DFTr studies of five- and six-residue cyclic-ß(1→4) cellulosic molecules.

Density functional (DFT) conformational in vacuo studies of cellobiose have shown that ϕ(H) -anti conformations are low in energy relative to the syn forms, while the ψ(H) -anti forms are higher in energy. Further, as the cellulosic fragments became larger than a disaccharide and new hydrogen bonding interactions between multiple residues become available, stable low energy ϕ(H) -anti, and ψ(H) -anti cellulosic structures became possible. To test the stability of cyclic anti-conformations, a number of ß-linked five- and six-residue molecules were created and then energy optimized in solvent (water, n-heptane) using the implicit solvation method COSMO at the B3LYP level of theory. The created symmetric cyclic structures were without distortion. Upon optimization some cyclic conformations were found to be of low energy when compared with linear five- and six-residue chains, after correcting the energy for the exclusion of a water molecule upon cyclization. It was also obvious from the hydrogen bonding network formed above and below the plane of the cyclic structure that these structures could exhibit strong synergistic tendencies. The conformational energy preferences for clockwise "c" and counter-clockwise "r" hydroxyl groups and preference for the hydroxymethyl rotamers is described. Because these structures contain energetically unfavorable flipped conformations in water, that is, dihedral angles of ∼180°/0° or ∼0°/180° in ϕ(H) /ψ(H) , it is clear that the synthesis of these compounds will be challenging.

DFT energy optimization of a large carbohydrate: cyclomaltohexaicosaose (CA-26).

CA-26 is the largest cyclodextrin (546 atoms) for which refined X-ray structural data is available. Because of its size, 26 D-glucose residues, it is beyond the scope of study of most ab initio or density functional methods and to date has only been computationally examined using empirical force fields. The crystal structure of CA-26 is folded like a figure "8" into two 10 D-glucoses long antiparallel left-handed V (Verkleisterung)-type helices with a "band-flip" and "kink" at the top and bottom of the helices. DFTr methods were applied to CA-26 to determine if a carbohydrate molecule of this size could be geometry optimized and if it would show structural variances from application of dispersion and/or solvation. The DFTr reduced basis set method developed by the authors uses 4-31G on the carbon atoms of the glucose rings and 6-31+G* on all other atoms. B3LYP is the density functional used to successfully optimize CA-26, and other density functionals were then applied, including a self-consistent charge density functional tight binding (SCC-DFTB) method and the B97D (dispersion-corrected) and B97D-PCM (dispersion + implicit solvent) methods. Heavy atom coordinates were taken from one X-ray structure, fitted with hydrogen atoms, and geometry optimized using PM3 followed by B3LYP/6-31+G*/4-31G optimization. After optimization, the heavy atom rms deviation of the optimized DFTr (B3LYP) structure to the crystal structure was 0.89 Å, the rmsd of the B97D optimization was 1.38 Å, that for B97D-PCM was 0.95 Å, and that for SCC-DFTB was 0.94 Å. These results are very good considering that no explicit water molecules were included in the computational analysis and there were ~32-38 water molecules around each CA-26 molecule in the crystal structure. Tables of internal coordinates and puckering parameters were compared to the X-ray structures, and close correspondence was found.

Male-specific sesquiterpenes from Phyllotreta flea beetles.

Flea beetles in several genera are known to possess male-specific sesquiterpenes, at least some of which serve as aggregation pheromones that attract both sexes. In continuing research on the chemical ecology of Phyllotreta flea beetles, six new male-specific sesquiterpenes were identified, one from P. striolata (hydroxyketone 9) and five from P. pusilla (aldehydes 10-12 and 14 and alcohol 13); both species are crop pests. The minute amounts from beetles provided mass spectra and chromatographic data but were insufficient for complete structure determination. However, it was discovered that the new compounds could all be produced by applying organic reactions to previously identified flea beetle sesquiterpenes, and the resulting, larger amounts of material permitted definitive structure analysis by NMR. Molecular modeling was used in conjunction with NMR to define relative configurations of several newly created stereogenic centers. The absolute configurations of natural 9-14 were established by chiral gas chromatography/mass spectrometry. In electrophysiological tests (GC-EAD) conducted with P. striolata, compound 9 was detected with high sensitivity by the beetle antennae, which is consistent with a pheromonal function. The research opens new possibilities for using behavioral chemicals to monitor or manage these pest species.

