Conformational analysis of levanbiose by molecular mechanics.

A relaxed conformational energy map for levanbiose, O-beta-D-fructofuranosyl-(2----6)-beta-D-fructofuranoside, was computed with the molecular mechanics program MM2(87). All torsion angles of the three linkage bonds were driven by 30 degrees increments while two primary alcohol groups were held at three staggered forms. The steric energy of all other parameters was optimized. The side groups were retained at the same relative positions on the two rings in this first part of the study so our results are directly applicable to the study of polymeric levan with identical repeating units. The low-energy dimers did not lead to viable polymers. The interresidue linkage torsion angles defined by C-6-O-2'-C-2'-C-1' (phi) and O-5-C-5-C-6-O-2' (omega) have minima at +60 degrees and -60 degrees, respectively, with accessible minima at other staggered forms. As observed in inulobiose, the preferred torsion angle at central linkage bond defined by C-5-C-6-O-2'-C-2' (psi) was antiperiplanar. An analysis of all conformations of staggered side groups showed that the C-1 and C-1' groups had little effect but the C-6' group showed a preference for chi-6'(O-5'-C-5'-C-6'-O-6') = -60 degrees. The fructofuranose rings were started at the low-energy 4(3)T conformation (angle of pseudorotation, phi 2 = 265 degrees) that was retained except when the linkage conformations created severe inter-residue conflict.     

Substituent effects on the PMR signals of methyl groups in methyl steroids

Proton magnetic resonance (PMR) signals were assigned to each of the methyl groups for a variety of C-3 oxygenated 4-monomethyl, 6-monomethyl, 4, 4-dimethyl, and 2, 2-dimethyl steroids related to 5α-cholestane series. The effects of various substituents (i.e. methyls at C-4, C-6, or C-2, functional groups at C-3, and unsaturated bonds) on the methyl proton resonances have been derived from the assignment. The application of such data to structural and stereo-chemical problems has also been discussed.     

Conformational analysis of inulobiose by molecular mechanics

Conformational energies for inulobiose [beta-D-fructofuranosyl-(2----1)-beta-D-fructofuranoside], a model for inulin, were computed with the molecular mechanics program MMP2(85). The torsion angles of the three linkage bonds were driven in 20 degree increments, and the steric energy of all other parameters was minimized. The linkage torsion angles defined by C-1'-C-2'-O-C-1 (phi) and O-C-1-C-2-O-2 (omega) have minima at +60 degrees and -60 degrees, respectively, regardless of side group orientation; accessible minima exist at other staggered conformations. The torsion angle at the central bond C-2'-O-1-C-1-C-2 (psi) was approximately 180 degrees in all the low-energy conformers. This appears to be generally true for rings linked by three bonds. The fructofuranose rings initially had low-energy 4/3T conformations (angle of pseudorotation, phi 2 = 265 degrees) that were retained except when the linkage conformations created severe inter-residue conflicts. In those cases, almost all puckerings of the furanose rings were found.     

Rearrangement of unsaturated 2,4-O-benzylidenehexitol derivatives into C-glycosylbenzene derivatives.

Acetolysis of (Z)-1,3-di-O-acetyl-2,4-O-benzylidene-6-C-(2,4-dichlorophenyl)-D-xylo-he x- 5-enitol (3) afforded (E)-1,2,3,4-tetra-O-acetyl-6-C-(2,4-dichlorophenyl)-D-xylo-hex-5-enit ol and 2-C-[(R)-acetoxy(2,4-dichlorophenyl)methyl]-3,4,6-tri-O-acetyl-2-deoxy- beta-L-galacto- and -beta-L-gulo-hexopyranosylbenzene. The mechanism of this new rearrangement was studied by exchanging the substituents at C-1 and C-3 in 3 and those of the aromatic ring attached to C-6.     

