Rotating Frame Nuclear Overhauser Effect Research Articles

Analysis of peptide rotational diffusion by homonuclear NMR.

The analysis of the rotational diffusion of a molecule using homonuclear NMR is investigated. The homonuclear longitudinal and transverse cross-relaxation rates, which can be quantitatively measured using off-Resonance Rotating frame nuclear Overhauser Effect Spectroscopy (ROESY), are used to build a distribution, which exhibits a solid-state-like pattern characteristic of the diffusion tensor. The distributions of the antimicrobial peptide ranalexin in water and in 30% of trifluoracetic acid (TFE) are compared, and the peptide rotational diffusion is shown to be more isotropic in water than in 30% TFE. This difference is further supported by the analysis of NMR ranalexin conformers in 30% TFE, and by the analysis of a molecular dynamics simulation of peptide in water.

Structural and serological relatedness of the O-antigens of Proteus penneri 1 and 4 from a novel Proteus serogroup O72.

O-specific polysaccharides (O-antigens) of the lipopolysaccharides (LPS) of Proteus penneri strains 1 and 4 were studied using sugar analysis, (1)H and (13)C NMR spectroscopy, including 2D COSY, H-detected (1)H,(13)C HMQC, and rotating-frame NOE spectroscopy (ROESY). The following structures of the tetrasaccharide (strain 1) and pentasaccharide (strain 4) repeating units of the polysaccharides were established: [reaction: see text]. In the polysaccharide of P. penneri strain 4, glycosylation with the lateral Glc residue (75%) and O-acetylation of the lateral GalNAc residue (55%) are nonstoichiometric. This polysaccharide contains also other, minor O-acetyl groups, whose positions were not determined. The structural similarity of the O-specific polysaccharides was consistent with the close serological relatedness of the LPS, which was demonstrated by immunochemical studies with O-antisera against P. penneri 1 and 4. Based on these data, it was proposed to classify P. penneri strains 1 and 4 into a new Proteus serogroup, O72, as two subgroups, O72a and O72a,b, respectively. Serological cross-reactivity of P. penneri 1 O-antiserum with the LPS of P. penneri 40 and 41 was substantiated by the presence of an epitope(s) on the LPS core region shared by all P. penneri strains studied.

A model for sequential threading of alpha-cyclodextrin onto a guest: a complete thermodynamic and kinetic study in water.

The first variable-temperature and variable-pressure stopped-flow spectrophotometric study of the sequential threading of alpha-cyclodextrin (alpha-CD) onto the guest dye Mordant Orange 10, S, is reported. Complementary (1)H one-dimensional (1D) variable-temperature kinetic studies and two-dimensional (2D) rotating-frame nuclear Overhauser effect spectroscopy (ROESY) and EXSY NMR studies are also reported. In aqueous solution at 298.2 K, the first alpha-CD threads onto S to form a 1:1 complex S.alpha-CD with a forward rate constant k(1,f) = 15 200 +/- 200 M(-1) s(-1) and dethreads with a reverse rate constant k(1,r) = 4.4 +/- 0.3 s(-1). Subsequently, S.alpha-CD isomerizes to S.alpha-CD (k(3,f) = 0.158 +/- 0.006 s(-1), k(3,f) = 0.148 +/- 0.006 s(-1)). This process can be viewed as a thermodynamically controlled molecular shuttle. A second alpha-CD threads onto S.alpha-CD to form a 1:2 complex, S.(alpha-CD)(2), with k(2,f) = 98 +/- 2 M(-1) s(-1) and k(2,r) = 0.032 +/- 0.002 s(-1). A second alpha-CD also threads onto S.alpha-CD to form another 1:2 complex, S.(alpha-CD)(2), characterized by k(4,f) = 9640 +/- 1800 M(-1) s(-1) and k(4,r) = 61 +/- 6 s(-1). Direct interconvertion between S.(alpha-CD)(2) and S.(alpha-CD)(2) was not detected; instead, they interconvert by dethreading the second alpha-CD and through the isomerization equilibrium between S.alpha-CD and S.alpha-CD. The reaction volumes, DeltaV(0), were found to be negative for the first three equilibria and positive for the fourth equilibrium. For the first three forward and reverse reactions, the volumes of activation are substantially more negative, indicating a compression of the transition state in comparison with the ground states. These data were used in conjunction with DeltaH, DeltaH degrees, DeltaS, and DeltaS degrees data to deduce the dominant mechanistic threading processes, which appear to be largely controlled by changes in hydration and van der Waals interactions, and possibly by conformational changes in both S and alpha-CD. The structure of the four complexes were deduced from (1)H 2D ROESY NMR studies.

