T-ROESY Experiments Research Articles

Comparative Proton Nuclear Magnetic Resonance Studies of Amantadine Complexes Formed in Aqueous Solutions with Three Major Cyclodextrins

Host-guest complexes of alpha-, beta-, and gamma-cyclodextrins (α-CD, β-CD, and γ-CD, respectively) with amantadine (1-aminoadamantane, AMA; an antiviral agent) were characterized in aqueous solutions using proton nuclear magnetic resonance (NMR) spectroscopy. Host-guest molecular interactions were manifested by changes in the chemical shifts of AMA protons. NMR Job's plots showed that the stoichiometry of all the studied complexes was 1:1. Two-dimensional T-ROESY experiments demonstrated that the complexes were formed by different degrees of incorporation of the adamantyl group of AMA into the CD cavity. The mode of AMA binding was proposed. The AMA molecule came into the α-CD cavity (the smallest size) or β-CD cavity (the intermediate size) through its wide entrance to become shallowly or deeply accommodated, respectively. In the complex of AMA with γ-CD (the largest cavity size), the adamantyl group was also quite deeply inserted into the CD cavity, but it arrived there through the narrow cavity entrance. It was found that the adamantyl group of AMA was best accommodated by the β-CD cavity. The binding constants Kaa of the studied complexes (in M(-1) ), determined from DOSY NMR, were fairly high; their values in an ascending order were: α-CD (183) < γ-CD (306) ≪ β-CD (5150).

Binding of reactive organophosphate by oximes via hydrogen bond

In this contribution, the ability of simple oximes to bind a well-known nerve agent simulant (dimethylmethylphosphonate, DMMP) via hydrogen bond is reported. UV/Vis measurements indicate the formation of 1:1 complexes. 1H-, 31P-NMR titrations and T-ROESY experiments confirm that oximes bind the organophosphate via hydrogen bond. The ability of simple oximes to bind a well-known nerve agent simulant (dimethylmethylphosphonate, DMMP) via hydrogen bond is reported. UV/Vis measurements indicate the formation of 1:1 complexes. 1H-, 31P-NMR titrations and T-ROESY experiments give evidence that oximes bind the organophosphate via hydrogen bond.

A β-cyclodextrin–resveratrol inclusion complex and the role of geometrical and electronic effects on its electronic induced circular dichroism

Complexation of trans-resveratrol with β-cyclodextrin results in an induced Cotton effect (ICD). On the basis of NMR T-ROESY experiments and molecular dynamics simulations a preferential orientation mode of resveratrol within the cyclodextrin is proposed, in which the dihydroxyl ring is oriented towards the narrow side of the β-cyclodextrin. TD-DFT calculations showed that the observed ICD is caused by orbital mixing on trans-resveratrol upon complexation with β-cyclodextrin, being geometry effects averaged out by the flexibility of resveratrol inside the CD cavity. Computations also show that Harata–Kodaka rules are not of direct applicability to systems like resveratrol where the chromophore is only partially buried into the cyclodextrin cavity.

Molecular Conformations in the Pentasaccharide LNF-1 Derived from NMR Spectroscopy and Molecular Dynamics Simulations

