The importance of including local correlation times in the calculation of inter-proton distances from NMR measurements: ignoring local correlation times leads to significant errors in the conformational analysis of the Glc alpha1-2Glc alpha linkage by NMR spectroscopy

The experimental determination of oligosaccharide conformations has traditionally used cross-linkage 1H-1H NOE/ROEs. As relatively few NOEs are observed, to provide sufficient conformational constraints this method relies on: accurate quantification of NOE intensities (positive constraints); analysis of absent NOEs (negative constraints); and hence calculation of inter-proton distances using the two-spin approximation. We have compared the results obtained by using 1H 2D NOESY, ROESY and T-ROESY experiments at 500 and 700 MHz to determine the conformation of the terminal Glc alpha1-2Glc alpha linkage in a dodecasaccharide and a related tetrasaccharide. For the tetrasaccharide, the NOESY and ROESY spectra produced the same qualitative pattern of linkage cross-peaks but the quantitative pattern, the relative peak intensities, was different. For the dodecasaccharide, the NOESY and ROESY spectra at 500 MHz produced a different qualitative pattern of linkage cross-peaks, with fewer peaks in the NOESY spectrum. At 700 MHz, the NOESY and ROESY spectra of the dodecasaccharide produced the same qualitative pattern of peaks, but again the relative peak intensities were different. These differences are due to very significant differences in the local correlation times for different proton pairs across this glycosidic linkage. The local correlation time for each proton pair was measured using the ratio of the NOESY and T-ROESY cross-relaxation rates, leaving the NOESY and ROESY as independent data sets for calculating the inter-proton distances. The inter-proton distances calculated including the effects of differences in local correlation times give much more consistent results.     

3 beta-Hydroxysialic acid glycosides. I. Calcium-binding ability and chemical and enzymatic stabilities.

Methyl alpha- and beta-glycosides of N-acetylneuraminic acid (Neu5Ac) and N-acetyl-3 beta-hydroxyneuraminic acid (Neu5Ac beta 3OH) (1-4) were prepared to evaluate their calcium-binding ability. (Methyl alpha-glucopyranosidonyl) alpha- and beta-, and 4-methylumbelliferyl alpha-glycosides of Neu5Ac and Neu5Ac beta 3OH (5-10) were also synthesized for the comparison of chemical and enzymatic stabilities, respectively. Methyl beta-glycosides of Neu5Ac and Neu5Ac beta 3OH, 3 and 4, respectively, showed intense calcium-binding abilities, while no such ability was observed in the corresponding alpha-glycosides, 1 and 2. The Neu5Ac beta 3OH glycosides, 6, 8, and 10, showed much stronger resistance to acidic hydrolysis and sialidase digestion than the corresponding Neu5Ac glycosides, 5, 7, and 9.     

Conformational properties of the macrocyclic trichothecene mycotoxin verrucarin A in solution

Phase-sensitive nuclear Overhauser enhancement spectroscopy (NOESY) experiments, (3)J couplings and computational molecular modeling (MM2* and MMFF force fields) were employed to examine the conformational properties of verrucarin A in chloroform solutions. The MMFF force field calculations resulted in a family of 12 low-energy structures along with their populations, the latter being determined by the NMR analysis of molecular flexibility in solution(NAMFIS) deconvolution analysis. The concluded model was capable of reproducing successfully the experimental NOESY cross-peak volumes and the proton-coupling constants. Among the 12 conformers, the one which was similar to the structure of verrucarin A in the solid state was the predominant accounting for 75% of the total relative population, although other low-energy conformations contributed to a lesser degree in order to explain the experimental data.     

Anomeric information obtained from a series of synthetic trisaccharides using energy resolved mass spectra

The majority of structural investigations of oligosaccharides based on mass spectrometry use naturally occurring oligosaccharides, which do not allow extracting any common feature associated with anomeric structures and linkage positions. In order to address the issue to find such characteristics possibly contained in oligosaccharide structure, a synthetic combinatorial trisaccharide library was analyzed. The trisaccharides used in the analysis consisted of L-fucose, D-galactose and D-glucose, in which individual glycosidic linkages existed in either alpha- or beta-anomers. The analysis of energy-resolved mass spectra (ERMS) and the scattered plot analysis of some parameters obtained from ERMS for a series of trisaccharides revealed that lower activation energy was required for the dissociation of alpha-glycosides of these sugars compared to those of the corresponding beta-anomers. It is suggested that this finding may be useful in structural analysis of natural oligosaccharides.     