Construction of a branched chain at C-3 of a hexopyranoside. Synthesis of miharamycin sugar moiety analogs

Synthesis of the conveniently protected epimer at C-3' of the miharamycin sugar moiety was accomplished starting from the corresponding 3,3'-spiroepoxide. Reaction of the epoxide with lithium cyanide, followed by hydrolysis and spontaneous cyclization, afforded the intermediate deoxylactone methyl 4,6-O-benzylidene-3-C-(carboxymethyl)-alpha-D-glucopyranoside-3',2-lacto ne (8). Stereoselective hydroxylation with MoO5 x py x HMPA, reduction with lithium aluminum hydride and cyclization with diethyl azodicarboxylate-triphenylphosphine gave the target molecule methyl 2,3'-anhydro-4,6-O-benzylidene-3-C-[(R)-1,2-dihydroxyethyl]-alpha -D-glucopyranoside (5). Direct reduction of 8 gave other analogs having no C-3' hydroxyl group together with having a C-3' hydroxyl group (hemiacetal). In addition, C-3' epimers were also synthesized through C-3', C-3' dihydroxy analogs. Wittig reaction of an appropriate ketosugar with [(ethoxycarbonyl)methylene]triphenylphosphorane leading to a 7:3 Z/E mixture, followed by hydroxylation with osmium tetroxide, reduction and cyclization afforded the target molecule 5 and the miharamycin sugar moiety methyl 2,3'-anhydro-4,6-O-benzylidene-3-C-[(S)-1,2-dihydroxyethyl]-alpha -D-glucopyranoside. Examination of X-ray data for 5 and its NMR spectroscopy data allowed us to explain a contradiction reported in the literature.     

Synthesis, structural studies, and cytostatic evaluation of 5,6-di-O-modified L-ascorbic acid derivatives

The 5,6-di-O-tosylated derivative of l-ascorbic acid was synthesized by selective protection and deprotection of 2,3- and 5,6-dihydroxy functional groups involving 5,6-ditosylation in the final step, while the novel 6-acetoxy, 6-hydroxy, and 6-chloro derivatives of 4,5-didehydro-l-ascorbic acid were obtained by reaction of ditosylated compound with nucleophilic reagents. The analysis of 3JH-4-H-5 homonuclear coupling constants shows that all l-ascorbic acid derivatives except for epoxy and 4,5-didehydro compounds exist in high population as gauche conformers across C-4-C-5 bonds, while 3JC-3-H-5 heteronuclear coupling constants in 4,5-didehydro derivatives indicate cis geometry along C-4-C-5 double bond. The X-ray crystal structure analysis of 2,3-di-O-benzyl-5,6-epoxy- and 5,6-isopropylidene-l-ascorbic acid shows that the oxygen atoms attached at positions 2 and 3 of the lactone ring are disposed in a synperiplanar fashion. Besides that, the dioxolane ring adopts half-chair conformation. The molecules of epoxy derivative are joined into infinite chains by one weak hydrogen bond of C-H...O type. Two O-H...O, and C-H...O hydrogen bonds link the molecules of 5,6-di-O-isopropylidene compound into two-dimensional network. 6-Chloro derivative of 2,3-di-O-benzyl-l-ascorbic acid showed the best cytostatic effects against all tested malignant tumor cells (IC50: approximately 18 microM).     

Microbial metabolism of pyridinium compounds. Radioisotope studies of the metabolic fate of 4-carboxy-1-methylpyridinium chloride

Extracts of Achromobacter D formed CO(2), methylamine, succinate and formate as metabolic end-products from N-methylisonicotinic acid (4-carboxy-1-methylpyridinium chloride). The origin of the CO(2) in the 4-carboxyl group and of the methylamine in the N-methyl group of N-methylisonicotinate was demonstrated with carboxyl-(14)C- and N-Me-(14)C-labelled substrates respectively. The carbon skeletons of formate and succinate were shown to arise from the C-2 and the C-3-C-6 atoms of the heterocyclic ring respectively by using N-methyl[2,3-(14)C(2)]isonicotinate. This result is consistent with ring cleavage by the organism between C-2 and C-3.     

