Derivation of 13C chemical shift surfaces for the anomeric carbons of oligosaccharides and glycopeptides using ab initio methodology.

The dependence between the anomeric carbon chemical shift and the glycosidic bond (phi, psi) dihedral angles in oligosaccharide and glycopeptide model compounds was studied by Gauge-Including Atomic Orbital (GIAO) ab initio calculations. Complete chemical shift surfaces versus phi and psi for D-Glcp-D-Glcp disaccharides with (1-->1), (1-->2), (1-->3), and (1-->4) linkages in both alpha- and beta-configurations were computed using a 3-21G basis set, and scaled to reference results from calculations at the 6-311G** level of theory. Similar surfaces were obtained for GlcNAcThr and GlcNAcSer model glycopeptides in alpha- and beta-configurations, using in this case different conformations for the peptide moiety. The results obtained for both families of model compounds are discussed. We also present the determination of empirical formulas of the form 13Cdelta = f (phi,psi) obtained by fitting the raw ab initio data to trigonometric series expansions suitable for use in molecular mechanics and dynamics simulations. Our investigations are consistent with experimental observations and earlier calculations performed on smaller glycosidic bond models, and show the applicability of chemical shift surfaces in the study of the conformational behavior of oligosaccharides and glycopeptides.

Design, synthesis, and biological evaluation of a series of simple and novel potential antimalarial compounds.

A series of compounds bearing an endocyclic -N-O- moiety with potential antimalarial activity based on simple derivatives of the tropolone purpurogallin was prepared by means of a hetero Diels-Alder reaction using nitrosobenzene as a dienophile. The rationale behind the design of these compounds is presented, together with the synthetic route to derivatives bearing aromatic and aliphatic esters of the C4'-position hydroxyl group of the purpurogallin framework, as well as biological data obtained from in vitro assays against Plasmodium falciparum and Trypanosoma cruzi. Several of the new compounds have activities in the 3-9 microM range, and provide leads for the development of a novel class of antiparasitic drugs with improved biological and pharmacological properties.

Detection of nascent polyproline II helices in solution by NMR in synthetic insect kinin neuropeptide mimics containing the X-Pro-Pro-X motif.

The conformations of three synthetic peptide analogs containing the dPro-dPro-dXaa motif (dXaa = dThr, dGlu, dAsn) in aqueous solution were studied by a combination of NMR and molecular modeling simulations. The three compounds were identified from a random D-amino acid tripeptide library on the basis of their ability to either mimic or block the diuretic activity of neuropeptides of the insect kinin family. TOCSY and ROESY correlations, as well as abnormal secondary chemical shifts for protons on the D-proline residues were employed to obtain conformational ensembles consistent with the experimental NMR data for the three analogs using an in vacuo simulated annealing protocol. Similar secondary structures were found for the three molecules after refinement, in agreement with the similarities observed between their NMR spectra. Unrestrained molecular dynamics simulations with explicit water representation indicate that the structural motifs found in vacuo are stable in aqueous solution. The three analogs can be considered initiators of right-handed poly D-proline II helices, mirror images of the poly L-proline II left-handed helical motifs normally found in proline-rich proteins. The role of these secondary folds on binding of the analogs to the kinin receptors is discussed.

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.

Comparison of active conformations of the insectatachykinin/tachykinin and insect kinin/Tyr-W-MIF-1 neuropeptide family pairs.

A comparison of solution conformations of active, restricted-conformation analogues of two sequence-similar insect/vertebrate neuropeptide family pairs shed light on the potential existence of molecular evolutionary relationships. Analogues of the locustatachykinins and the mammalian tachykinin substance P, containing a sterically hindered Aib-NMePhe/Tyr residue block, share similar low-energy turn conformations incorporating a cis peptide bond. Conversely, restricted conformation analogues of the insect kinins and the mammalian opiate peptide Tyr-W-MIF-1, with near identical C-terminal tetrapeptide sequences, adopt different conformations. The insect kinins adopt a cisPro 1-4 beta-turn, in which the Phe1 is critical for bioactivity. Tyr-W-MIF-1 prefers a transPro 2-5 turn, and an additional N-terminal Phe severely inhibits mu-opiate receptor binding. Comparisons of the chemical/conformational requirements for receptor interaction are consistent with a distant evolutionary relationship between the insectatachykinins and tachykinins, but not between the insect kinins and Tyr-W-MIF-1. Therefore, analogues of the insect kinins with pest control potential can be readily designed to avoid mammalian interactions.