Preparation of alpha-C-glycosides from glycals

[reaction: see text]. A novel approach to simple C-glycosides is reported. Reductive ring opening of 1,2-anhydro sugars with titanocene(III) chloride produces an anomeric radical that can be trapped with a variety of agents. The reaction stereospecifically affords alpha-glycosides and produces a free C-2 hydroxyl group allowing for further elaboration.     

Stereoselective tris-glycosylation to introduce beta-(1-->3)-branches into gentiotetraose for the concise synthesis of phytoalexin-elicitor heptaglucoside

Dodecyl thioglycosides (3, 4, 5) were prepared by conventional transformation of d-glucose and used as new glycosyl donors for a short-step synthesis of phytoalexin elicitor heptaglucoside. A gentio-tetraoside derivative (6) having three hydroxyl groups was synthesized by NIS-TfOH promoted glycosylate in more than 90% yield followed by selective removal of temporary protective groups. Undesired formation of alpha-glycosides at the introduction of beta-(1-->3)-branches into gentio-oligosaccharides was found to be suppressed by use of a thiophilic reagent system, BSP (1-benzenesulfinyl piperidine)-Tf2O, giving the heptaglucoside in only four glycosylation steps.     

Filtered backprojection for the reconstruction of a high-resolution (4,2)D CH3-NH NOESY spectrum on a 29 kDa protein

Projection-reconstruction (PR) NMR enables rapid collection of multidimensional NMR data. NOESY represents a particularly difficult challenge for currently existing reconstruction algorithms, as it requires the quantitative reconstruction of an unknown number of peaks, at full sensitivity. We have demonstrated the successful application of PR-NMR to NOESY by determining the 4D methyl/amide NOESY spectrum of a 29 kDa protein, human carbonic anhydrase II, from 2D projections, using filtered backprojection for reconstruction. Compared with a 3D control spectrum, all expected peaks were faithfully reconstructed, with correct volumes and with no artifacts. Filtered backprojection thus provides a way to obtain high-resolution 4D NOESY data in the time required for conventional 3D data collection.     

Improved resolution in two-dimensional 1H NMR spectra of peptides by band-selective, homonuclear decoupling during both the evolution and acquisition periods: application to characterization of the binding of peptides by heparin

Two-dimensional 1H NMR experiments that achieve band-selective, homonuclear decoupling in both the indirectly detected F1 and directly detected F2 dimensions were used to assign the highly overlapped 1H NMR spectrum of the peptide Ac-SRGKARVRAKVKDQTK-NH2, both free in solution and bound to heparin. Band-selective, homonuclear decoupling during the evolution period was achieved using a double pulsed field gradient spin-echo (DPFGSE) with semi-selective shaped pulses; band-selective, homonuclear decoupling during the acquisition period was achieved by time-shared semi-selective shaped pulse decoupling. Regular TOCSY, ROESY and NOESY spectra and TOCSY, ROESY and NOESY spectra measured with band-selective, homonuclear decoupling in the evolution (F1) dimension (BASHD-TOCSY, ROESY and NOESY spectra) and with band-selective, homonuclear decoupling in both the F1 and F2 dimensions (D-(or Double)-BASHD-TOCSY, ROESY and NOESY spectra) are reported and compared for the peptide and its heparin complex. Complete assignment of the 1H-NMR spectra of the free and heparin-complexed peptide was achieved with the high resolution of the D-BASHD-TOCSY, ROESY and NOESY spectra. Characterization of the heparin-complexed peptide is of interest because of the ability of the peptide to neutralize the anticoagulant activity of heparin.     

Complete 1H and 13C NMR assignments of two phytosterols from roots of Piper nigrum

Using modern NMR techniques, including DQF-COSY, NOESY, HETCOR and HMBC, the full assignments of all 1H and 13C chemical shifts were made for stigmastane-3,6-dione and stigmast-4-ene-3,6-dione, isolated from the roots of Piper nigrum (Piperaceae). Their stereochemistry was resolved by a combination of 2D NOESY data and analysis of coupling constants. The two compounds were isolated from the genus Piper for the first time.     

Stereospecific entry to [4.5]spiroketal glycosides using alkylidenecarbene C-H insertion

[reaction: see text] A novel method for the stereospecific preparation of [4.5]spiroketal glycosides utilizing the 1,5 C-H bond insertion of alkylidenecarbenes is described. Treatment of 2-oxopropyl beta-pyranosides A with lithium (trimethylsilyl)diazomethane in THF at -78 degrees C afforded 1,6-dioxaspiro[4,5]decenes B in good yield. Submission of the corresponding alpha-glycosides C to the same reagent gave the isomeric insertion products D in moderate to high yield.     