Solution structure of hyaluronic acid oligomers by experimental and theoretical NMR, and molecular dynamics simulation.

The conformational properties of hyaluronic acid (HA) oligomers in aqueous solution were investigated by combining high-resolution NMR experimental results, theoretical simulation of NMR two-dimensional (2D) spectra by Complete Relaxation Matrix Analysis (CORMA), and molecular dynamics calculations. New experimental findings recorded for the tetra- and hexasaccharides enabled the stiffness of the HA and its viscoelastic properties to be interpreted. In particular, rotating frame nuclear Overhauser effect spectroscopy spectra provided new information about the arrangement of the glycosidic linkage. From (13)C NMR relaxation the rotational correlation time (tau(c)) were determined. The tau(c) were employed in the calculation of geometrical constraints, by using the MARDIGRAS algorithm. Restrained simulated annealing and 1 ns of unrestrained molecular dynamic simulations were performed on the hexasaccharide in a box of 1215 water molecules. The beta(1 --> 3) and beta(1 --> 4) glycosidic links were found to be rigid. The lack of rotational degree of freedom is due to direct and/or water-mediated interresidue hydrogen bonding. Both single or tandem water bridges were found between carboxylate group and N-acetil group. The carboxylate group of glucuronic acid is not involved in a direct link with the amide group of N-acetyl glucosamine and this facilitated bonding between the residue and the water molecules.

The rotating frame nuclear Overhauser effect spectroscopy experiment as an aid to determining solution structures of DNA oligomers

Important intrinsic characteristics of the rotating frame nuclear Overhauser effect spectroscopy (ROESY) experiment were found to be advantageous in DNA solution structure determination. In a ROESY experiment, the different mechanisms of relaxation result in different signs of cross peaks, enabling a clear distinction between H2′ resonances and H2″ resonances of the DNA sugar backbone. This method is of particular importance in crowded spectra, for purine resonances whose H2′, H2″ protons typically resonate closely, as well as in conditions where line broadening makes coupling constants in a correlated spectroscopy experiment impossible to determine. By observing the signs of cross peaks in the base proton to H2′, H2′′ sugar proton region, the ROESY spectrum can be used to distinguish A-form, B-form, and Z-form DNA. © 2000 John Wiley & Sons, Inc. Biopoly 54: 35–43, 2000

An NMR study of conformations of substituted dipeptides in dodecylphosphocholine micelles: implications for drug transport.

Efficient transport of intact drug (solute) across the intestinal epithelium is typically a requirement for good oral activity. In general, the membrane permeability of a solute is a complex function of its size, lipophilicity, hydrogen bond potential, charge, and conformation. In conjunction with theoretical/computational and in vitro drug transport studies, seven dipeptide (R(1)-D-Xaa-D-Phe-NHMe) homologues were each dissolved in a micellar d(38)-dodecylphosphocholine solvent system. In this homologous dipeptide series, factors such as size, lipophilicity, hydrogen-bond potential, and charge were either tightly controlled or well-characterized by other methods in order to investigate by nmr how conformational factors relate to transport. Nuclear Overhauser effect spectroscopy experiments and amide-NH-H(2)O chemical exchange rates showed that the five more lipophilic dipeptides were predominately associated with micelle, whereas the two less lipophilic analogues were not. Rotating frame nuclear Overhauser effect spectroscopy derived interproton distance restraints for each analogue, along with (3)J(HH)-derived dihedral restraints, were used in molecular dynamics/simulated annealing computations. Our results suggest that-other factors being equal-flexible dipeptides having a propensity to fold together nonpolar N- and C-terminal moieties allow greater segregation of polar and nonpolar domains and may possess enhanced transport characteristics. Dipeptides that were less flexible or that retained a less amphiphilic conformation did not have comparably enhanced transport characteristics. We suggest that these conformational/transport correlations may hold true for small, highly functionalized solutes (drugs) in general.