27 ps DFT molecular dynamics simulation of alpha-maltose: A reduced basis set study.

DFT molecular dynamics simulations are time intensive when carried out on carbohydrates such as alpha-maltose. In a recent publication (Momany et al., J. Mol. Struct. THEOCHEM, submitted) forces for dynamics were generated from B3LYP/6-31+G* electronic structure calculations. The implicit solvent method COSMO was applied to simulate the solution environment. Here we present a modification of the DFT method that keeps the critical aspects of the larger basis set (B3LYP/6-31+G*) while allowing the less-essential atom interactions to be calculated using a smaller basis set, thus allowing for faster completion without sacrificing the interactions dictating the hydrogen bonding networks in alpha-maltose. In previous studies, the gg'-gg-c solvated form quickly converged to the "r" form during a 5 ps dynamics run. This important conformational transition is tested by carrying out a long 27 ps simulation. The trend for the "r" conformer to be most stable during dynamics when fully solvated, is confirmed, resulting in approximately 20/80% c/r population. Further, the study shows that considerable molecular end effects are important, the reducing end being fairly stable, the O6-H pointing at the O5, while the nonreducing end moves freely to take on different conformations. Some "kink" and transition state forms are populated during the simulation. The average H1'...H4 distance of 2.28 A confirms that the syn form is the primary glycosidic conformation, while the average C1'-O1'-C4 bond angle was 118.8 degrees , in excellent agreement with experimental values. The length of this simulation allowed the evaluation of vibrational frequencies by Fourier transform of the velocity correlation function, taken from different time segments along the simulation path.

DFT conformation and energies of amylose fragments at atomic resolution. Part 1: Syn forms of alpha-maltotetraose.

DFT optimization studies of 90 syn alpha-maltotetraose (DP-4) amylose fragments have been carried out at the B3LYP/6-311++G** level of theory. The DP-4 fragments studied include V-helix, tightly bent conformations, a boat, and a (1)C(4) conformer. The standard hydroxymethyl rotamers (gg, gt, tg) were examined at different locations in the residue sequence, and their influence on the bridge conformations phi/psi values and conformer energy is described. Hydroxyl groups were considered to be homodromic, that is, they are either in the all clockwise, 'c', or all counterclockwise, 'r'. Energy differences between conformations are examined in order to assess the stability of the different conformations and to identify the sources of energy that dictate amylose polymer formation. A small nearly cyclic compact structure is of low energy as one would expect when these flexible molecules are studied in vacuo. Many conformations in which the only differences are a single hydroxymethyl variation in the residue sequence show similar energies and bridge conformations, with trends being a result of the hydroxymethyl as well as hydroxyl orientation. In general the 'c' structures are of lower energy than the 'r' structures, although this is only true for the in vacuo state. The solvent dependence on conformational preference of several low-energy DP-4 structures was investigated via the continuum solvation method COSMO. These results suggest that the 'r' structures may be favored for fully solvated molecules.

DFT conformation and energies of amylose fragments at atomic resolution. Part 2: 'Band-flip' and 'kink' forms of alpha-maltotetraose.

In Part 2 of this series of DFT optimization studies of alpha-maltotetraose, we present results at the B3LYP/6-311++G** level of theory for conformations denoted 'band-flips' and 'kinks'. Recent experimental X-ray studies have found examples of amylose fragments with conformations distorted from the usual syn forms, and it was of interest to examine these novel structural motifs by the same high-level DFT methods used in Part 1. As in Part 1, we have examined numerous hydroxymethyl rotamers (gg, gt, and tg) at different locations in the residue sequence, and include the two hydroxyl rotamers, the clockwise 'c' and counterclockwise 'r' forms. A total of fifty conformations were calculated and energy differences were found to attempt to identify those sources of electronic energy that dictate stressed amylose conformations. Most stressed conformations were found to have relative energies considerably greater (i.e., approximately 4 to 12 kcal/mol) than the lowest energy syn forms. Relative energy differences between 'c' and 'r' forms are somewhat mixed with some stressed conformations being 'c' favored and some 'r' favored, with the lowest energy 'kink' form being an all-gg-r conformation with the 'kink' in the bc glycosidic dihedral angles. Comparison of our calculated structures with experimental results shows very close correspondence in dihedral angles.

DFT conformational studies of alpha-maltotriose.