New insights into the interactions between dendrimers and surfactants by two dimensional NOE NMR spectroscopy

The interactions between polyamidoamine dendrimers and different surfactants including sodium dodecyl sulfate (SDS) and dodecyltrimethyl ammonium bromide in aqueous solutions have been investigated by a NOESY NMR technique. Strong NOE cross-peaks between hydrophobic chain protons of SDS and methylene protons of cationic dendrimers were found, suggesting a strong tendency for the long hydrophobic tails of SDS to associate with the hydrophobic pockets of dendrimers. The hydrophilic head of SDS localizes near the core or the boundary of each generation of dendrimers, and the hydrophobic chain of SDS localizes in the relative nonpolar pockets of dendrimers. The encapsulation of surfactant monomers by dendrimers is dependent on the charge type of the surfactants, the surface functionality, and the generation of dendrimers. The NOESY analysis provides a new insight into interactions between dendrimers and surfactants in comparison with previous investigations.     

Study of the complexation of different methacrylates with cyclodextrins employing a combination of electrophoretic, chromatographic, and NMR-spectroscopic methods

The present study describes the application of capillary electromigration techniques; CEC and micellar EKC (MEKC), and the application of spectroscopic methods; 1H NMR and 1H NOESY spectroscopy to investigate interactions between CDs (alpha-CD, statistically methylated beta-CD, hydroxypropyl-beta-CD, and 2-hydroxypropyl-gamma-CD) and different methacrylates (adamantyl, isobornyl, cyclohexyl, and phenyl methacrylate). It is shown that these methods complement each other. While CD-mediated MEKC is a rapid screening technique for comparing complex stabilities in aqueous media, 1H NMR chemical shift analysis provides quantitative data for very strong methacrylate-CD complexes and CD-mediated CEC provides quantitative data for complexes with lower complex forming constants. CD-mediated MEKC did not prove to be suitable for the calculation of complex forming constants. Reasons are discussed. 1H NOESY spectra were used to study spatial relationships between host and guest atoms.     

Homonuclear Selective One-Dimensional Gradient NMR Experiments in the Structure Determination of Natural Diterpene Derivatives

Summary.  The solution structure of two natural diterpene derivatives, the secondary metabolites esulatin-A and esulatin-B of Euphorbia esula, was investigated by homonuclear NMR experiments. Since the spectral dispersion of the ¹H NMR spectra at 500 MHz was sufficient to separate several skeletal protons of the title compounds, they were selectively excited with a double pulsed field gradient spin-echo (DPFGSE) sequence using 180°Gaussian pulses sandwiched between sine shaped gradients. With the use of selective excitation, scalar as well as dipolar interactions of the selected spins were monitored through one-dimensional (1D) COSY, TOCSY, and NOESY experiments. The chemical shifts of the coupling partners could be accurately extracted from the 1D COSY and TOCSY spectra recorded with high digital resolution. The selective TOCSY experiment provided an excellent opportunity to identify spins belonging to the same scalarly coupled spin system. The solution state conformation was investigated by selective gradient enhanced NOESY experiments. Proton–proton distances were evaluated from the cross-relaxation rates obtained from a quantitative analysis of the NOESY spectra recorded with different mixing times. The NMR derived distances were compared to the results of solid state X-ray diffraction measurements. Corresponding author. E-mail: pforgo@chem.u-szeged.hu Received November 21, 2001. Accepted (revised) January 9, 2002     

Solution structure of folic acid. Molecular mechanics and NMR investigation

The structure of folic acid in solution was investigated by nuclear magnetic resonance (NMR) and theoretical calculations. Dynamical information and geometrical constraints were obtained by carbon-13 relaxation study, homo-nuclear NOESY spectra and hetero-nuclear 1H-13C NOE experiments. This set of experimental data was used for the molecular mechanics and molecular dynamic calculations. The accuracy of the final structure was established by the R(NMR) factor, which was calculated comparing the experimental NOESY cross-peaks intensities and the corresponding values simulated by using the complete relaxation matrix analysis (CORMA) approach.     

NMR研究cis-Pt(NH_3)_2(Guo)_2~(2+)的溶液构象

本文应用NMR研究了cis-Pt(NH_3)_2(Guo)_2~(2+)的溶液构象。通过质子—质子之间扭转角φ_(HH)计算发现:配位以后糖环的构象有~2E→~3E转变的趋势;C(4′)—C(5′)之间旋转仍主要采取gg构象。通过~1H—~1H NOESY分析,发现碱基构象在Anti-式优势的前提下,存在Anti-Syn-平衡。     

Two new cembrane-based diterpenoids from the marine soft coral Sinularia crassa

Two new cembrane diterpenes, sicrassarines A and B, were isolated from the Taiwanese soft coral Sinularia crassa. The structures of the new metabolites were determined on the basis of extensive spectroscopic analysis, particularly mass spectroscopy and 2D NMR (1H--1H COSY, HMQC, HMBC, and NOESY) spectroscopy.     