The conformational dynamics of the human milk oligosaccharide lacto-N-fucopentaose (LNF-1), α-L-Fucp-(1 → 2)-β-D-Galp-(1 → 3)-β-D-GlcpNAc-(1 → 3)-β-D-Galp-(1 → 4)-D-Glcp, has been analyzed using NMR spectroscopy and molecular dynamics (MD) computer simulations. Employing the Hadamard (13)C-excitation technique and the J-HMBC experiment, (1)H,(13)C trans-glycosidic J coupling constants were obtained, and from one- and two-dimensional (1)H,(1)H T-ROESY experiments, proton-proton cross-relaxation rates were determined in isotropic D(2)O solution. In the lyotropic liquid-crystalline medium consisting of ditetradecylphosphatidylcholine, dihexylphosphatidylcholine, N-cetyl-N,N,N-trimethylammonium bromide, and D(2)O, (1)H, (1)H and one-bond (1)H, (13)C residual dipolar couplings (RDCs), as well as relative sign information on homonuclear RDCs, were determined for the pentasaccharide. Molecular dynamics simulations with explicit water were carried out from which the internal isomerization relaxation time constant, τ(N), was calculated for transitions at the ψ torsion angle of the β-(1 → 3) linkage to the lactosyl group in LNF-1. Compared to the global reorientation time, τ(M), of ∼0.6 ns determined experimentally in D(2)O solution, the time constant for the isomerization relaxation process, τ(N(scaled)), is about one-third as large. The NMR parameters derived from the isotropic solution show very good agreement with those calculated from the MD simulations. The only notable difference occurs at the reducing end, which should be more flexible than observed by the molecular simulation, a conclusion in complete agreement with previous (13)C NMR relaxation data. A hydrogen-bond analysis of the MD simulation revealed that inter-residue hydrogen bonds on the order of ∼30% were present across the glycosidic linkages to sugar ring oxygens. This finding highlights that intramolecular hydrogen bonds might be important in preserving well-defined structures in otherwise flexible molecules. An analysis including generalized order parameters obtained from nuclear spin relaxation experiments was performed and successfully shown to limit the conformational space accessible to the molecule when the number of experimental data are too scarce for a complete conformational analysis.

Synthesis and conformational aspects of 20- and 40-membered macrocyclic mono and dinuclear uranyl complexes incorporating salen and ( R)-BINOL units

20- and 40-membered macrocyclic mono and dinuclear uranyl complexes 11– 13 incorporating salen and ( R)-BINOL units have been designed and synthesized starting from 4- tert-butylphenol in eight steps. NMR measurements (COSY, NOESY/EXSY) indicate that such complexes in solution are involved in conformational equilibria, which for the 20-membered derivatives are observable already at room temperature ( k 1=300 s −1 at 300 K) and which are probably related to a flipping motion of salicylaldehyde units. T-ROESY experiments suggest that in solution, the dinuclear complexes do not assume structures wrapped around the metal ions.

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 Glcα1–2Glcα 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.

A Conformational Dynamics Study of α-l-Rhap-(1→2)[α-l-Rhap-(1→3)]-α-l-Rhap-OMe in Solution by NMR Experiments and Molecular Simulations

The conformational preference of alpha-l-Rhap-(1-->2)[alpha-l-Rhap-(1-->3)]-alpha-l-Rhap-OMe in solution has been studied by NMR spectroscopy using one-dimensional (1)H,(1)H T-ROESY experiments and measurement of trans-glycosidic (3)J(C,H) coupling constants. Molecular dynamics (MD) simulations with a CHARMM22 type of force field modified for carbohydrates were performed with water as the explicit solvent. The homonuclear cross-relaxation rates, interpreted as effective proton-proton distances, were compared to those obtained from simulation. Via a Karplus torsional relationship, (3)J(C,H) values were calculated from simulation and compared to experimental data. Good agreement was observed between experimental data and the MD simulation, except for one inter-residue T-ROE between protons in the terminal sugar residues. The results show that the trisaccharide exhibits substantial conformational flexibility, in particular along the psi glycosidic torsion angles. Notably, for these torsions, a high degree of correlation (77%) was observed in the MD simulation revealing either psi(2)(+) psi(3)(+) or psi(2)(-)psi(3)(-) states. The simulations also showed that non-exoanomeric conformations were present at the phi torsion angles, but to a limited extent, with the phi(3) state populated to a larger extent than the phi(2) state. Further NMR analysis of the trisaccharide by translational diffusion measurements and (13)C T(1) relaxation experiments quantified global reorientation using an anisotropic model together with interpretation of the internal dynamics via the "model-free" approach. Fitting of the dynamically averaged states to experimental data showed that the psi(2)(+)psi(3)(+) state is present to approximately 49%, psi(2)(-) psi(3)(-) to approximately 39%, and phi(3) (non-exo) to approximately 12%. Finally, using a dynamic and population-averaged model, (1)H,(1)H T-ROE buildup curves were calculated using a full relaxation matrix approach and were found to be in excellent agreement with experimental data, in particular for the above inter-residue proton-proton interaction between the terminal residues.