Model peptide studies of sequence repeats derived from the intracrystalline biomineralization protein, SM50. II. Pro,Asn-rich tandem repeats.

In the biomineralization process, a number of Pro-rich proteins participate in the formation of three-dimensional supramolecular structures. One such protein superfamily, the Pro,Gly-rich sea urchin intracrystalline spicule matrix proteins, form protein-protein supramolecular assemblies that modify the microstructure of the inorganic mineral phase (calcite) within embryonic sea urchin spicules and adult sea urchin spines. These proteins represent a useful model for understanding Pro sequence usage and the resulting generation of extended or "open" structures for protein-protein and/or protein-crystal recognition. In the sea urchin spicule matrix protein, SM50 (Strongylocentrotus purpuratus), there exists an unusual 20-residue Pro,Asn-containing repeat, &bond;PNNPNNPNPNNPNNPNNPNPbond which links the upstream 15-residue C-terminal domain and the downstream 211-residue beta-spiral repeat domain. To define the structural preferences of this 20-residue repeat, we created a 20-residue N- and C-terminal "capped" peptidomimetic of this sequence. Using far-uv CD dichroism, CH(alpha) and alpha-(15)N conformational shifts, (3)J(NH-CHalpha) coupling constants, sequential d(NN(i, i + 1)) rotating frame nuclear Overhauser effect connectivities, d(alphaN(i, i + 1))/d(NN(i, i + 1)) intensity ratios, amide temperature shift coefficients, amide solvent exchange, and simulated annealing refinement protocols, we have determined that this 20-residue repeat motif adopts an extended "twist" structure consisting of turn- and coil-like regions. These findings are consistent with previous studies, which have shown that Pro-rich tandem repeats adopt extended, flexible structures in solution. We hypothesize that this 20-residue repeat may fulfill the role of a mineral-binding domain, a protein-protein docking domain, or as an internal "molecular spacer" for the SM50 protein during spicule biocomposite formation.

Structure of a 2-aminoethyl phosphate-containing O-specific polysaccharide of Proteus penneri 63 from a new serogroup O68.

Lipopolysaccharide of Proteus penneri strain 63 was degraded by mild acid to give a high molecular mass O-specific polysaccharide that was isolated by gel-permeation chromatography. Sugar and methylation analyses and NMR spectroscopic studies, including two-dimensional 1H, 1H COSY, TOCSY rotating-frame NOE spectroscopy, H-detected 1H,13C and 1H,31P heteronuclear multiple-quantum coherence (HMQC), and 1H, 13C HMQC-TOCSY experiments, demonstrated the following structure of the polysaccharide: where FucNAc is 2-acetamido-2,6-dideoxygalactose and PEtn is 2-aminoethyl phosphate. The polysaccharide studied shares some structural features, such as the presence of D-GlcNAc6PEtn and an alpha-L-FucNAc-(1-->3)-D-GlcNAc disaccharide, with other Proteus O-specific polysaccharides. A marked cross-reactivity of P. penneri 63 O-antiserum with P. vulgaris O12 was observed and substantiated by a structural similarity of the O-specific polysaccharides of the two strains. In spite of this, the polysaccharide of P. penneri 63 has the unique structure among Proteus O-antigens, and therefore a new, separate serogroup, O68, is proposed for this strain.

D-configuration of serine is crucial in maintaining the phalloidin-like conformation of viroisin.