Solution-phase hexasaccharide synthesis using glucosyl iodides

[reaction: see text] Oligosaccharides composed of 1,6-glucosyl residues have been prepared from glucosyl iodides. The reactions are highly stereoselective, giving the alpha-glycosides as the only isolated products in yields ranging from 84% to 94%. Oligomer synthesis can take place in an iterative 1 + 1 + 1 fashion or in a convergent manner where dimer iodides serve as donors for higher order acceptors.     

Influence of NMR Data Completeness on Structure Determinations of Homodimeric Proteins

The structure determination of homodimeric proteins by NMR using conventional NOESY experiments is still challenging due to the degeneracy of the chemical shifts in the identical monomers, which causes ambiguity in the NOE assignments. Residues involved in the interface between two monomers provide essential intermolecular NOEs for the structure determinations of homodimeric proteins. Hence NMR data, such as NOE peak lists and chemical shift assignments of these interface residues, play a crucial role for the successful structure determination of homodimeric proteins. This paper extends our previous report (Lin, Y.‐J.; Kirchner, D. K.; Güntert, P. J. Magn. Reson.­ 2012 , 222, 96) and investigates the influence of incomplete NOESY peak lists combined with incomplete ¹H chemical shift assignments of the interface residues on the structure determination of homodimeric proteins using the program CYANA. Data incompleteness was simulated by random omission of both NOESY cross peaks and interface ¹H chemical shifts. Our results for three proteins with different percentages of interface residues reveal that the algorithm can tolerate about 40–50% NOESY peak omission with complete interface chemical shift assignments, which indicates that partial NOESY peak omission does not cause severe problems when the interface chemical shifts are completely assigned. Combining NOESY peak omission with incomplete interface chemical shift assignments, the tolerance for interface chemical shift omission decreases with the extent of omitted NOESY peaks. The tolerance for unassigned interface side chain, methyl and aromatic chemical shifts is affected more strongly by NOESY peak omission than that for the omission of general interface ¹H chemical shifts including the backbone. In general about 10–30% peaks omission is tolerated in conjunction with missing chemical shift assignments. If more NOESY peaks are omitted calculations gradually become unstable and tend not to tolerate any missing interface chemical shifts. A large amount of omitted NOESY peaks, for instance 30% omission in our calculations, could decrease the tolerance for missing aromatic or methyl interface ¹H chemical shifts to as few as 2–4 missing chemical shifts, suggesting that complete aromatic and methyl ¹H chemical shift assignments are important when the NOESY peak data is significantly incomplete. Finally, for homodimeric proteins with a low percentage of interface residues, our results reveal that the omission of NOESY peaks, even at an extent of only 10%, can result in no tolerance against the omission of interface ¹H chemical shifts, suggesting that the completeness of both interface ¹H chemical shift assignments and NOESY peaks are important for the successful structure determination of proteins with a small homodimer interface.     

Substrate flexibility of vicenisaminyltransferase VinC involved in the biosynthesis of vicenistatin

A glycosyltransferase VinC is involved in the biosynthesis of antitumor beta-glycoside antibiotic vicenistatin. It catalyzes a glycosyl transfer reaction between dTDP-alpha-D-vicenisamine and vicenilactam. Previous identification of its broad substrate specificity toward various glycosyl acceptors enabled us to explore the potential of VinC for glycodiversification. In vitro study of the substrate specificity toward several dTDP-sugars with vicenilactam established that VinC displayed activities with alpha-anomers of several dTDP-2-deoxy-D-sugars such as mycarose, digitoxose, olivose, and 2-deoxyglucose to afford respective beta-glycosides. Notably, beta-anomers of dTDP-2-deoxy-D-sugars also appeared to be accepted by VinC to form alpha-glycosides. Furthermore, VinC is capable of catalyzing glycosyl transfer reactions from both the alpha-anomer and beta-anomer of dTDP-l-mycarose, respectively, into beta-glycoside and alpha-glycoside. These results indicate that VinC is a unique glycosyltransferase possessing broad substrate specificity. The mechanism of this axially oriented glycosidic bond formation from the equatorially oriented dTDP-sugar might be explained by conformational change of dTDP-sugar to a boat conformation during the glycosyl transfer reaction. To apply these features of VinC for glycodiversification, 22 sets of structurally diverse glycosides were constructed using unnatural glycosyl donors and acceptors.