Synthesis of stereospecifically labeled 3,6-dideoxyhexoses

Preparations of ascarylose (3,6-dideoxy-L-arabino-hexose), abequose (3,6-dideoxy-D-xylo-hexose), and paratose (3,6-dideoxy-D-ribo-hexose) with stereospecific deuterium labeling at C-3 are discussed. The methods used to synthesize these sugars, such as the hydrogenation of olefins, the displacement of halides, the reduction of epoxides, and the substitution of tosyl esters, illustrate a variety of strategies leading to stereospecific deuterium incorporation. Many of the techniques described here should be of general utility for the synthesis of other deuterium-labeled sugars.     

Evaluation of 3,6-dibutanoylmorphine as an analgesic in vivo: comparison with morphine and 3,6-diacetylmorphine

Dibutanoylmorphine (DBM), a synthetic diester of morphine, was compared with morphine (M) and diacetylmorphine (DAM) for analgesic efficacy, potency and duration of action following I.V. administration in rats. Analgesia was assessed in groups of eight animals using both tail-flick and hot-plate testing methods following random administration of five different doses of each drug. DBM was found to be substantially more potent than M, but less potent than DAM in both tail-Flick and hot-plate tests of nociception. Similarly, assessment of duration of action at the ED50 for each drug revealed that DBM has a duration of analgesia which is intermediate between the durations of M and DAM. Thus, in rats in vivo, DBM is an effective analgesic and has a reasonable duration of action release to other opioids.     

Monodeoxy-1,4:3,6-dianhydrohexitol nitrates

Monodeoxy-1,4:3,6-dianhydrohexitol nitrates with (5) or without (6) an additional chloro substituent were synthesized, starting from 1,4:3,6-dianhydrohexitol-monoacetates, via 3 as key intermediates. Attempts to generate an unsaturated nitrate ester resulted in a DBN alkylation product (9).Both the endo- and exo-configurated monodeoxy-1,4:3,6-dianhydrohexitol nitrates have been prepared. Attempts to generate an unsaturated nitrate ester resulted in a DBN alkylation produkt.     

Biosynthesis of dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose

Derivatives of 3-amino-3,6-dideoxyhexoses are widespread in Nature. They are part of the repeating units of lipopolysaccharide O-antigens, of the glycan moiety of S-layer (bacterial cell surface layer) glycoproteins and also of many antibiotics. In the present study, we focused on the elucidation of the biosynthesis pathway of dTDP-alpha-D-Quip3NAc (dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose) from the Gram-positive, anaerobic, thermophilic organism Thermoanaerobacterium thermosaccharolyticum E207-71, which carries Quip3NAc in its S-layer glycan. The biosynthesis of dTDP-alpha-D-Quip3NAc involves five enzymes, namely a transferase, a dehydratase, an isomerase, a transaminase and a transacetylase, and follows a pathway similar to that of dTDP-alpha-D-Fucp3NAc (dTDP-3-acetamido-3,6-dideoxy-alpha-D-galactose) biosynthesis in Aneurinibacillus thermoaerophilus L420-91(T). The ORFs (open reading frames) of interest were cloned, overexpressed in Escherichia coli and purified. To elucidate the enzymatic cascade, the different products were purified by HPLC and characterized by NMR spectroscopy. The initiating reactions catalysed by the glucose-1-phosphate thymidylyltransferase RmlA and the dTDP-D-glucose-4,6-dehydratase RmlB are well established. The subsequent isomerase was shown to be capable of forming a dTDP-3-oxo-6-deoxy-D-glucose intermediate from the RmlB product dTDP-4-oxo-6-deoxy-D-glucose, whereas the isomerase involved in the dTDP-alpha-D-Fucp3NAc pathway synthesizes dTDP-3-oxo-6-deoxy-D-galactose. The subsequent reaction steps of either pathway involve a transaminase and a transacetylase, leading to the specific production of nucleotide-activated 3-acetamido-3,6-dideoxy-alpha-D-glucose and 3-acetamido-3,6-dideoxy-alpha-D-galactose respectively. Sequence comparison of the ORFs responsible for the biosynthesis of dTDP-alpha-D-Quip3NAc revealed homologues in Gram-negative as well as in antibiotic-producing Gram-positive bacteria. There is strong evidence that the elucidated biosynthesis pathway may also be valid for LPS (lipopolysaccharide) O-antigen structures and antibiotic precursors.     