NMR studies have revealed that the conformation of the monocyclic viroisin is dissimilar to that of the corresponding monocyclic derivative of phalloidin, dethiophalloidin, but has much similarity with the conformation of the bicyclic phalloidin. Obviously, one of three structural features found exclusively in the virotoxins is able to compensate for the conformational strain that in the bicyclic phallotoxins maintains the toxic conformation. Synthetic work on virotoxin analogues has shown that both the additional hydroxy group in allo-hydroxyproline and the methylsulfonyl moiety in the 2'-position of tryptophan are unlikely to represent the structural element in question, leaving the D-serine moiety as the supposed key element. In this study we asked whether it is the hydroxy group of this amino acid or its D-configuration that is responsible for the effect. We synthesized four viroisin analogues and submitted them to conformational analysis by NMR as well as to an actin binding assay. While the rotating-frame nuclear Overhauser effect (ROESY) spectra of the analogues with L-configured amino acids showed several sets of signals, indicating the existence of conformers interconverting more slowly than the NMR time scale, the spectra of the analogues with D-configured amino acids showed only one set of signals. Remarkably, the two viroisin analogues with D-serine and D-alanine also had distinctly higher affinities for filamentous actin than their L-configured counterparts, suggesting that the high biological activity may be correlated with the absence of multiple and slowly interconverting conformers. Anyhow, D-configuration of serine is the structural element that maintains the phalloidin-like structure, while the hydroxy group does not contribute to conformational stability but is likely to be in contact with the actin surface.

Conformation in solution and dynamics of a structurally constrained linear insect kinin pentapeptide analogue.

The preferred conformations of the active diuretic insect kinin pentapeptide analogue Phe-Phe-Aib-Trp-Gly-NH2 were studied using nmr spectroscopy and molecular modeling. Structure sets consistent with rotating frame nuclear Overhauser effect spectroscopy distance constraints obtained by restrained simulated annealing in vacuo indicate a predominant population of a type II beta-turn involving the Phe1-Trp4 region. An equilibrium between this type II and a type I beta-turn formed by residues Phe2 and Gly5 was observed in a 5 ns restrained molecular dynamics simulation using the implicit generalized Born solvent accessible surface area (GB/SA) solvation model. When subjected to 500 ps dynamics with explicit water both beta-turn folds were conserved throughout the simulations. The results obtained with implicit and explicit solvation models are compared, and their consistency with the nmr observations is discussed. The behavior of the linear pentapeptide in this study is in agreement with an earlier report on the consensus conformation of the insect kinin active core derived from analysis of cyclic active analogues.

Conformation in solution and dynamics of a structurally constrained linear insect kinin pentapeptide analogue

The preferred conformations of the active diuretic insect kinin pentapeptide analogue Phe–Phe–Aib–Trp–Gly–NH2 were studied using nmr spectroscopy and molecular modeling. Structure sets consistent with rotating frame nuclear Overhauser effect spectroscopy distance constraints obtained by restrained simulated annealing in vacuo indicate a predominant population of a type II β-turn involving the Phe1–Trp4 region. An equilibrium between this type II and a type I β-turn formed by residues Phe2 and Gly5 was observed in a 5 ns restrained molecular dynamics simulation using the implicit generalized Born solvent accessible surface area (GB/SA) solvation model. When subjected to 500 ps dynamics with explicit water both β-turn folds were conserved throughout the simulations. The results obtained with implicit and explicit solvation models are compared, and their consistency with the nmr observations is discussed. The behavior of the linear pentapeptide in this study is in agreement with an earlier report on the consensus conformation of the insect kinin active core derived from analysis of cyclic active analogues. © 1999 John Wiley & Sons, Inc. Biopoly 49: 403–413, 1999

Model peptide studies of sequence repeats derived from the intracrystalline biomineralization protein, SM50. I. GVGGR and GMGGQ repeats.

