Rotating Frame Nuclear Overhauser Enhancement Spectroscopy Research Articles

NMR Spectroscopic Analysis on the Chiral Recognition of Noradrenaline by β-Cyclodextrin ( β-CD) and Carboxymethyl- β-cyclodextrin (CM- β-CD)

<TEX>${\beta}$</TEX>-CD and CM- <TEX>${\beta}$</TEX>-CD as chiral NMR shift agents were used to resolve the enantiomers of noradrenaline (NA). The stoichiometry of each complex formed between the CDs and the enantiomers of NA was found to be 1 : 1 through the continuous variation plots. The binding constants (K) of the complexes were determined from <TEX>$^1H$</TEX> NMR titration curves. This result indicated that both <TEX>${\beta}$</TEX>-CD and CM- <TEX>${\beta}$</TEX>-CD formed the complexes with the S(+)-NA more preferentially than its R(-)-enantiomer. The K values for the complexes with <TEX>${\beta}$</TEX>-CD (<TEX>$K_{S(+)}$</TEX> = 537 <TEX>$M^{-1}$</TEX> and <TEX>$K_{R(-)}$</TEX> = 516 <TEX>$M^{-1}$</TEX> was larger than those with CM- <TEX>${\beta}$</TEX>-CD (<TEX>$K_{S(+)}$</TEX> = 435 <TEX>$M^{-1}$</TEX> and <TEX>$K_{R(-)}$</TEX> = 313 <TEX>$M^{-1}$</TEX>), however, enantioselectivity (<TEX>${\alpha}$</TEX>) of S(+)- and R(-)-NA to CM- <TEX>${\beta}$</TEX>-CD ( <TEX>${\alpha}$</TEX> = 1.38) was larger than that to <TEX>${\beta}$</TEX>-CD ( <TEX>${\alpha}$</TEX> = 1.04), indicating that CM- <TEX>${\beta}$</TEX>-CD was the better chiral NMR solvating agents for the recognition of the enantiomers of NA. Two dimensional rotating frame nuclear Overhauser enhancement spectroscopy (ROESY) experiments were also performed to explain the binding properties in terms of spatial fitting of the NA molecule into the macrocyclic cavities.

Retarding effects of cyclodextrins on the decomposition of organic isothiocyanates in an aqueous solution.

One- and two-dimensional (1)H nuclear magnetic resonance ((1)H NMR) spectra were measured in order to estimate the dissociation constants (K(diss)) and molecular geometries of cyclodextrin (CD) with three organic isothiocyanates (ITC), allyl-ITC, 3-butenyl-ITC, and 4-pentenyl-ITC, in an aqueous solution (pH 5.0, I(c)=0.75 M). In every ITC, the K(diss) values decreased in the order of alpha-CD>beta-CD>gamma-CD, and the three rod-like linear molecules of ITCs were accommodated with the best fit into the smallest cavity of alpha-CD. By rotating-frame nuclear Overhauser enhancement spectroscopy, cross peaks were found between the H-3 of alpha-CD and the H-1 of AITC. From the result, the molecular geometry of alpha-CD-AITC complex presume that the isothiocyanate group of AITC is located some where around the widening rim and a hydrophobic allyl group is included within the hydrophobic cavity of alpha-CD.

Transferred nuclear Overhauser enhancement (NOE) and rotating-frame NOE experiments reflect the size of the bound segment of the Forssman pentasaccharide in the binding site of Dolichos biflorus lectin.

A complex between the Forssman pentasaccharide alpha-D-GalNAc-(1-->3)-beta-D-GalNAc-(1-->3)-alpha-D-Gal-(1-->4)-beta-D- Gal-(1-->4)-D-Glc and the seed lectin from Dolichos biflorus was studied using transfer-NOESY and transfer rotating frame NOE spectroscopy (ROESY) experiments. The evolution of transferred NOEs and ROEs as a function of the pentasaccharide/lectin ratio was different for the non-reducing disaccharide moiety alpha-D-GalNAc-(1-->3)-beta-D-GalNac compared to the rest of the molecule, which reflects distinct relaxation properties and effects of exchange broadening of the corresponding ligand resonances. Significantly, several intermolecular transferred NOEs were observed between protons of the nonreducing disaccharide moiety alpha-D-GalNAc-(1-->3)-beta-D-GalNAc and aliphatic as well as aromatic amino acid side chain protons in the binding pocket of the lectin. It is concluded that the non-reducing disaccharide fragment is buried in the lectin-binding pocket, whereas the reducing trisaccharide portion alpha-D-Gal-(1-->4)-beta-D-Gal-(1-->4)-D-Glc has no immediate contacts with the protein. The experimental transfer NOE data were qualitatively compared to theoretical proton-proton distances from a model that was based on a previous homology modeling study of a complex between the disaccharide fragment alpha-D-GalNAc-(1-->3)-beta-D-GalNAc and D. biflorus lectin. It was found that all intermolecular transferred NOEs matched short interatomic distances between ligand protons and aliphatic or aromatic amino acid side chain protons predicted by the theoretical model.