Quantum mechanical and NMR spectroscopy studies on the conformations of the hydroxymethyl and methoxymethyl groups in aldohexosides

The potential energy surfaces of the hydroxymethyl and methoxymethyl groups in methyl hexopyranosides have been extensively studied, employing quantum mechanical calculations and high resolution NMR data. The structure and energy of the C-5-C-6 rotamers were calculated at the B3LYP level of the density functional theory (DFT). For all, geometry optimizations were carried out for 264 conformers of 16 methyl D-gluco- and methyl D-galactopyranoside derivatives 1-16 at the B3LYP/6-31G** level. For all calculated minima, single-point calculations were performed at the B3LYP/6-311++G** level. Solvent effects were considered using a self-consistent reaction field method. Values of the vicinal coupling constants 3J(H-5-H-6R), 3J(H-5-H-6S), 3J(C-4-H-6R), and 3J(C-4-H-6S) for methyl D-glucopyranosides, methyl D-galactopyranosides and their 6-O-methyl derivatives 9-16 were measured in two solvents, methanol and water. The calculated gg, gt, and tg rotamer populations of the hydroxymethyl and methoxymethyl groups in 9-16 agreed well with experimental data. The results clearly showed that the population of gg, gt, and tg rotamers is sensitive to solvent effects. It was concluded that the preference of rotamers in 1-16 is due to the hydrogen bonding and solvent effects.     

Intramolecular aldol cyclization of C-4-ulopyranosyl-2'-oxoalkanes controlled by steric effects. Asymmetric synthesis of substituted 8-oxabicyclo[3.2.1]octanones and -octenones and cyclopentenones

Whereas C-2- and 4-ulopyranosyl compounds (C-2- and C-4-ulosides) can be converted to cyclopentenones under base conditions through beta-elimination and ring contraction, base-initiated beta-elimination of C-glycosyl 2'-aldehydes and 2'-ketones results in the formation of acyclic alpha,beta-unsaturated aldehydes or ketones. By combining both molecular features we synthesized 1-C-(4-ulopyranosyl)-2-oxoalkanes 6, 13, and 20 and investigated their reactions when they were treated with base. Both alpha- and beta-anomers of C-(4-ulopyranosyl)acetaldehydes 6 and 13 underwent a fast intramolecular aldol reaction between the C-5 enolate and 2'-aldehyde to form optically pure 8-oxabicyclo[3.2.1]octanones, which further transformed to 8-oxabicyclo[3.2.1]octenones 14 and 15 by beta-elimination. However, this aldol reaction did not occur when 1-C-(4-ulopyranosyl)propan-2-one 20 was treated with base because of steric hindrance exerted by the additional methyl group. Instead, an alternate C-3 enolization led to beta-elimination and further electro-ring opening to form an acyclic enol, which was then converted through a disrotatory intramolecular aldol cyclization to a cis-substituted cyclopentenone 21.     

Production of a co-polyester of 3-hydroxybutyric acid and 3-hydroxyvaleric acid from succinic acid by Rhodococcus ruber: biosynthetic considerations

The biosynthesis of the 3-hydroxyvalerate (3HV) monomer of polyhydroxyalkanoate by Rhodococcus ruber from succinic acid was investigated using nuclear magnetic resonance analysis. Polymer produced from [2,3-¹³C]- and [1,4-¹³C]succinate showed that the C-1-C-2 and C-4-C-5 fragments of 3HV were derived from carbons 2 and 3 of succinate, essentially without bond cleavage, and carbon 3 of 3HV was derived from a carboxyl carbon of succinate. Using [1,2-¹³C]succinate it was demonstrated that the C-1-C-2 bond of succinate was cleaved during polymer biosynthesis. Methylmalonyl-coenzyme A (CoA) mutase activity was detected in cell-free extracts of R. ruber by enzyme assay and HPLC analysis of reaction products. A pathway, involving the known methylmalonyl-CoA pathway for propionate formation in Propionibacteria, followed by the established pathway for PHA biosynthesis from propionyl-CoA and acetyl-CoA, is proposed for the biosynthesis of 3HV from succinate by R. ruber. Correspondence to: A. J. Anderson     