NMR Study of the Amide Bond-formation Using a Heterocyclic Amine and Acid Receptor

Abstract Recently, a tricyclic receptor capable of binding a carboxylic acid or an amine has been described. A kinetic study of the reaction between benzoic acid and benzylamine to give the amide bond using this heterocyclic receptor has been performed. The results suggest that the receptor plays a significant role in accelerating the amide bond-formation. To gain further information on the complex formation between the host, benzoic acid and benzylamine, NOESY and T-ROESY experiments were performed.

NMR and molecular modeling studies of the interaction between wheat germ agglutinin and the beta-D-GlcpNAc-(1-->6)-alpha-D-Manp epitope present in glycoproteins of tumor cells.

The beta-D-GlcpNAc-(1-->6)-alpha-D-Manp disaccharide is a constituent of highly branched cell-surface glycoconjugates that are malignancy markers. The conformational preference of the disaccharide beta-D-GlcpNAc-(1-->6)-alpha-D-Manp-OMe in solution has been studied by molecular modeling and NMR spectroscopy including 1D (1)H,(1)H T-ROESY experiments and analysis of (3)J(H,H) of the hydroxymethyl group being part of the glycosidic linkage of the disaccharide, which revealed the relative populations of the omega torsion angle as gt = 0.60, gg = 0.35, and tg = 0.05. Good agreement was obtained between the effective proton-proton distances from the experiment and those obtained by molecular modeling when the flexibility at the omega torsion angle was taken into account. Molecular modeling of the disaccharide in the binding sites of the lectin wheat germ agglutinin indicates that several conformations could be adopted in the bound state. (1)H NMR and transfer NOESY experiments confirmed that binding took place, and trans-glycosidic proton-proton interactions indicated that a conformational preference was present in the bound state, as observed by the relative change of the NOEs from H1' to H6(pro-R) and H6(pro-S). STD NMR experiments showed that binding occurred in the region of the N-acetyl group of the terminal sugar residue. In addition, the O-methyl group received saturation transfer because of the proximity to the protein. (1)H,(1)H NOEs indicated that the two methyl groups were close in space, as observed in only one of the predicted bound conformations. Experimental and theoretical data therefore agree that one conformation with a gt conformation of the hydroxymethyl group and a negative sign for the psi torsion angle is indeed selected by the lectin upon binding.

Determination of the Conformational Flexibility of Methyl α-Cellobioside in Solution by NMR Spectroscopy and Molecular Simulations

The conformational flexibility of methyl α-cellobioside in water and dimethyl sulfoxide solutions was investigated by 1D 1H,1H T-ROESY experiments. In combination with molecular dynamics simulat ...

A conformational study of α- d-Man p-(1 → 2)-α- d-Man p-(1 → O)- l-Ser by NMR 1H, 1H T-ROESY experiments and molecular-dynamics simulations

The conformational preference of α- d-Man p-(1 → 2)-α- d-Man p-(1 → O)- l-Ser has been investigated by one-dimensional 1H, 1H T-ROESY experiments and molecular-dynamics simulations with CHARMM22 type of force fields and water as explicit solvent. Proton–proton distances were obtained from the simulations and subsequently experimentally determined distances could be derived. Measurements were performed on the title compound as well as on selectively deuterium-substituted analogues synthesized as part of this study to alleviate possible NMR spectroscopic difficulties. A very good agreement was present between the separate NMR experiments. In the subsequent analysis a key nuclear Overhauser effect between the anomeric protons in the two sugar residues was used to assess the conformational dynamics revealed by the molecular simulations. The combined results support a model in which two states are significantly populated as a result of flexibility around the bond defined by the glycosidic torsion angle ψ.