Recent DFT optimization studies on alpha-maltose improved our understanding of the preferred conformations of alpha-maltose. The present study extends these studies to alpha-maltotriose with three alpha-D-glucopyranose residues linked by two alpha-[1-->4] bridges, denoted herein as DP-3's. Combinations of gg, gt, and tg hydroxymethyl groups are included for both "c" and "r" hydroxyl rotamers. When the hydroxymethyl groups are for example, gg-gg-gg, and the hydroxyl groups are rotated from all clockwise, "c", to all counterclockwise, "r", the minimum energy positions of the bridging dihedral angles (phi(H) and psi(H)) move from the region of conformational space of (-, -), relative to (0 degrees , 0 degrees), to a new position defined by (+, +). Further, it was found previously that the relative energies of alpha-maltose gg-gg-c and "r" conformations were very close to one another; however, the DP-3's relative energies between hydroxyl "c" or "r" rotamers differ by more than one kcal/mol, in favor of the "c" form, even though the lowest energy DP-3 conformations have glycosidic dihedral angles similar to those found in the alpha-maltose study. Preliminary solvation studies using COSMO, a dielectric solvation method, point to important solvent contributions that reverse the energy profiles, showing an energy preference for the "r" forms. Only structures in which the rings are in the chair conformation are presented here.

Differential EI fragmentation pathways for peracetylated C-glycoside ketones as a consequence of bicyclic ketal ring structures.

Several C-glycoside ketones and peracetylated C-glycoside ketones have been synthesized from 13 structurally-diverse aldoses sugars (including isotope labeled [1-(13)C]Glc, [U-(13)C]Glc, and [6, 6'-(2)H(2)]Glc) via an aqueous-based Knoevanagel condensation with aliphatic 1,3-diketones. Sodium adduct molecular ions observed by MALDI-TOF MS confirmed that the reactions are essentially quantitative, and that the acetylation products are the expected peracetylated C-glycoside ketones, rather than cyclized ketofurans. Analysis of the peracetylated C-glycoside ketones by gas chromatography-EI-MS show characteristic fragment ions that have been assigned to four distinct fragmentation pathways. Peracetylated aldohexose-, aldopentose-, and 6-deoxyaldohexose-C-glycoside ketones fragment via gas phase furanoid intermediates. These data, and DFT calculations, indicate that the furanoid intermediates arise because the peracetylated C-glycoside ketones adopt a bicyclic structure containing a 5-member ketal ring. This ketal ring is the precursor of the furanoid rings in the gas phase. The 2-deoxyhexose-C-glycoside ketones are unable to form an intramolecular 2-ketal bond, and therefore undergo ion fragmentations via nonfuranoid pathways.

DFT studies of the disaccharide, alpha-maltose: relaxed isopotential maps.

The disaccharide, alpha-maltose, forms the molecular basis for the analysis of the structure of starch, and determining the conformational energy landscape as the molecule oscillates around the glycosidic bonds is of importance. Thus, it is of interest to determine, using density functionals and a medium size basis set, a relaxed isopotential contour map plotted as a function of the phi(H) and psi(H) dihedral angles. The technical aspects include the method of choosing the starting conformations, the choice of scanning step size, the method of constraining the specific dihedral angles, and the fitting of data to obtain well defined contour maps. Maps were calculated at the B3LYP/6-31+G( *) level of theory in 5 degrees intervals around the (phi(H),psi(H))=(0 degrees ,0 degrees ) position, out to approximately +/-30 degrees or greater, for gg-gg'-c, gg-gg'-r, gt-gt'-c, gt-gt'-r, tg-tg'-c, and tg-tg'-r conformers, as well as one-split gg(c)-gg'(r) conformer. The results show that the preferred conformation of alpha-maltose in vacuo depends strongly upon the hydroxyl group orientations ('c'/'r'), but the energy landscape moving away from the minimum-energy position is generally shallow and transitions between conformational positions can occur without the addition of significant energy. Mapped deviations of selected parameters such as the dipole moment; the C1-O1-C4', H1-C1-O1, and H4'-C4'-O1 bond angles; and deviations in hydroxymethyl rotamers, O5-C5-C6-O6, O5'-C5'-C6'-O6', C5-C6-O6-H, and C5'-C6'-O6'-H', are presented. These allow visualization of the structural and energetic changes that occur upon rotation about the glycosidic bonds. Interactions across the bridge are visualized by deviations in H(O2)...O3', H(O3')...O2, and H1...H4' distances and the H(O2)-O2-C2-C1 and H'(O3')-O3'-C3'-C4' hydroxyl dihedral angles.