Isolation and charaterization of hexasaccharides derived from heparin. Analysis by HPLC and elucidation of structure by 1H NMR

Four hexasaccharides representing major structural sequences of heparin were isolated and characterized after degradation of heparin by heparinase. The structures were determined from two-dimensional 1H NMR spectroscopy including TOCSY (total correlated spectroscopy), COSY (correlated spectroscopy), and ROESY (rotating frame nuclear Overhauser enhancement spectroscopy) methods, providing new data on hexasaccharides. One of the hexasaccharides, the last eluting component from anion exchange chromatography, was derived from the tri-sulfated repeating disaccharide, α- l-idopyranosyluronic acid 2- sulfate-(1 → 4)-2- amino-2-deoxy- d-glucopyranose 6,N-disulfate, and having the structure δ UA p2 S-(1 → 4)-α- d-GlcN p2 S6S-(1 → 4)-α- l-IdoA p2 S-(1 → 4)-α- d-GlcN p2 S6S-(1 → 4)-α- l-IdoA p2 S-(1 → 4)-α- d-GlcN p2 S6S . The second hexasaccharide contained a nonsulfated d-glucuronic acid unit instead of the l-iduronic acid adjacent to the reducing end, and having the structure δUA p2 S-(1 → 4)-α- d-GlcN p2 S6S-(1 → 4)-α- l-IdoA p2 S-(1 → 4)-α- d-GlcN p2 S6S-(1 → 4)-β- d-GlcA p-(1 → 4)-α- d-GlcN p2 S6S . The last two hexasaccharides were obtained in lower yield and they have not been isolated and characterized before. The structure of the third saccharide corresponded to a trimer of the repeating disaccharide except for the lack of a 6- O-sulfate group at the reducing end glucosamine residue; δ UA p2 S-(1 → 4)-α- d-GlcN p2 S6S-(1 → 4)-α- l-IdoA p2 S-(1 → 4)-α- d-GlcN p2 S6S-(1

Assignment of the 1H‐nmr resonances of the four rotamers of β‐casomorphin‐5 in DMSO

We report the complete assignment of the 1H-nmr spectrum of beta-casomorphin-5 in DMSO-d6 solution. With a combination of one-dimensional, double quantum filtered correlated spectroscopy, homonuclear Hartmann-Hahn, and rotating frame nuclear Overhauser enhancement spectroscopy (ROESY) spectra, we were able to differentiate the four conformers originating from two Xxx-Pro bonds present in the sequence. Exchange peaks in the ROESY spectra confirmed the presence of four interchanging conformational isomers. Based on integrations, the relative populations of the four species were estimated, while characteristic sequential nuclear Overhauser enhancements (NOEs) were used to determine the orientation of the Xxx-Pro bonds. This orientation was also shown to correlate with the chemical shift changes for the alpha protons of both the Xxx and Pro residues. Finally, interresidue NOEs indicate conformational preferences for the aromatic side chains, especially in the all-trans conformer.

The determination of the conformational properties of nucleic acids in solution from NMR data

A program, NUCFIT, has been written for simulating the effects of conformational averaging on nuclear Overhauser enhancement (NOE) intensities for the spin systems found in nucleic acids. Arbitrary structures can be generated, and the NOE time courses can be calculated for truncated one-dimensional NOEs, two-dimensional NOE and rotating frame NOE spectroscopy (NOESY and ROESY) experiments. Both isotropic and anisotropic molecular rotation can be treated, using Woessner's formalism (J. Chem. Phys. (1962) 37, 647–654). The effects of slow conformational averaging are simulated by taking population-weighted means of the conformations present. Rapid motions are allowed for by using order parameters which can be supplied by the user, or calculated for specific motional models using the formalism of Tropp (J. Chem. Phys. (1980) 72, 6035–6043). NOE time courses have been simulated for a wide variety of conformations and used to determine the quality of structure determinations using NMR data for nucleic acids. The program also allows grid-searching with least-squares fitting of structures to experimental data, including the effects of spin-diffusion, conformational averaging and rapid internal motions. The effects of variation of intra and internucleotide conformational parameters on NOE intensities has been systematically explored. It is found that (i) the conformation of nucleotides is well determined by realistic NOE data sets, (ii) some of the helical parameters, particularly the base pair roll, are poorly determined even for extensive, noise-free data sets, (iii) conformational averaging of the sugars by pseudorotation has at most second-order influence on the determination of other parameters and (iv) averaging about the glycosidic torsion bond also has, in most cases, an insignificant effect on the determination of the conformation of nucleotides.