Stereochemistry of the methoxymercuration of glycal acetates

The methoxymercuration of the acetates of d-glucal, d-galactal, l-arabinal and d-xylal was studied by n.m.r. spectroscopy, and by stereoselective demercuration with thiourea, as well as by reductive demercuration with sodium borohydride. It is considered to be a trans-addition to the double bond to yield isomeric methyl glycosides having mercury attatched to C-2. The proportion of diaxial to diequatorial addition is strongly affected by the protecting groups at C-4 and C-5.     

Heterocyclization of 3-deoxy-D-erythro-hexos-2-ulose-1,2-bis(thiosemicarbazone). Crystal structure of the major diastereomer

Both thiosemicarbazone groups of the derivative 1 of 3-deoxy-D-erythro-hexos-2-ulose underwent, on acetylation, a heterocyclization process to give (5R,5'R)-2,2'-diacetamido-4,4'-di-N-acetyl-5'-(1-deoxy-2,3,4-tri-O-acetyl-D-erythritol-1-yl)-5,5'-bis(1,3,4-thiadiazoline) (2) as a major product. The X-ray diffraction data of a single crystal of 2 indicated the R,R configuration for the stereocenters of the thiadiazoline rings (C-5 and C-5'). In the solid state, 2 adopts a sickle conformation (by clockwise rotation of the C-2-C-3 axis of the sugar chain) which has a S//O 1,3-parallel interaction. In solution, as determined by (1)H NMR spectroscopy which included NOE experiments, a similar sickle conformation was observed. From the reaction mixture of acetylation of 1 was isolated the bis(thiadiazoline) 3 as a by-product. The configuration of the C-5 and C-5' stereocenters of 3 were respectively assigned as S,R by comparison of the physical and spectroscopic data of this compound with those of 2.     

X-ray crystal structure of galabiose, O-alpha-D-galactopyranosyl-(1---4)-D-galactopyranose

O-alpha-D-Galactopyranosyl-(1---4)-D-galactopyranose, C12H22O11, Mr = 342.30, crystallises in the orthorhombic space group P2(1)2(1)2(1), and has alpha = 5.826(1), b = 13.904(3), c = 17.772(4) A, Z = 4, and Dx = 1.579 g.cm-3. Intensity data were collected with a CAD4 diffractometer. The structure was solved by direct methods and refined to R = 0.063 and Rw = 0.084 for 2758 independent reflections. The glycosidic linkage is of the type 1-axial-4-axial with torsion angles phi O-5' (O-5'-C-1'-O-1'-C-4) = 98.1(2) degrees, psi C-3 (C-3-C-4-O-1'-C-1') = -81.9(3) degrees, phi H (H-1'-C-1'-O-1'-C-4) = -18 degrees, and psi H (H-4-C-4-O-1'-C-1') = 35 degrees. The conformation is stabilised by an O-3 . . . O-5' intramolecular hydrogen-bond with length 2.787(3) A and O-3-H . . . O-5' = 162 degrees. The glycosidic linkage causes a folding of the molecule with an angle of 117 degrees between the least-square planes through the pyranosidic rings. The crystal investigated contained 56(1)% of alpha- and 44(1)% of beta-galabiose as well as approximately 70% of the gauche-trans and approximately 30% of the trans-gauche conformers about the exocyclic C-5'-C-6' and C-5-C-6 bonds. The crystal packing is governed by hydrogen bonding that engages all oxygen atoms except the intramolecular acceptor O-5' and the glycosidic O-1' oxygen atoms.     

Structural elucidation of the 3-deoxy-D-manno-octulosonic acid containing meningococcal 29-e capsular polysaccharide antigen using carbon-13 nuclear magnetic resonance.

