4.0 x 10(-5) s-1 at pH*7.04 and 30 degrees C) is retarded by > 10(6)-fold relative to an exposed histidine. The pKa of His 156 is unperturbed at approximately 6.5, as measured from the pH dependence of the 15N- and 1H-NMR spectra of BCX. In contrast, His 149 has a pKa < 2.3, existing in the neutral N epsilon 2H tautomeric state under all conditions examined. BCX unfolds at low pH and 30 degrees C, and thus His 149 is never protonated significantly in the context of the native enzyme. The structural importance of this buried histidine is confirmed by the destablizing effect of substituting a phenylalanine or glutamine at position 149 in BCX.

Proton-fluorine wideline separation experiments on poly(vinylidene difluoride)

Two-dimensional heteronuclear proton-fluorine correlation solid-state spectroscopy is demonstrated on a sample of poly(vinylidene difluoride) (PVDF). This allows the correlation of structural information contained in the fluorine chemical shift in one dimension with information on mobility from proton wideline spectra in the second dimension. The effect of the heteronuclear fluorine-proton dipolar coupling on the proton linewidth is investigated separately. The incorporation of magnetization filters into the preparation period enables one to measure the wideline separation (WISE) spectra for the amorphous or the crystalline parts of the sample separately.

Analysis of the1H and13C NMR Spectra of 5-(Endo)-isopropylsulfonyl-5-(exo)-phenyl-2-norbornene-7-spiro-cyclopentane and Di[cyclopentane -spiro-cyclopentadiene] Through the Application of Two-Dimensional NMR Techniques

Abstract Analysis of 13C and H NMR spectra of cycloadducts 1 and 2 was achieved using a concerted application of two-dimensional homonuclear and heteronuclear chemical shift correlations.

Structural Analysis of the Mannan Region of Lipoarabinomannan from Mycobacterium bovis BCG.: HETEROGENEITY IN PHOSPHORYLATION STATE

Lipoarabinomannan (LAM) is a major antigen of mycobacterial cell walls, involved in host-Mycobacterium interactions. In a previous work, LAM from the vaccine strain, Mycobacterium bovis BCG, was found to exhibit mannooligosaccharides at its arabinan nonreducing ends (ManLAM). The present report concerns the mannan core structure of this ManLAM. After partial hydrolysis of ManLAM, two populations of mannans (Ma1 and Ma2) were obtained by gel filtration chromatography. Their structural features were defined by means of two-dimensional homo- and heteronuclear (1H-13C) NMR sequences and methylation analysis. They were both found to be composed of an alpha-(1-->6)-linked mannan backbone with alpha-(1-->2)-Manp-linked side chains. They are highly branched, and Ma2 presents a higher frequency of branching than Ma1. Moreover, chemical analysis indicates that only Ma1 is phosphorylated. By a two-dimensional heteronuclear 1H-31P total correlation experiment, the phosphate was found to be involved in a phosphodiester bond between inositol C-1 and glycerol C-3. Then, the molecular mass of mannan was established by mass spectrometry, which revealed a molecular mass of 3517 Da for the major molecular species of Ma1. Likewise, analysis of unfractionated mannans showed the occurrence of other, quantitatively minor molecular species, endowed with two phosphates. This study clearly indicates that the mannan region of M. bovis BCG ManLAM exists as a heterogeneous population of molecules whose structures differ in their degree of glycosylation, level of branching, and phosphorylation state. The hypothesis that the relative abundance of these different molecules modulates the biological functions of LAM is discussed.

Reassignment of 13C NMR peaks of SDS micellar system by HMQC-NMR spectroscopy

The 13C NMR chemical shifts of sodium dodecyl sulphate (SDS) were measured at high concentrations by two-dimensional heteronuclear multiple quantum correlation (HMQC) spectroscopy to give accurate assignments for various signals obtained in the 13C NMR spectra of an SDS aggregated system.

Estimation of31P–1H and1H–1H vicinal coupling constants along the DNA backbone by 2D HELCO measurements

An accurate method for simultaneous estimation of 31P–1H and 1H–1H vicinal coupling constants along the backbone of DNA fragments is described using the two-dimensional heteronuclear long-range correlation (2D HELCO) experiment.

An NMR and conformational investigation of the trans-syn cyclobutane photodimers of dTpdU.