DFT study of alpha- and beta-D-allopyranose at the B3LYP/6-311++G ** level of theory.

One hundred and two conformations of alpha- and beta-D-allopyranose, the C-3 substituted epimer of glucopyranose, were geometry optimized using the density functional, B3LYP, and the basis set, 6-311++G **. Full geometry optimization was performed on different ring geometries and on the hydroxymethyl rotamers (gg/gt/tg). Analytically derived Hessians were used to calculate zero point energy, enthalpy, and entropy. The lowest energy and free energy conformation found is the alpha-tg(g-)-4C1-c conformation, which is only slightly higher in electronic (approximately 0.2 kcal/mol) and free energy than the lowest energy alpha-D-glucopyranose. The in vacuo calculations showed a small (approximately 0.3 kcal/mol) energetic preference for the alpha- over the beta-anomer for allopyranose in the 4C1 conformation, whereas in the 1C4 conformation a considerable (approximately 1.6 kcal/mol) energetic preference for the beta- over the alpha-anomer for allopyranose was encountered. The results are compared to previous aldohexose calculations in vacuo. Boat and skew forms were found that remained stable upon gradient optimization although many starting boat conformations moved to other skew forms upon optimization. As found for glucose, mannose, and galactose the orientation and interaction of the hydroxyl groups make the most significant contributions to the conformation/energy relationship in vacuo. A comparison of different basis sets and density functionals is made in the Discussion section, confirming the appropriateness of the level of theory used here.

Determination of the preferred conformation of the bicyclic Galerucella pheromone using density functional theory optimization and calculations of chemical shifts.

A pheromone from the beetle, Galerucella calmariensis, was recently isolated and identified (Bartelt, R. J. et al. J. Chem. Ecol. 2006, 32, 693-712) as a 14-carbon, bicyclic dimethylfuran lactone, with the systematic name 12,13-dimethyl-5,14-dioxabicyclo[9.2.1]tetradeca-1(13),11-dien-4-one. The main 12-membered lactone ring is very flexible; as a result, there exist multiple possible conformations. The preferred conformation cannot be deduced solely from room-temperature NMR measurements. Using density functional (DFT) studies, 26 unique conformers with energies within 10.0 kcal/mol of the global minimum-energy structure were found. A mirror-image plane exists so that each conformer has an "inverse" structure with the same energy, for which the dihedral angles around the flexible ring have opposite sign. The isotropic 1H and 13C NMR chemical shifts of the DFT-optimized structures were calculated using the gauge-including atomic orbital (GIAO) method. By considering the relative energies of the conformers and the calculated and observed NMR spectra, we concluded that the molecule exists primarily as a mixture of two distinct conformers at room temperature, each being present with its mirror-image inverse. Structural interconversions among these likely occur on a time scale that is fast compared to the NMR experiments. Using mode-following and dihedral-driving techniques, several potential pathways were found for the conversion of the lowest-energy conformer to its mirror-image structure. Ab initio molecular dynamics (AIMD) using the 4-31G basis set was carried out for 50 ps to test the availability of various low-energy minima and the transition states found from the searches noted above.

Structural characterization of alpha-zein.

A variety of published physical measurements, computational algorithms, and structural modeling methods have been used to create a molecular model of 19 kDa alpha-zein (Z19). Zetaeins are water-insoluble storage proteins found in corn protein bodies. Analyses of the protein sequence using probability algorithms, structural studies by circular dichroism, infrared spectroscopy, small-angle X-ray scattering (SAXS), light scattering, proton exchange, NMR, and optical rotatory dispersion experiments suggest that Z19 has approximately 35-60% helical character, made up of nine helical segments of about 20 amino acids with glutamine-rich "turns" or "loops". SAXS and light-scattering experiments suggest that in alcohol/water mixtures alpha-zein exists as an oblong structure with an axial ratio of approximately 6:1. Furthermore, ultracentifugation, birefringence, dielectric, and viscosity studies indicate that alpha-zein behaves as an asymmetric particle with an axial ratio of from 7:1 to 28:1. Published models of alpha-zein to date have not been consistent with the experimental data, and for this reason the structure was re-examined using molecular mechanics and dynamics simulations creating a new three-dimensional (3D) structure for Z19. From the amino acid sequence and probability algorithms this analysis suggested that alpha-zein has coiled-coil tendencies resulting in alpha-helices with about four residues per turn in the central helical sections with the nonpolar residue side chains forming a hydrophobic face inside a triple superhelix. The nine helical segments of the 19 kDa protein were modeled into three sets of three interacting coiled-coil helices with segments positioned end to end. The resulting structure lengthens with the addition of the N- and C-terminal sections, to give an axial ratio of approximately 6 or 7:1 in agreement with recent experiments. The natural carotenoid, lutein, is found to fit into the core of the triple-helical segments and help stabilize the configuration. Molecular dynamics simulations with explicit methanol/water molecules as solvent have been carried out to refine the 3D structure.