We report solution-state pulsed field gradient nmr studies of a native sequence-derived 23-residue peptidomimetic, N alpha-acetyl-QPGVGGRQPGMGGQPGVGGRQPG-C alpha-amid, that incorporates the prevalent GVGGR and GMGGQ repeats found in the sea urchin embryo intracrystalline spicule matrix protein, SM50 (Strongylocentrotus purpuratus). These repeats are sequence homologues of elastin protein repeats (VPGVG, VGGVG, and APGVGV) and spider dragline silk protein repeats (GPGG, GQGG, and QPGYG). Using rotating frame nuclear Overhauser effect (ROE) connectivities, CH alpha proton conformational shifts, 3JNH-CH alpha coupling constants, amide temperature shift coefficients, and pulsed field gradient ROE spectroscopy solvent exchange measurements, we find that the 23-mer peptidomimetic possesses a multiple beta-turn structure in aqueous solution, in equilibria with an extended or coil structure (60% beta-turn: 40% random coil). The GVGGR sequence adopts a double beta-turn conformation that is stabilized by two hydrogen bonds (R7-->V4, R20-->V17; G6-->G3, G19-->G16). The GMGGQ region adopts a single beta-turn conformation that is stabilized by a hydrogen bond involving residues Q14 and M11. Repeating beta-turn structures, or beta-spirals, may play an important role with regard to matrix assembly, protein stability, molecular elasticity, and/or protein-crystal recognition within the spicule mineralized matrix.

Model peptide studies of sequence repeats derived from the intracrystalline biomineralization protein, SM50. I. GVGGR and GMGGQ repeats

We report solution-state pulsed field gradient nmr studies of a native sequence-derived 23-residue peptidomimetic, Nα–acetyl–QPGVGGRQPGMGGQPGVGGRQPG–Cα–amide, that incorporates the prevalent GVGGR and GMGGQ repeats found in the sea urchin embryo intracrystalline spicule matrix protein, SM50 (Strongylocentrotus purpuratus). These repeats are sequence homologues of elastin protein repeats (VPGVG, VGGVG, and APGVGV) and spider dragline silk protein repeats (GPGG, GQGG, and QPGYG). Using rotating frame nuclear Overhauser effect (ROE) connectivities, CHα proton conformational shifts, 3JNH-CHα coupling constants, amide temperature shift coefficients, and pulsed field gradient ROE spectroscopy solvent exchange measurements, we find that the 23-mer peptidomimetic possesses a multiple β-turn structure in aqueous solution, in equilibria with an extended or coil structure (60% β-turn: 40% random coil). The GVGGR sequence adopts a double β-turn conformation that is stabilized by two hydrogen bonds (R7 → V4, R20 → V17; G6 → G3, G19 → G16). The GMGGQ region adopts a single β-turn conformation that is stabilized by a hydrogen bond involving residues Q14 and M11. Repeating β-turn structures, or β-spirals, may play an important role with regard to matrix assembly, protein stability, molecular elasticity, and/or protein–crystal recognition within the spicule mineralized matrix. © 1999 John Wiley & Sons, Inc. Biopoly 49: 303–312, 1999

Structure of the O-specific polysaccharide of a serologically separate strain Proteus penneri 2 from a new proposed serogroup O66.

O-specific polysaccharide chain of Proteus penneri strain 2 lipopolysaccharide was studied by full and partial acid hydrolysis, Smith degradation, methylation analysis, and NMR spectroscopy, including two-dimensional rotating-frame NOE spectroscopy (ROESY) and 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments. Together with D-glucose and 2-acetamido-2-deoxy-D-glucose, the polysaccharide was found to contain two rarely occurring sugars, 6-deoxy-L-talose (L-6dTal) and 2,3-diacetamido-2,3,6-trideoxy-L-mannose (L-RhaNAc3NAc), and the following structure of a non-stoichiometrically O-acetylated tetrasaccharide repeating unit was established: [equation: see text] The O-specific polysaccharide studied has a unique composition and structure and, accordingly, P. penneri 2 is serologically separate among Proteus strains. Therefore, we propose for P. penneri 2 a new Proteus O-serogroup O66 where this strain is at present the single representative.