The capsular polysaccharide antigen from Neisseria meningitidis serogroup 29-e contains equimolar quantities of 2-acetamido-2-deoxy-D-galactose and 3-deoxy-D-manno-octulosonic acid (KDO), the latter of which is rarely found in biopolymers other than lipopolysaccharides. Carbon-13 nuclear mangetic resonance in conjunction with other chemical data indicated that the polysaccharide is composed of an alternating sequence of these two residues, the linkages being at C-3 of galactosamine and C-7 of KDO in the alpha-D and beta-D configuration, respectively. The native 29-e polysaccharide is O-acetylated, the O-acetyl groups being located at C-4 and C-5 of the KDO residues. Assignments of signals in the 13C nuclear magnetic resonance spectrum of the 29-e polysaccharide were made by consideration of those in the spectra of the monomer models, which necessitated the first recorded syntheses of methyl-alpha- and beta-D-3-deoxy-manno-octulopyranosonic acid. Like the methyl alpha- and beta-D-ketosides of sialic acid (Na+ salts), the equivalent methyl alpha- and beta-D-ketosides of KDO exhibit large chemical shift differences in the exocyclic C-8 position dependent on anomeric configuration. This can again be attributed to hydrogen bonding between the axial carboxylate group of the methyl beta-D anomer of KDO (C1 conformation) and the primary hydroxyl group at C-8. This phenomenon is also exhibited by the beta-D-linked KDO units of the 29-e polysaccharide.     

Structural requirements for binding to the sugar-transport system of the human erythrocyte

The structural requirements for binding to the glucose/sorbose-transport system in the human erythrocyte were explored by measuring the inhibition constants, K(i), for specifically substituted analogues of d-glucose when l-sorbose was the penetrating sugar. Derivatives in which a hydroxyl group in the d-gluco configuration was inverted, or replaced by a hydrogen atom, at C-1, C-2, C-3, C-4 or C-6 of the d-glucose molecule, all bound to the carrier, confirming that no single hydroxyl group is essential for binding to the carrier. The binding and transport of 1-deoxy-d-glucose confirmed that the sugars bind in the pyranose form. The relative inhibition constants of d-glucose and its deoxy, epimeric and fluorinated analogues are consistent with the combination of beta-d-glucopyranose with the carrier by hydrogen bonds at C-1, C-3, probably C-4, and possibly C-6 of the sugar. Both polar and non-polar substituents at C-6 enhance the affinity of d-glucose derivatives relative to d-xylose, and d-galactose derivatives relative to l-arabinose, and it is suggested that the carrier region around C-6 of the sugar may contain both hydrophobic and polar binding groups. The spatial requirements at C-1, C-2, C-3, C-4 and C-6 were explored by comparing the relative binding of d-glucose and its halogeno and O-alkyl substituents. The carrier protein closely approaches the sugar except at C-3 in the d-gluco configuration, C-4 and C-6. d-Glucal was a good inhibitor, showing that a strict chair form is not essential for binding. 3-O-(2',3'-Epoxypropyl)-d-glucose, a potential substrate-directed alkylating agent, bound to the carrier, but did not inactivate it.     

Homologation of methyl 2-azido- and 2-acetamido-3,4-di-O-benzyl-2-deoxy-D-hexopyranosides with allyloxymethylmagnesium chloride.

Methyl 2-azido-2-deoxy-hexodialdo-1,5-pyranosides of the alpha-, beta-D-gluco and alpha-D-manno configuration as well as methyl 2-acetamido-2-deoxy-hexodialdo-1,5-pyranosides of the alpha- and beta-D-gluco configuration, protected at positions 3 and 4 with O-benzyl groups were reacted with an excess of allyloxymethylmagnesium or (phenyldimethylsilyl)methylmagnesium chlorides to afford mixtures of C-6 stereoisomeric heptopyranosides. Configuration of the products separated by column chromatography was assigned by 1H NMR data.     