DFT study of alpha- and beta-D-galactopyranose at the B3LYP/6-311++G** level of theory.

Forty-one conformations of alpha- and beta-d-galactopyranose were geometry optimized using the B3LYP density functional and 6-311++G** basis set. Full geometry optimization was performed on different ring geometries and different hydroxymethyl rotamers (gg/gt/tg). Analytically derived Hessians were used to calculate zero point energy, enthalpy, and entropy. The lowest energy and free-energy conformation found is the alpha-gg-(4)C(1)-c chair conformation, which is of lower electronic and free energy than the lowest energy alpha-d-glucopyranose conformer because of favorable hydrogen-bonding interactions. The in vacuo calculations showed considerable ( approximately 2.2kcal/mol) energetic preference for the alpha over the beta anomer for galactopyranose in both the (4)C(1) and (1)C(4) chair conformations. Results are compared to glucopyranose and mannopyranose calculations in vacuo. Boat and skew-boat forms were found that remained stable upon gradient optimization, although many starting conformations moved to other boat forms upon optimization. As with glucopyranose and mannopyranose, the orientation and interaction of the hydroxyl groups make the most significant contributions to the conformation-energy relationship in vacuo.

B3LYP/6-311++G** geometry-optimization study of pentahydrates of alpha- and beta-D-glucopyranose.

Five water molecules were placed in 37 different configurations around alpha- and beta-D-glucopyranose in the gt, gg, and tg conformational states, and the glucose-water complexes were geometry optimized using density functionals at the B3LYP/6-311++G** level of theory. The five water molecules were organized in space and energy minimized using an empirical potential, AMB02C, and then further geometry optimized using DFT algorithms to minimum energy positions. Electronic energy, zero point vibrational energy, enthalpy, entropy, stress energy on glucose and the water cluster, hydrogen-bond energy, and relative free energy were obtained for each configuration using thermodynamic procedures and an analytical Hessian program. The lowest energy complex was that of a clustering of water molecules around the 1- and 6-hydroxyl positions of the beta-gt anomer. Configurations in which the water molecules created a favorable network completely around and under glucose were found to have low energy for both alpha and beta anomers. Calculation of the alpha/beta anomeric ratio using the zero point corrected energy gave, approximately 32/68%, highly favoring the beta anomer in agreement with the experimental approximately 36/64% value. This ratio is better than the approximately 50/50% ratio found in our previous monohydrate study. An approximate hydroxymethyl population was obtained by noting average relative energies among the three conformational states, gt, gg, and tg. In the beta anomer complexes the gt conformation was favored over the gg state, while in the alpha anomer complexes the gg state was favored over the gt conformation, with the tg conformations all being of higher energy making little or no contribution to the rotamer population. Some geometry variances, found between glucose in vacuo and glucose after interaction with water molecules, are described and account for some observed C-5-C-6 bond length anomalies reported by us previously for the vacuum glucose structures.

Modeling bacterial UDP-HexNAc: polyprenol-P HexNAc-1-P transferases.