Synthesis, biological activity, and conformational studies of insect allatostatin neuropeptide analogues incorporating turn-promoting moieties.

Allatostatins are 6-18 amino acid peptides synthezed by insects to control production of juvenile hormones, which in turn regulate functions including metamorphosis and egg production. Four insect allatostatin neuropeptide analogues incorporating turn-promoting pseudopeptide moieties in the region responsible for biological activity were prepared by solid phase peptide synthetic methods. Bioassay indicated that activities approached those of the natural neuropeptides, and molecular models based on NMR data showed similar conformations and the presence of a beta-turn in the active core region for the four analogues. Differences in activity are believed to be due to differences in bulk and relative position of atoms in the unnatural portion of the analogues, and their differing degrees of conformational freedom. The studies support the feasibility of development of neuropeptide-based insect control agents resistant to peptidase deactivation.

Comparison of ring current methods for use in molecular modeling refinement of NMR derived three-dimensional structures.

A comparison between three different methods commonly used to estimate ring current effects on chemical shifts is presented. Haigh-Mallion, Johnson-Bovey, and classical point-dipole approximations were used to estimate the ring current contribution to chemical shifts for protons in several proteins for which both detailed X-ray crystal structures and chemical shift assignments were available. For the classical point-dipole model, new proportionality constants were calculated by fitting to ring current estimations from both the quantum-mechanical Haigh-Mallion and semiclassical Johnson-Bovey methods and compared with the previously used point-dipole constant of Perkins and Dwek. Statistical analysis of the predictions obtained by all methods indicates that the point-dipole approximation parametrized against quantum-mechanical data is superior to the previously used classical model, comparable to Johnson-Bovey calculations, and slightly poorer than predictions from the Haigh-Mallion theory. The implementation of a pseudoenergy penalty term for use in structure refinement from chemical shift data based on the classical point-dipole model is described, and its usefulness in cases where other NMR information is limited is discussed with a specific example.

Conformational studies of paclitaxel analogs modified at the C-2' position in hydrophobic and hydrophilic solvent systems.

The conformations of two paclitaxel analogs modified at the C-2' position, 2'-deoxypaclitaxel and 2'-methoxypaclitaxel, were studied in hydrophobic and hydrophilic solvent systems by a combination of NMR spectroscopy, CD measurements, and molecular modeling. Both analogs have hydrophobic and hydrophilic conformations that resemble those of paclitaxel itself in the same media. Since the two have diminished biological activities in a number of bioactivity assays and the hydrogen-bonding capability of the 2'-hydroxyl group has been eliminated, we postulate that this group is involved in hydrogen bonding with tubulin and plays an important role in molecular recognition. The results of this study are in agreement with our earlier report on paclitaxel 2'-acetate, an analog in which the 2'-hydroxyl group hydrogen-bonding capacity has also been eliminated.

Development of Weiner et al. force field parameters suitable for conformational studies of [1,4]-benzodiazepines and related compounds.

A set of force field parameters capable of reproducing the preferred conformations of the biologically important [1,4]-benzodiazepines was developed for AMBER and other molecular modeling programs that utilize the Weiner et al. force field. Equilibrium parameters were obtained from representative model compounds found in the Cambridge Structural Database, and bond stretching and torsion potential force constants were estimated using AM1 and PM3 semiempirical Hamiltonians. Parameters obtained with the two semiempirical methods and the existing linear interpolation method are compared. Molecular mechanics and dynamic simulations showed that AM1 derived parameters, together with MNDO ESP fitted atomic charges, predicted the X-ray structure of a number of representative [1,4]-benzodiazepines within 0.01 A, 0.8 degree, and 5 degrees, from observed bond lengths, bond angles, and bond torsions, respectively.