Labdane diterpenes of Leonurus sibiricus.

Seven new labdane diterpenes, sibiricinones A-E (1-4, 6) and 15-epi-sibiricinones D and E (5 and 7), and the flavone genkwanin were isolated from the aerial parts of Leonurus sibiricus. Sibiricinone D (4) and 15-epi-sibiricinone D (5), and sibiricinone E (6) and 15-epi-sibiricinone E (7), respectively, were isolated as C-15 epimeric pairs. These secondary metabolites were identified on the basis of 1D and 2D NMR including (1)H-(1)H COSY, HSQC, and HMBC spectroscopic techniques. The stereochemical configurations of compounds 4-7 were assigned through 2D T-ROESY and selective NOE experiments.

Complexation of monosulfonated triphenylphosphine oxides with β-cyclodextrin: spectroscopic study and consequence on the behaviour of cyclodextrins in aqueous-phase organometallic catalysis

Interactions between the β-cyclodextrin and the monosulfonated isomers of triphenylphosphine oxide were investigated in aqueous solution by NMR, UV-vis and ESI mass spectroscopy. Titration and continuous variation plots obtained from 31P and 1H NMR data indicate the formation of 1 ∶ 1 inclusion complexes. The structures of these inclusion complexes were proposed from T-ROESY experiments. All inclusion complexes were enthalpy stabilized, but entropy destabilized. Formation of such complexes cannot decrease the efficiency of cyclodextrins in aqueous-phase organometallic catalysis.

Conformational studies of the two anomeric methyl glycosides of α-D-Manp-(1→2)-D-Glcp by molecular simulations and NMR 1H,1H T-ROESY experiments

A conformational study has been performed of the two disaccharides α-D-Manp-(1→2)-α-D-Glcp-OMe (1) and α-D-Manp-(1→2)-β-D-Glcp-OMe (2) differing only in the anomeric configuration at the methyl glucoside. Molecular simulations were carried out with five different potential energy functions ranging from pure in vacuo to explicit solvent treatment. For comparison, 1H,1H T-ROESY and heteronuclear trans-glycosidic correlation NMR experiments were performed which resulted in proton–proton distances and 3JC,H values, respectively. In addition, interpretation of 13C NMR glycosylation shifts indicated slightly different average conformations of the two disaccharides. From the molecular simulations selected parameters were calculated for comparison to experiments. The combined analysis revealed the conformational region of the disaccharides and showed that the average ψ torsion angle in 2 was more eclipsed than in 1. Moreover, the translational diffusion coefficients of the disaccharides were determined at 25 °C in D2O by the Longitudinal Eddy-Delayed experiment which resulted in Dt = 0.37 × 10−9 m2 s−1. These values were compared to those obtained from molecular dynamics simulations with explicit water as solvent. The relative differences in translational diffusion between the disaccharides and water were similar for experiment and simulation. However, it is well-known that the TIP3P water model overestimates Dt, so also in these simulations.

Oligosaccharides display both rigidity and high flexibility in water as determined by 13C NMR relaxation and 1H,1H NOE spectroscopy: evidence of anti-phi and anti-psi torsions in the same glycosidic linkage.