Two trans-syn cyclobutane photodimers of thymidylyl (3'-5') deoxyuridine were formed by deamination of the corresponding trans-syn cyclobutane photodimers of thymidylyl (3'-5') deoxycytidine and were examined by 1H-, 13C-, and 31P-nmr spectroscopy. Correlation spectroscopy, nuclear Overhauser enhancement spectroscopy, and one-dimensional heterodecoupling experiments allowed a more complete assignment of the 1H spectra, compared with previous reports by Koning et al. [(1991) European Journal of Biochemistry, Vol. 195, pp. 29-40] and Liu and Yang [(1978) Biochemistry, Vol. 17, pp. 4865-4876]. Deoxyribose ring conformations were calculated from 1H coupling constants by pseudorotational analysis, and rotamer distributions of exocyclic bonds were calculated from the observed homonuclear and heteronuclear coupling constants. The cyclobutane ring configuration (CB) of each isomer was identified, using arguments based upon observed scalar and dipolar couplings. Glycosidic bond conformation was ascertained from nuclear Overhauser enhancements observed between base and deoxyribose protons. Isomer I (S-type class; CB-; SYN-ANTI) and isomer II (N-type class; CB+; ANTI-SYN) exhibit markedly different conformational features. 31P chemical shifts and exocyclic bond rotamer distributions indicate diminished backbone flexibility for both photoproducts relative to parent thymidylyl (3'-5') deoxyuridine. Isomer I (SYN-ANTI) is particularly rigid, while isomer II (ANTI-SYN) maintains some flexibility. Also, 13C spectra were acquired and assigned unequivocally with the aid of short- and long-range two-dimensional heteronuclear shift correlation experiments.

Complete Analysis of the1H and13C NMR Spectra of 5-Isopropylsulfonyl-2-Norbornenes Through the Application of Two-Dimensional NMR Techniques

Abstract Total assignment of 13C and 1H NMR spectra of the 5-isopropylsulfonyl-2-norbornenes 2 was achieved using the concerted application of two-dimensional homonuclear and heteronuclear chemical shift correlations. The stereochemistry of both the diastereoisomers endo 2a and exo 2b have been established using the magnitude of the proton coupling constants.

5117186 Method for increasing resolution in two-dimensional solid-state NMR heteronuclear correlation spectra

A new RF pulse sequence is applied to selected nuclei in the evolution period of a 2D NMR heteronuclear correlation experiment to more effectively suppress heteronuclear interactions. The new pulse sequence is designed to be effective with the existing BLEW-12 pulse sequence so that both homonuclear and heteronuclear interactions are suppressed. In addition, the new pulse sequence effectively suppresses homonuclear interactions so that it can be used with a variety of nuclear species.

An improved experiment for heteronuclear-correlation 2D NMR in Solids

Techniques have been proposed in the past (I, 2) for the measurement of twodimensional heteronuclear-correlation spectra of solids. However, they have not been widely used in practice, and seem to be capable of producing useful results only for a few, carefully selected materials. In this Communication an improved heteronuclear chemical-shit&on-elation experiment is introduced which can be applied to the analysis of ‘H13C and other heteronuclear correlations in a wide variety of crystalline and amorphous materials of practical interest, such as polymers, pharmaceutical products, and foods. The dominant interaction between proton and 13C nuclei in most solids is the direct heteronuclear dipolar coupling. Because this interaction depends entirely on the distance between the nuclei, regardless of chemical bonding, heteronuclear correlation by two-dimensional NMR provides a method for studying the geometry of molecules in the solid state. This includes both the stereochemistry of individual molecules and the positioning of adjacent molecules relative to each other. Another advantage of heteronuclear correlation is that it separates the proton resonances over the much larger 13C chemical-shift range. Therefore, the technique can provide well-resolved proton chemical-shift information for samples where it is impossible to resolve the proton resonances with any standard, one-dimensional spectroscopic technique. The complete experiment is illustrated in Fig. 1. It has the classic four-part structure (3) of preparation, evolution, mixing, and detection which is common to virtually all 2D NMR. In this case, the spin system is prepared by a single pulse, following which the protons are allowed to evolve in order to “label” them according to their chemical shifts. After the evolution period, a special mixing pulse sequence is applied to transfer polarization selectively from the protons to the carbon spins via the heteronuclear dipolar interaction. Finally, the 13C FID is acquired during cw proton decoupling. During the entire experiment, the sample is rotated about the magic angle in order to suppress broadening due to chemical-shift anisotropy. Phase cycling may be added to the basic experiment to suppress potential artifacts. The phase-cycling scheme used to obtain all data presented here is shown in Table 1. The 180” phase alternation of the initial pulse P is an implementation of the wellknown “spin-temperature alternation” method for eliminating pulse dead-time effects.

Sensitivity improvement in proton-detected two-dimensional heteronuclear correlation NMR spectroscopy

The sensitivity of proton-detected two-dimensional heteronuclear correlation NMR spectroscopy can be increased by as much as a factor of √2 relative to that of the conventional methods, for heteronuclei with a single attached proton. The enhanced sensitivity is obtained by refocusing and detecting two orthogonal in-phase proton magnetization components, rather than the single component recorded conventionally. The two magnetization components are deconvoluted to produce two pure-phase spectra that are added together to produce a spectrum with an enhanced signal-to-noise ratio. Methods for improving the sensitivity of the main classes of heteronuclear correlation experiments are presented and the effect of relaxation during the new experiments on the sensitivity enhancement is discussed. The new methods are demonstrated by comparing conventional and sensitivity-enhanced 1H- 15N heteronuclear correlation spectra of bovine pancreatic trypsin inhibitor at 15N natural abundance.