Protein N-glycosylation in eukaryotes and peptidoglycan biosynthesis in bacteria are both initiated by the transfer of a D-N-acetylhexosamine 1-phosphate to a membrane-bound polyprenol phosphate. These reactions are catalyzed by a family of transmembrane proteins known as the UDP-D-N-acetylhexosamine: polyprenol phosphate D-N-acetylhexosamine 1-phosphate transferases. The sole eukaryotic member of this family, the d-N-acetylglucosamine 1-phosphate transferase (GPT), is specific for UDP-GlcNAc as the donor substrate and uses dolichol phosphate as the membrane-bound acceptor. The bacterial translocases, MraY, WecA, and WbpL, utilize undecaprenol phosphate as the acceptor substrate, but differ in their specificity for the UDP-sugar donor substrate. The structural basis of this sugar nucleotide specificity is uncertain. However, potential carbohydrate recognition (CR) domains have been identified within the C-terminal cytoplasmic loops of MraY, WecA, and WbpL that are highly conserved in family members with the same UDP-N-acetylhexosamine specificity. This review focuses on the catalytic mechanism and substrate specificity of these bacterial UDP-D-N-acetylhexosamine: polyprenol phosphate D-N-acetylhexosamine 1-P transferases and may provide insights for the development of selective inhibitors of cell wall biosynthesis.

DFT study of alpha- and beta-D-mannopyranose at the B3LYP/6-311++G** level.

Thirty-five conformations of alpha- and beta-d-mannopyranose, the C-2 substituted epimer of glucopyranose, were geometry optimized using the density functional (B3LYP), and basis set (6-311++G**). Full geometry optimization was performed on the hydroxymethyl rotamers (gg/gt/tg) and an analytical hessian program was used to calculate the harmonic vibrational frequencies, zero point energy, enthalpy, and entropy. The lowest energy conformation investigated is the beta-tg in the (4)C(1) chair conformation. The in vacuo calculations showed little energetic preference for either the alpha or beta anomer for mannopyranose in the (4)C(1) chair conformation. Results are compared to similar glucopyranose calculations in vacuo where the alpha anomer is approximately 1kcal/mol lower in electronic energy than the beta anomer. In the case of the generally higher energy (1)C(4) chair conformations, one low-energy, low-entropy beta-gg-(1)C(4) chair conformation was identified that is within approximately 1.4kcal/mol of the lowest energy (4)C(1) conformation of mannopyranose. Other (1)C(4) chair conformations in our investigation are approximately 2.9-7.9kcal/mol higher in overall energy. Many of the (3,O)B, B(3,O), (1,4)B, and B(1,4) boat forms passed through transitions without barriers to (1)S(3), (5)S(1), (1)S(5) skew forms with energies between approximately 3.6 and 8.9kcal/mol higher in energy than the lowest energy conformation of mannopyranose. Boat forms were found that remained stable upon gradient optimization. As with glucopyranose, the orientation and interaction of the hydroxy groups make a significant contribution to the conformation/energy relationship in vacuo.

Alternansucrase acceptor reactions with D-tagatose and L-glucose.

Alternansucrase (EC 2.4.1.140) is a d-glucansucrase that synthesizes an alternating alpha-(1-->3), (1-->6)-linked d-glucan from sucrose. It also synthesizes oligosaccharides via d-glucopyranosyl transfer to various acceptor sugars. Two of the more efficient monosaccharide acceptors are D-tagatose and L-glucose. In the presence of d-tagatose, alternansucrase produced the disaccharide alpha-d-glucopyranosyl-(1-->1)-beta-D-tagatopyranose via glucosyl transfer. This disaccharide is analogous to trehalulose. We were unable to isolate a disaccharide product from L-glucose, but the trisaccharide alpha-D-glucopyranosyl-(1-->6)-alpha-d-glucopyranosyl-(1-->4)-l-glucose was isolated and identified. This is analogous to panose, one of the structural units of pullulan, in which the reducing-end D-glucose residue has been replaced by its L-enantiomer. The putative L-glucose disaccharide product, produced by glucoamylase hydrolysis of the trisaccharide, was found to be an acceptor for alternansucrase. The disaccharide, alpha-D-glucopyranosyl-(1-->4)-L-glucose, was a better acceptor than maltose, previously the best known acceptor for alternansucrase. A structure comparison of alpha-D-glucopyranosyl-(1-->4)-L-glucose and maltose was performed through computer modeling to identify common features, which may be important in acceptor affinity by alternansucrase.

Conformational analysis of chirally deuterated tunicamycin as an active site probe of UDP-N-acetylhexosamine:polyprenol-P N-acetylhexosamine-1-P translocases.