The trisaccharide beta-D-Glcp-(1-->2)-beta-D-Glcp-(1-->3)-alpha-D-Glcp-OMe has been investigated by molecular dynamics (MD) simulations and NMR experiments in water. 13C spin-lattice (T1) and spin-spin (T2) relaxation times, together with 1H,13C NOE data were measured at two magnetic field strengths (9.4 and 14.1 T) in a 277 K D2O solution. Relaxation data interpreted by means of the model-free formalism revealed a rigid (S2 approximately 0.9) oligosaccharide tumbling in solution. 1H,1H Cross-relaxation rates were determined at 600 MHz by 1D DPFGSE NOESY and T-ROESY experiments, which provided high quality data and subsequently proton-proton distances within the trisaccharide. The presence of anti conformers at both torsions of a glycosidic linkage is demonstrated for the first time. MD simulations were carried out to facilitate analysis of the NOE data. In total, 15 simulations-starting from five different conformational states--were performed, with production runs of up to 10 ns, resulting in 83 ns of oligosaccharide dynamics in water. anti Conformers were populated to different degrees in the simulations, especially at the phi2 torsion angle. By combining the results from the NOE experiments and the MD simulations, the anti conformers at the (1-->2)-linkage were quantified as 7% anti-phi2 and 2% anti-psi2, revealing a highly flexible trisaccharide in which large conformational changes occur. From the MD simulations, interresidue hydrogen bonding, from HO2" to O2 or O3, was significantly populated (approximately 40%) in both of the anti conformational states. The contentious issue over rigidity versus flexibility in oligosaccharides has thus been thoroughly examined, showing that the dynamics should be taken into account for a relevant description of the molecular system.

A conformational study of the trisaccharide beta-D-Glcp-(1-->2)[beta-D-Glcp-(1-->3)]alpha-D-Glcp-OMe by NMR NOESY and TROESY experiments, computer simulations, and X-ray crystal structure analysis.

Proton-proton cross-relaxation rates have been measured for the trisaccharide beta-D-Glcp-(l --> 2)[beta-D-Glcp-(1 --> 3)]alpha-D-Glcp-OMe in D2O as well as in D2O/[D6]DMSO 7:3 solution at 30 degrees C by means of one-dimensional NMR pulsed field gradient 1H,1H NOESY and TROESY experiments. Interatomic distances for the trisaccharide in D2O were calculated from the cross-relaxation rates for two intraresidue and three interglycosidic proton pairs, using the isolated spin-pair approximation. In the solvent mixture one intraresidue and three interglycosidic distances were derived without the use of a specific molecular model. In this case the distances were calculated from the cross-relaxation rates in combination with "model-free" motional parameters previously derived from 13C relaxation measurements. The proton-proton distances for interglycosidic pairs were compared with those averaged from Metropolis Monte Carlo and Langevin Dynamics simulations with the HSEA, PARM22, and CHEAT95 force fields. The crystal structure of the trisaccharide was solved by analysis of X-ray data. Interresidue proton pairs from the crystal structure and those observed by NMR experiments were similar. However, the corresponding proton-proton distances generated by computer simulations were longer. For the (1 --> 2) linkage the glycosidic torsion angles of the crystal structure were found in a region of conformational space populated by all three force fields, whereas for the (1 --> 3) linkage they occupied a region of low population density, as seen from the simulations.

The influence of macrocyclic polyether constitution upon ammonium ion/crown ether recognition processes

Secondary dialkylammonium (R2NH2+) ions are bound readily by dibenzo[24]crown-8 (DB24C8) to form threaded complexes, namely [2]pseudo-rotaxanes. The effect of replacing one or both of the catechol rings in DB24C8 with resorcinol rings upon the crown ether's ability to bind R2NH2+ ions has now been investigated. When only one aromatic ring is changed from catechol to resorcinol, a crown ether with a [25]crown-8 constitution is created-namely benzometaphenylene[25]crown-8 (BMP25C8). A [2]pseudorotaxane is formed in the solid state when BMP25C8 is co-crystallized with dibenzylammonium hexafluorophosphate, as evidenced by its X-ray crystal structure. Furthermore, this crown ether has been shown to bind R2NH2+ ions in solution, an observation which has been exploited in the synthesis of the first BMP25C8-containing [2]rotaxane. The methodology employed to generate this [2]rotaxane--the reaction of an amine with an isocyanate to form a urea--was tested initially on a system incorporating DB24C8 and was shown to work efficiently. Both [2]rotaxanes have been fully characterized by 1H and 13C NMR spectroscopies, FAB mass spectrometry and X-ray crystallography. Interestingly, the unsymmetrical nature of the dumbbell-shaped component in each of the two [2]rotaxanes renders each face of the encircling macrocyclic polyether diastereotopic, a feature that is apparent upon inspection of their 1H NMR spectra. The resonances associated with the diastereotopic protons on each face of the macrorings are well enough resolved to enable the faces of the crown ethers to be readily identified with respect to their protons by 1H NMR spectroscopy. Unambiguous assignments can be made as a result of the fact that the protons on each face of the macrocyclic polyether experience a unique set of through-space interactions, as evidenced by T-ROESY experiments. Additionally, the two-dimensional NMR analyses are in agreement with the X-ray crystallographic studies performed on these [2]rotaxanes, indicating that the crown ethers are located intimately around the NH2+ centers as expected. Replacement of both catechol rings in the DB24C8 constitution with resorcinol rings results in a crown ether with a [26]crown-8 constitution--namely bismetaphenylene[26]crown-8 (BMP26CS). All the evidence to date points to the fact that this further change in constitution results in a crown ether that does not bind R2NH2+ ions in either the solution or solid states.