Boc-Glu-Thr-Ile-His-OMe/Zn 2+ in terms of the zinc-binding sites of proteases

Reaction of Boc-Glu-Thr-Ile-His-OMe (Boc: butoxy carbonyl) (H 2 1), and Zn(ClO 4) 2·6H 2O in N,N-dimethylformamide (DMF) was studied using NMR and mass spectrometry with respect to the active sites of proteases. The study of H 21/Zn 2+ = 1:1 mixture in DMF-d 7 revealed that Zn 2+ is bound to the His-imidazolyl group. The results of IR experiments on the 1:1 dried material suggested that the Glucarboxyl group still remains in the form of -COOH. Mass spectrometry showed that Zn 2+ in H 2 1/Zn 2+ attracts water molecules under the ionization conditions of fast atom bombardment. The temperature ( T) dependence of 1H NMR of H 2 1/Zn 2+ = 1:1 in DMF-d 7, studied in the temperature range 218–323 K, revealed that the Zn 2+ unit undergoes protonation and hydration at low temperatures (below 253 K). The chemical shifts ( δ) of the amide protons were nevertheless linearly dependent on temperature over the range 218–323 K, meaning that the 1:1 solution obeys the common reaction equilibrium and that the averaged main-chain folding of the peptide moiety is conserved. Based on this finding, the distance geometry and restrained molecular dynamics analyses were carried out using the distance information on 61 1H— 1H pairs, extracted from the rotating frame nuclear Overhauser effect spectroscopy experiment on this solution (300 K). These analyses revealed that the averaged main-chain folding of the Glu-Thr-Ile-His is just superimposed on that of the Zn 2+ binding sites of proteases forming a single helix with the Glu carboxyl group situated in the vicinity of the His imidazole group, or is directly but distally bound to Zn 2+ (in the case of γ-COO Glu −). The hydrogen bond between γ-COO Glu − and the H 2O on Zn 2+ was also found in comparatively higher energy area. Gel chromatography of the dried material obtained from H 2 1/Zn 2+ = 1:1 mixture gave only [(H 1) 2Zn] ( 2), as a discrete compound. The coordination atoms to Zn 2+ in 2 were assigned as Glu amide (N −) and His imidazole nitrogens.

Structure of the O polysaccharide and immunochemical relationships between the lipopolysaccharides of Pseudomonas syringae pathovar tomato and pathovar maculicola.

O polysaccharides (OPS) of the lipopolysaccharides (LPS) of Pseudomonas syringae pathovar tomato GSPB 483 and pathovar maculicola IMV 381 were studied by 1H-NMR and 13C-NMR spectroscopy, including two-dimensional COSY, rotating-frame NOE spectroscopy (ROESY), and H-detected 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments. The OPS from both strains were shown to be chemically identical. Two structurally different types of repeating units (O repeats 1 and 2) were elucidated in each OPS. The minor O repeat is a pentasaccharide having the structure 2. The same structure has been reported earlier for some other OPS of P. syringae. The major O repeat is a hexasaccharide with the novel structure 1 which is distinguished from 2 by the presence of the second lateral residue of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc) and by a different position of substitution of one of the L-rhamnose (Rha) residues in the backbone. [structures: see text] Structural and serological diversity of OPS of strains belonging to P. syringae pv. tomato and pv. maculicola and phylogenetic relatedness of the strains are discussed.

Structure of the acidic polysaccharide chain of the lipopolysaccharide of Shewanella alga 48055

A lipopolysaccharide (LPS) with an acidic polysaccharide chain was isolated from the bacterium Shewanella alga strain 48055 and cleaved selectively at the glycosidic linkage of N-acetylneuraminic acid to give a tetrasaccharide. Studies of the tetrasaccharide and the O-deacylated LPS by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, NOESY, rotating-frame NOE spectroscopy (ROESY), and H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC) experiments, revealed the following structure of the polysaccharide repeating unit: →3)- β- d-Gal pA6GroN-(1⃗3)- β- d-Glc pNAc-(1⃗3)- α- d-Gal pA6GroN-(1⃗4)- α-Neu p5Ac-(2⃗ where GroN is an amidically linked residue of 2-amino-1,3-propanediol (2-amino-2-deoxyglycerol). A similar structure, but with 2-acetamido-2,6-dideoxy- d-glucose instead of 2-acetamido-2-deoxy- d-glucose, has been reported previously for the polysaccharide chain of a non-O1 Vibrio cholerae H11 LPS [E.V. Vinogradov, O. Holst, J.E. Thomas-Oates, K.W. Broady, and H. Brade, Eur. J. Biochem., 210 (1992) 491–498].