Structural variation in the antithrombin III binding site region and its occurrence in heparin from different sources

A tetrasaccharide possessing a biosynthetically permissible structural variability in and adjacent to the antithrombin III (ATIII) binding site has been isolated from heparin lyase depolymerized bovine lung heparin by using strong anion-exchange high-pressure liquid chromatography (SAX-HPLC). On the basis of two-dimensional 500-MHz 1H NMR experiments, including phase-sensitive correlated spectroscopy (COSY) and rotating frame nuclear Overhauser enhancement spectroscopy (ROESY), and fast-atom bombardment mass spectrometry (FAB-MS), the primary structure of this tetrasaccharide was unambiguously established as delta UAp2S (1----4)-alpha-D-GlcNp2S6S(1----4)-beta-D-GlcAp(1----4)-alph a-D-GlcNp2S3S6S (where delta UA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid). The 1H NMR ROESY experiment proved to be particularly valuable in offering sequence information. Heparins from a variety of species and tissue sources were examined by oligosaccharide mapping using SAX-HPLC and gradient polyacrylamide gel electrophoresis. Two of these heparins are used as anticoagulants; they are porcine intestinal mucosal heparin and bovine lung heparin. The predominant ATIII-binding site in porcine heparin contained an N-acetylated glucosamine residue. We now report the structure of the predominant ATIII-binding site in bovine heparin as----4)-alpha-D-GlcNp2S6S(1----4)-beta-D-GlcAp(1----4)-alph a-D- GlcNp2S3S6S(1----4)-alpha-L-IdoAp2S(1----4)-alpha-D-GlcNp 2S6S(1----. This study shows the presence of one or both types of ATIII-binding-site variants in all of the heparins that were examined.

Rotating-frame nuclear overhauser enhancement spectroscopy of aqueous solutions with elimination of the water resonance by transverse relaxation

We report here that the water resonance can be completely and selectively eliminated from ROESY spectra by the WATR (water attenuation by transverse relaxation) method. Elimination of the water resonance by the WATR method is achieved by selectively decreasing the transverse relaxation time of the water protons by addition of a water proton exchange reagent, and then measuring the spectrum with a delay for transverse relaxation prior to acquisition of the free induction decay

Sequencing of peracetylated oligosaccharides by rotating-frame nuclear Overhauser enhancement spectroscopy

A combination of two-dimensional total correlation spectroscopy (TOCSY) and rotating-frame nuclear Overhauser enhancement spectroscopy (ROESY) is suggested as the optimum strategy for determining the primary structure of peracetylated oligosaccharides.

Sequence determination in oligosaccharides by relayed NOE experiments in the rotating frame

An hybrid experiment, composed of 1H-NMR total correlation spectroscopy (TOCSY) and rotating frame nuclear Overhauser enhancement spectroscopy (ROESY) steps, makes it possible to determine both the partial (disaccharide) sequences, and the sequential order, of whole linear and branched oligosaccharide chains.

Conformational analysis of cyclic peptides in solution.

The strategy and tactics of conformational analysis of cyclic peptides in solution is demonstrated by the example of cyclo(-D-Pro-Phe-Thr-Phe-Trp-Phe-). Spin-locked experiments like rotating frame nuclear Overhauser enhancement spectroscopy (ROESY), ROTO, and TOCSY are successfully applied to assign all proton signals and to obtain distance information. A crude conformational model was built using the nmr data. This starting model was refined by restrained molecular dynamics (MD) calculations using ROE derived distances and fixed bond angles as determined from homo- and heteronuclear coupling constants. To mimic the solvent and to reduce artifacts in an in vacuo calculation the charges of the solvent-exposed NH protons were gradually reduced according to the temperature gradients. The thus obtained "conformation" (mean of a 40 ps MD trajectory) shows very close similarity to x-ray structures in an orthorhombic and in two monoclinic crystal modifications of the same compound. The main difference is the breaking of an intermolecular hydrogen bond of the threonine hydroxyl group on dissolution of the crystal and forming an intramolecular hydrogen bond in solution.

Scope and limitations of rotating-frame nuclear overhauser enhancement spectroscopy applied to oligosaccharides

Two-dimensional rotating-frame nuclear Overhauser enhancement spectroscopy has been applied to milk oligosaccharides containing the blood-group determinants Le a, Le d, or Le b. The results indicate a large number of dipolar interactions that are consistent with the preferred conformations described for these blood-group determinants. Despite careful experimental conditions, coherent transfer processes are not suppressed completely, allowing only a qualitative evaluation of the nuclear Overhauser data. The 1H- and 13C-n.m.r. spectra are assigned by comparison with literature data and by application of two-dimensional 13C- 1H shift-correlation spectroscopy.