Room-temperature (1H,13C) and variable-temperature (1H) NMR studies on spinosin

Spinosin, a C-glycoside flavonoid, was isolated from the plant Desmodium tortuosum (Sw.) DC. The 1H and 13C NMR data acquired at room temperature exhibited doubling of signals, suggesting the presence of two rotamers in solution. Variable-temperature 1H NMR experiments confirmed this hypothesis. T-ROESY experiments in conjunction with theoretical (MM2) calculations supported the proposal that the two rotamers interchange via rotation about the C-6— C-1′′ bond. The spectral data were completely assigned using 1H– 1H COSY, HMQC, HSQC and HMBC experiments. Copyright © 2000 John Wiley & Sons, Ltd.

Conformational Flexibility of Carbohydrates: A Folded Conformer at the φ Dihedral Angle of a Glycosidic Linkage

The trisaccharide β-d-Glcp-(1→2)-β-d-Glcp-(1→3)-α-d-Glcp-OMe has been studied by Langevin dynamics (LD) simulations and NMR spectroscopy including measurement of transglycosidic 3JC,H coupling constants, 3JH,OH coupling constants, temperature dependence of the chemical shift of hydroxyl protons, and 2D NOESY and T-ROESY experiments as well as their 1D DPFGSE analogues. A folded conformer at the φH dihedral angle of the (1→2)-linkage observed in the LD simulation was corroborated by experimental NMR data, in particular spatial proximity of hydroxyl protons in nonadjacent sugar residues as deduced from a 2D T-ROESY experiment. The results from the present study with an “anti” φH conformer show that oligosaccharides exhibit large conformational flexibility under certain conditions and that this inherent property needs to be taken into account in the analysis of carbohydrate structure.

Oligosaccharides corresponding to biological repeating units of Shigella flexneri variant Y polysaccharide Part 5. Conformational analysis of a heptasaccharide hapten utilizing a combined molecular dynamics and NMR spectroscopic protocol

The conformational analysis of a heptasaccharide, α- L- Rhap-(1 → 2)- α- L- Rhap-(1 → 3)- α- L- Rhap-(1 → 3)- β- D- GlcpNAc-(1 → 2) - α- L- Rhap-(1 → 2)- α- L- Rhap-(1 → 3)- α- L- Rhap- OPr (ABCDA'B'C') corresponding to the O-antigen of Shigella flexneri variant Y is described. ROESY, T-ROESY and NOESY experiments have been used to derive proton-proton distances. These distances have been used as initial constraints to select a starting conformation for molecular dynamics (MD) calculations with the PIMM91 and CHARMm22 force fields in vacuum and in water, with and without the distance constraints. The time frames of the constrained and unconstrained dynamics simulations were employed to compute ROE build-up curves with crosrel, a program that performs a full-matrix relaxation treatment of cross relaxation. Experimental and calculated build-up curves agree favorably and are used to infer a model of conformation. Extension of this model to the polysaccharide leads to differences from the currently accepted model.