Three-dimensional structure of moenomycin A--a potent inhibitor of penicillin-binding protein 1b.

The first three-dimensional structure of moenomycin A in aqueous solution based on NMR-derived distance constraints and molecular dynamics simulations is presented. The antibiotic moenomycin A was obtained from the FlavomycinR complex by ultrafiltration, chromatography on a DEAE-cellulose ion exchanger and reverse-phase chromatography in 98% purity. In contrast to the previously reported behaviour, the compound gave rise to well-resolved NMR spectra in standard solvents. Using several two-dimensional experiments, a complete assignment of proton and carbon chemical shifts was achieved in (CD3)2SO, CD3OD and H2O/D2O (9:1, pH 7.3). A total of 175 interproton distances were determined from 600-MHz rotating-frame NOE (ROE) spectra and were used as restraints in molecular dynamics calculations. These restraints included 84 ROEs between protons of the moenocinol part leading to a very well-defined structure of the lipid part of the molecule. The relative orientation of the subunits was determined by 66 ROEs among different sugar rings and between the sugar rings and moenocinol. As a result of the molecular dynamics calculation, rings D, E, and F as well as the moenocinol part are very well-defined (average rms deviation over all heavy atoms 0.48 A) whereas rings A, B and C display a higher degree of conformational flexibility which might be an artefact due to the lower number of ROEs in this part of the molecule. A three-dimensional pharmacophore hypothesis comprising functional groups of rings E and F and the carboxyl group of glyceric acid is presented on the basis of the degradation and derivatization studies of Welzel and coworkers [Welzel, P., Kunisch, F., Kruggel, F., Stein, H., Scherkenbeck, J., Hiltmann, A., Duddeck, H., Müller, D., Maggio, J. E., Fehlhaber, H.-W., Seibert, G., van Heijenoort, Y. & van Heijenoort, J. (1987) Moenomycin A: Minimum structural requirements for biological activity, Tetrahedron 43, 585-598; Welzel, P. (1993) Transglycosylase inhibition, in Antibiotics and antiviral compounds--Chemical synthesis and modifications (Krohn, K., Kirst, H. A. & Maag, H., eds) pp. 373-378, VCH Weinheim].

Conformer selection and differential restriction of ligand mobility by a plant lectin--conformational behaviour of Galbeta1-3GlcNAcbeta1-R, Galbeta1-3GalNAcbeta1-R and Galbeta1-2Galbeta1-R' in the free state and complexed with galactoside-specific mistletoe lectin as revealed by random-walk and conformational-clustering molecular-mechanics.