A simple algorithm for superimposing sets of NMR derived structures: its application to the conformational study of cephalomannine in lipophobic and lipophilic solution.

A simple iterative method for superimposing sets of NMR derived structures and calculation of the root mean square deviation (RMSD) of the sets is described. It was compared to the commonly used algorithm involving pairwise best fitting in the conformational study of the taxoid anticancer drug cephalomannine in lipophobic and lipophilic solvents. Lower RMSD values were obtained, indicating a better superposition of the structures in the sets. The conformations of cephalomannine in the two solvent systems reported are in good agreement with earlier conformational studies on other active taxoids.

NMR and molecular modeling study of the conformations of taxol 2'-acetate in chloroform and aqueous dimethyl sulfoxide solutions.

Taxol 2'-acetate, an analog of the antitumor drug taxol, displays no significant in vitro microtubule polymerization activity, thus underscoring the importance of a free 2'-OH group to the biological activity of taxol. Previous work had suggested that the inactivity of taxol 2'-acetate is not due to steric interference by the acetyl group. The present study examined the conformations of taxol 2'-acetate in deuteriochloroform and (2)H2O-deuteriodimethyl sulfoxide solutions and found them to be essentially the same as the respective conformations adopted by taxol itself. Thus, neither destabilization of an active taxol conformation by the acetyl group nor the formation of an important taxol conformation determining role for the 2'-OH group appears likely. The implication of these findings is that the taxol 2'-OH group interacts directly with a protein residue in the taxol-microtubule complex, perhaps as a hydrogen bond donor.

Diversity in cyclic sesquiterpene production by Gossypium hirsutum.

Major sesquiterpene components of oil of Texas Race Stock 810 of Gossypium hirsutum were alpha- and beta-selinene. This is the seventh cyclic terpene type found to date in this genus. Both alpha- and beta-selinene, along with aromadendrene, were found but only as minor components of extracts of several domestic cultivars of G. hirsutum.

Metabolism of benzbromarone in man: structures of new oxidative metabolites, 6-hydroxy- and 1'-oxo-benzbromarone, and the enantioselective formation and elimination of 1'-hydroxybenzbromarone.

1. The uricosuric drug benzbromarone is extensively metabolized in man and two main metabolites are formed: the previously characterized 1'-hydroxybenzbromarone (metabolite M1) and an arylhydroxybenzbromarone (metabolite M2) of unknown structure. A dimethyl derivative was isolated from urine after methylation and was characterized by gas chromatography-mass spectrometry (g.l.c.-m.s.) and high resolution nuclear magnetic resonance spectroscopy as 4''-O-methyl-6-methoxybenzbromarone; the structure of M2 therefore is 6-hydroxybenzbromarone. 2. A minor metabolite was similarly characterized as 1'-oxobenzbromarone by comparison with authentic synthetic samples and is a product of biodegradation and not an artifact derived from the in vitro oxidation of 1'-hydroxybenzbromarone. Further minor metabolites were detected and were provisionally characterized by g.l.c.-m.s. after derivatization and include: 2'-hydroxybenzbromarone (an isomer of 1'-hydroxybenzbromarone); 1',6-dihydroxybenzbromarone; dihydroxy-aryl-benzbromarone; and two structure isomers of 6-hydroxybenzbromarone. Debrominated metabolites were not detectable. 3. Benzbromarone is hydroxylated in vivo at the prochiral centre C1' to 1'-hydroxybenzbromarone; analysis of 1'-hydroxybenzbromarone from plasma and urine extracts by h.p.l.c. using a chiral column revealed that two peaks were eluted which showed a mean enantiomeric ratio of 2.1 for plasma and 7.3 for urine; these data demonstrate that the formation and elimination of this metabolite is enantioselective; the absolute configuration of the 1'-chiral centre is presently unknown.