Synthetic explorations towards 3-deoxy-3-fluoro derivatives of D-perosamine

Based on a literature precedent, preparation of methyl 4-azido-3,4,6-trideoxy-3-fluoro-alpha-D-mannopyranoside (18) was attempted via fluorination of methyl 4-azido-2-O-benzyl-4,6-dideoxy-alpha-D-altropyranoside with diethylaminosulfur trifluoride (DAST). Contrary to expectations, the reaction took place with retention of configuration at the site of the fluorination yielding methyl 4-azido-2-O-benzyl-3,4,6-trideoxy-3-fluoro-alpha-D-altropyranoside. Treatment with DAST of methyl 4-azido-2-O-benzyl-4,6-dideoxy-alpha-D-allopyranoside (8), or its 2-(p-methoxybenzyl) analog 9 resulted in fluorination with inversion of configuration at position 3, to give the corresponding 3-deoxy-3-fluoro glucopyranosides 10 and 11, respectively. Accordingly, compound 18 was prepared from 11, by de-p-methoxybenzylation at O-2, followed by inversion of configuration at C-2 in the resulting methyl 4-azido-3,4,6-trideoxy-3-fluoro-alpha-D-glucopyranoside. The 2-O-methyl analog of 18 (19) was prepared by methylation of 18. Compounds 18 and 19 were converted, conventionally, into the 3-fluoro analogs of the terminal determinants of the O-PS of Vibrio cholerae O:1, serotype Inaba and Ogawa, respectively.     

Cardiac effects of six actodigin (AY-22,241) - related semisynthetic glycosides

Six actodigin (AY-22,241) - related semisynthetic glycosides (with the C-3 natural linkage of the lactone ring to the steroid nucleus, transposed to C-2) were assayed in failing hearts of canine heart-lung preparations. Two compounds, with additional small modifications in the steroid nucleus, were incapable of reversing heart failure. Two others, an isodigoxigenin and an isogitoxigenin derivative, were only slightly active. However, the two other actodigin-derived compounds, with methyl groups at the lactane's C-4, were very active. Compound 5 had the methyl group in beta position and was 2.5 times more potent than actodigin. Compound 6, with the methyl group in alpha position, had a potency similar to that of actodigin. In the anesthetized and vagotomized dog, their toxic effects (to the point of atrioventricular dissociation) were short lasting and completely reversible. Both of these agents had a wide margin of safety (relationship between the minimal therapeutic, irregularity and lethal doses).     

Preparation of methyl glycosides of di- and higher oligosaccharides from glycosaminoglycuronans by solvolysis with dimethyl sulfoxide containing methanol

Solvolysis of chondroitin 4- or 6-sulfate (pyridinium salt) with dimethyl sulfoxide containing 10% of methanol for 18 h at 95° resulted in the cleavage of the 2-amino-2-deoxy-D-glucoside bonds together with initial desulfation to give methyl β-glycosides of N-acetylchondrosine as a main product and, in addition, higher oligosaccharides, without any loss of uronic acid. Dermatan sulfate was also depolymerized to yield methyl glycosides of di- and higher oligosaccharides under the same conditions. Hyaluronic acid (free acid) was depolymerized by the same solvent in the presence of an equimolar amount of pyridine-sulfur trioxide or pyridinium sulfate per disaccharide unit to give methyl glycosides of di- and higher oligosaccharides. In contrast N-desulfated, N-acetylated heparin was stable under these solvolytic conditions and did not yield heparin oligosaccharides.     

Base-catalyzed oligomerization of levoglucosenone

Heating levoglucosenone in aqueous triethylamine gives a dimer and two trimers in yields of 8, 18, and 56%, respectively. These compounds have been isolated crystalline, and their structures and stereochemistry have been investigated by ¹³C- and ¹H-n.m.r. and other spectroscopic methods. These data indicate that the dimer is apparently formed by Michael addition to provide a C-3-C-4 linkage. Similar reactions provide a non-olefinic, C-3-C-4-linked, cyclic trimer and an olefinic, cyclic trimer containing two C-3-C-4 linkages and one C-2-C-3 linkage.