To study conformational parameters of ligands before and after complex formation with the galactoside-binding agglutinin of Viscum album L. (VAA) in solution, combined computer-assisted random walk molecular mechanics (RAMM) calculations extended by conformational clustering analysis (CCA), molecular dynamics (MD) simulations as well as two-dimensional rotating-frame nuclear Overhauser effect (ROE) and two-dimensional nuclear Overhauser effect (NOE) spectroscopy NMR experiments were employed. Derivatives of the naturally occurring disaccharides Galbeta1-3GlcNAcbeta1-R and Galbeta1-3GalNAcbeta1-R as well as of a synthetic high-affinity binding partner, i.e. the disaccharide Galbeta1-2Galbeta1-R', were chosen as ligands in this study. The disaccharides displayed inherent flexibility in the valley of the global minimum between phi/psi combinations of (40 degrees/60 degrees) and (40 degrees/-60 degrees). Calculations of the de-N-acetylated sugars revealed that presence of this group did not markedly influence the distribution of low-energy conformers in the phi, psi, epsilon plot. Occupation of side minima at phi/psi (180 degrees/0 degrees) or (0 degrees/180 degrees) is either unlikely or low according to the results of MD simulations and RAMM calculations extended by CCA. Notably, these side minima define conformations which are not stable during a MD simulation. Transitions to other minima occur already a few picoseconds after the start of the simulation. NMR experiments of the free-state ligand confirmed the validity of the data sets obtained by the calculations. Following the description of the conformational space in the free-state NMR experiments were performed for these disaccharides complexed with VAA. They yielded two interresidual contacts for Galbeta1-3GlcNAcbeta1-R and Galbeta1-2Galbeta1-R'. The ligand conformations in the complex did not deviate markedly from those of a minimum conformation in the free state. One- and two-dimensional transferred nuclear Overhauser enhancement (TRNOE) experiments at different mixing times excluded the influence of spin-diffusion effects. When the NOE build-up curves in the three studied cases were compared, the residual mobility of the penultimate carbohydrate unit of Galbeta1-3GalNAcbeta1-R was observed to be higher than that of the respective hexopyranose unit of the other two bound ligands. Due to the availability of the conformational parameters of Galbeta1-2Galbeta1-R' in association with a galectin, namely the beta-galactoside-binding protein from chicken liver, it is remarkable to note that this ligand displays different conformations in the binding sites of either the plant or the animal lectin. They correspond to local energy-minimum conformations in the phi,psi, epsilon plot and substantiate differential conformer selection by these two lectins with identical nominal monosaccharide specificity.

Screening of cyclodextrins by nuclear magnetic resonance for the design of chiral capillary electrophoresis separations

High-field one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy and capillary electrophoresis (CE) are examined to investigate the process of chiral recognition occurring between different cyclodextrins (CDs) and the racemic anti-schistosomiasis drug, oxamniquine. Five neutral CDs (α-CD, β-CD, γ-CD, hydroxypropyl-β-CD and hydroxyethyl-β-CD) and two anionic CDs, carboxymethyl-β-CD (CM-β-CD) and sulphobutyl ether-β-CD (SBE-β-CD) were selected for these NMR and CE studies. Three batches of the anionic CD [SBE-β-CD(7), SBE-β-CD(4) and SBE-β-CD(1)] differing in their degree of substitution (6.5, 4.5 and 1.0, respectively), were also examined in these studies. In the 1D NMR studies, the shift displacement values (Δδ Hz), after addition of each of the nine CDs in a 1:1 molar ratio, the diagnostic aromatic singlets indicated interaction between oxamniquine and each of the CDs. With regard to shift non-equivalence (Δδ*) for the two aromatic singlets, the anionic CDs were the only agents to show any interaction. It was also found that by manipulating the sample pH value, the ionic state of both selector and selectand had an influence on the enantiorecognition process. The complex between oxamniquine and the SBE-β-CD(7) was subsequently examined using 2D rotating frame nuclear Overhauser effect spectroscopy (ROESY), and the complexation process was found to involve an inclusion mechanism. Spin-lattice relaxation time (T1) measurements confirmed this observation. In the CE studies, each of the nine CDs was examined as a chiral electrolyte additive for the enantioseparation of racemic oxamniquine. Complete separation was not found when using any of the neutral CDs, but in contrast to the anionic CDs, CM-β-CD and the SBE-β-CD(4), displayed a 95 and 100% separation, respectively. Interestingly, the higher and lower derivatised SBE-β-CD selectors yielded 10% and zero separation, respectively. This is strong evidence that the degree of substitution has a critical effect on the enantiorecognition process. Good correlation between the NMR shift non-equivalence data for oxamniquine and the degree of enantioseparation was observed in CE. The neutral CDs displayed no shift non-equivalence for oxamniquine and no enantioseparation was found. However, for the anionic CDs a significant shift non-equivalence was observed and this corresponded to a significant enantioseparation in CE.