Tunicamycins are potent inhibitors of UDP-N-acetyl-D-hexosamine:polyprenol-phosphate N-acetylhexosamine-1-phosphate translocases (D-HexNAc-1-P translocases), a family of enzymes involved in bacterial cell wall synthesis and eukaryotic protein N-glycosylation. Structurally, tunicamycins consist of an 11-carbon dialdose core sugar called tunicamine that is N-linked at C-1' to uracil and O-linked at C-11' to N-acetylglucosamine (GlcNAc). The C-11' O-glycosidic linkage is highly unusual because it forms an alpha/beta anomeric-to-anomeric linkage to the 1-position of the GlcNAc residue. We have assigned the (1)H and (13)C NMR spectra of tunicamycin and have undertaken a conformational analysis from rotating angle nuclear Overhauser effect (ROESY) data. In addition, chirally deuterated tunicamycins produced by fermentation of Streptomyces chartreusis on chemically synthesized, monodeuterated (S-6)-[(2)H(1)]glucose have been used to assign the geminal H-6'a, H-6'b methylene bridge of the 11-carbon dialdose sugar, tunicamine. The tunicamine residue is shown to assume pseudo-D-ribofuranose and (4)C(1) pseudo-D-galactopyranosaminyl ring conformers. Conformation about the C-6' methylene bridge determines the relative orientation of these rings. The model predicts that tunicamycin forms a right-handed cupped structure, with the potential for divalent metal ion coordination at 5'-OH, 8'-OH, and the pseudogalactopyranosyl 7'-O ring oxygen. The formation of tunicamycin complexes with various divalent metal ions was confirmed experimentally by MALDI-TOF mass spectrometry. Our data support the hypothesis that tunicamycin is a structural analogue of the UDP-D-HexNAc substrate and is reversibly coordinated to the divalent metal cofactor in the D-HexNAc-1-P translocase active site.

Oxidation and metal-ion affinities of a novel cyclic tetrasaccharide.

The cyclic tetrasaccharide, cyclo-(-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->), was oxidized in high yield to a dicarboxylic acid, cyclo-(-->6)-alpha-D-Glcp-(1-->3)-alpha-D-GlcpA-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-GlcpA-(1-->). The parent and oxidized compound were then screened for the ability to form stable complexes with 20 metal cations. Ion-exchange thin-layer chromatography was utilized to survey binding in aqueous and 50% methanolic solutions. The screening identified Pb2+, Fe2+ and Fe3+ as forming strong metal chelates with the oxidized cyclic tetrasaccharide. The stoichiometry of the oxidized cyclic tetrasaccharide and Pb2+ complex was determined to be 1:1 using aqueous gel-permeation chromatography. Perturbations between the free and complexed structure were examined using NMR spectroscopy. Molecular simulations were used to identify a probable structure of oxidized cyclic tetrasaccharide complexed with Pb2+.

Ab initio computational study of beta-cellobiose conformers using B3LYP/6-311++G**.

The molecular structure of 27 conformers of beta-cellobiose were studied in vacuo through gradient geometry optimization using B3LYP density functionals and the 6-311++G** basis set. The conformationally dependent geometry changes and energies were explored as well as the hydrogen-bonding network. The lowest electronic energy structures found were not those suggested from available crystallographic and NMR solution data, where the glycosidic dihedral angles fall in the region (phi, psi) approximately (40 degrees, -20 degrees ). Rather, 'flipped' conformations in which the dihedral angles are in the range (phi, psi) approximately (180 degrees, 0 degrees ) are energetically more stable by approximately 2.5 kcal/mol over the 'experimentally accepted' structure. Further, when the vibrational free energy, deltaG, obtained from the calculated frequencies, is compared throughout the series, structures with (phi, psi) in the experimentally observed range still have higher free energy ( approximately 2.0 kcal/mol) than 'flipped' forms. The range of bridging dihedral angles of the 'normal' conformers, resulting from the variance in the phi dihedral is larger than that found in the 'flipped' forms. Due to this large flat energy surface for the normal conformations, we surmise that the summation of populations of these conformations will favor the 'normal' conformations, although evidence suggests that polar solvent effects may play the dominant role in providing stability for the 'normal' forms. Even though some empirical studies previously found the 'flipped' conformations to be lowest in energy, these studies have been generally discredited because they were in disagreement with experimental results. Most of the DFT/ab initio conformations reported here have not been reported previously in the ab initio literature, in part because the use of less rigorous theoretical methods, i.e. smaller basis sets, have given results in general agreement with experimental data, that is, they energetically favored the 'normal' forms. These are the first DFT/ab initio calculations at this level of theory, apparently because of the length and difficulty of carrying out optimizations at these high levels.