Metabolic pathway of 3,6-anhydro-L-galactose in agar-degrading microorganisms

Recently, agarose-containing macroalgae have gained attention as possible renewable sources for bioethanol-production because of their high polysaccharide content. Complete hydrolysis of agarose produces two monomers, D-galactose (D-Gal) and 3,6-anhydro-L-galactose (L-AnG). However, at present, bioethanol yield from agarophyte macroalgae is low due to the inability of bioethanolproducing microorganisms to convert non-fermentable sugars, such as L-AnG, to bioethanol. Therefore, to increase the bioethanol productivity of agarophytes, it is necessary to determine how agar-degrading microorganisms metabolize L-AnG, and accordingly, construct recombinant microorganisms that can utilize both D-Gal and L-AnG. Previously, we isolated a novel microorganism belonging to a new genus, Postechiella marina M091, which hydrolyzes and metabolizes agar as the carbon and energy source. Here, we report a comparative genomic analysis of P. marina M091, Pseudoalteromonas atlantica T6c, and Streptomyces coelicolor A3(2), of the classes Flavobacteria, Gammaproteobacteria, and Actinobacteria, respectively. In this bioinformatic analysis of these agarolytic bacteria, we found candidate common genes that were believed to be involved in L-AnG metabolism. We then experimentally confirmed the enzymatic function of each gene product in the L-AnG cluster. The formation of two key intermediates, 2-keto-3-deoxy-L-galactonate and 2-keto-3-deoxy-D-gluconate, was also verified using enzymes that utilize these molecules as substrates. Combining bioinformatic analysis and experimental data, we showed that L-AnG is metabolized to pyruvate and D-glyceraldehyde-3-phosphate via six enzymecatalyzed reactions in the following reaction sequence: 3,6-anhydro-L-galactose → 3,6-anhydro-L-galactonate → 2-keto-3-deoxy-L-galactonate → 2,5-diketo-3-deoxy-L-galactonate → 2-keto-3-deoxy-D-gluconate → 2-keto-3-deoxy-6-phospho-D-gluconate → pyruvate + D-glyceraldehyde-3- phosphate. To our knowledge, this is the first report on the metabolic pathway of L-AnG degradation.     

Synthetische polysaccharide mit D-glucose- und 3,6-anhydro-d-glucoseresten

D-glucopyranose in anhydrous hydrogen fluoride at 20° forms oligosaccharides and low-molecular-weight polysaccharides composed of d-glucose and 3,6-anhydro-d-glucose residues in ratios of 10:1 to 5:1 depending on reaction conditions.     

Fluorescence decay studies of the DNA-3,6-diaminoacridine complexes

The interaction of several 3,6-diaminoacridines with DNAs of various base composition has been studied by steady-state and transient fluorescence measurements. The acridine dyes employed are of the following two classes: class I - proflavine, acriflavine and 10-benzyl proflavine; class II - acridine yellow, 10-methyl acridine yellow and benzoflavine. It is found that the fluorescence decay kinetics follows a single-exponential decay law for free dye and the poly[d(A-T)]-dye complex, while that of the dye bound to DNA obeys a two-exponential decay law. The long lifetime (tau 1) for each complex is almost the same as the lifetime for the poly[d(A-T)]-dye complex, and the amplitude alpha 1 decreases with increasing GC content of DNA. The fluorescence quantum yields (phi F) of dye upon binding to DNA decrease with increasing GC content; the phi F values for class I are nearly zero when bound to poly(dG) X poly(dC), but those for class II are not zero. This is in harmony with the finding that GMP almost completely quenches the fluorescence for class I, whereas a weak fluorescence arises from the GMP-dye complex for class II. The fluorescence spectra of the DNA-dye complexes gradually shift toward longer wavelengths with increasing GC content. In this connection, the fluorescence decay parameters show a dependence on the emission wavelength; alpha 1 decreases with an increase in the emission wavelength. In view of these results, it is proposed that the decay behavior of the DNA-dye complexes has its origin in the heterogeneity of the emitting sites; the long lifetime tau 1 results from the dye bound to AT-AT sites, while the short lifetime tau 2 is attributable to the dye bound in the vicinity of GC pairs. Since GC pairs almost completely quench the fluorescence for class I, partly intercalated or externally bound dye molecules may play an important role in the component tau 2.     

Metabolism and disposition of 3,6-dibutanoylmorphine in rat brain

In previous studies from this laboratory it was found that dibutanoylmorphine (DBM) was more potent than morphine as an analgesic in rats and that it was less active than acetyl esters of morphine on behaviour. As DBM is a morphine prodrug, the aim of this work was to determine if rat brain homogenates were capable of deacylating DBM and monobutanoylmorphine (MBM) and to determine relative proportions of parent drug to metabolites in the brain in vivo. In 10% (w/v) brain homogenates, DBM was eliminated with a half-life of about 70 min (corrected for dilution), while MBM was eliminated 10 times as quickly. DBM and its metabolites were found in both blood and brain as early as 1 min after i.v. administration of DBM. After 5 min, the predominant form in blood was MBM and in brain it was DBM. Thus, rat brain possesses the capacity to metabolize DBM by deesterification and the parent drug, MBM, and morphine were found in blood and brain in vivo.     

Structural and spectroscopic study of 3,6-dibutanoic-1,2,4,5-tetroxane

This paper deals with the synthesis of 3,6-dibutanoic-1,2,4,5-tetroxane and the theoretical study of its IR and UV spectra as well as the determination of its optimized molecular structure. Theoretical calculations are performed at the molecular dynamics (MD), molecular mechanics, semi empirical, ab initio and density functional theory (DFT) levels. The different structural and electronic effects determining the molecular stability of the conformers are discussed in a comparative fashion.     

Efficient synthesis of a 3,6-branched mannose hepta- and octasaccharide

Alpha-D-Manp-(1-->3)-[alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)]-alpha-D-Manp-(1-->3)-[alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)]-D-Manp and alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->3)-[alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)]-alpha-D-Manp-(1-->3)-[alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)]-D-Manp, were synthesized as their methyl glycosides in a regio- and stereoselective way.     

Synthesis of Novel Bicyclic Nucleosides with 3,6-Anhydro Sugar Moiety

Based on the biological importance of conformationally restricted nucleoside analogues, we have efficiently synthesized 3,6-anhydro sugar moiety with 3-C-hydroxymethyl substituent from 1,2;5,6-di-O-isopropylidene-D-glucose and condensed 15 with silylated nucleobases to afford the bicyclic nucleoside with 3,6-anhydro skeleton as potential antiviral agent.