Complete and regioselective deacetylation of peracetylated uridines using a lipase

Lipase-catalysed alcoholysis and hydrolysis of 2',3',5'-tri-O-acetyluridine (1a) and 2',3',5'-tri-O-acetyl-2'-C-methyluridine (1b) were studied. Conditions for full and regioselective deacetylation of 1aand 1b are shown in the present work. New compound 2',3'-di-O-acetyl-2'-C-methyluridine (3b) was prepared by regioselective lipase-catalysed deacetylation.     

Different actions of mono- and disaccharides on rat liver mitochondria

Mitochondria, isolated with 0.3M disaccharide (sucrose, maltose, trehalose) solutions, showed significantly lower specific activities both in uncoupler-stimulated adenosinetriphosphatase and succinate dehydrogenase activities than organelles prepared in parallel from the same livers with isosomolar media based on mannitol, glucose or sorbitol. Furthermore, the glutamate content and the inulin impermeable space appeared markedly reduced by 0.3M disaccharides. These effects of the disaccharides were dependent on the concentration of the solute, and were not discernible at a concentration of 0.2M. On the basis of these results, one might suggest the avoidance of further use of sucrose in the preparation of liver mitochondria.     

Enzymatic regioselective and complete deacetylation of two arabinonucleosides

Candida antarctica lipase B (CAL-B)-catalysed regioselective deacetylation of 2′,3′,5′-tri-O-acetyl-1-β-d-arabinofuranosyluracil (1) and 2′,3′,5′-tri-O-acetyl-9-β-d-arabinofuranosyladenine (2) was studied. The choice of the reaction medium allowed the regioselective formation of products bearing different degree of acetylation: in isopropanol, CAL-B catalysed the formation of the corresponding 2′-O-acetylated arabinonucleosides, while hydrolyses afforded the 2′,3′-di-O-acetylated products. In particular, the procedure herein described allows a simple and efficient preparation of the reported vidarabine prodrug 2′,3′-di-O-acetyl-9-β-d-arabinofuranosyladenine, avoiding the utilisation of protective groups. Moreover, to achieve full deacetylation of the assayed substrates, a set of commercial hydrolases and fungal keratinases from Doratomyces microsporus (DMK) and Paecilomyces marquandii (PMK) were tested. While only PMK and DMK catalysed the quantitative complete deacetylation of 1, DMK accomplished full deacetylation of 2 in shorter time than the other assayed enzymes.     

Ultraviolet difference spectroscopy study of peanut lectin binding to mono- and disaccharides

Ultraviolet difference spectroscopy was used to study the interaction of peanut (Arachis hypogaea) lectin with complementary carbohydrates. A correlation was observed between variations of ultraviolet spectra during the binding of sugars to the lectin and the specificity and the strength of the binding. The association constant, free energy, enthalpy and entropy for peanut lectin-lactose interactions were calculated over the temperature range 10–30°C. The binding constants for 10 mono- and disaccharides containing a D-galactopyranosyl or a D-talopyranosyl residue were calculated. Comparing their effectiveness to interact with peanut lectin, methyl $\text{α-}\text{D}\text{-galactopyranoside}$ appeared to have a more marked affinity than lactose; D-galacturonic acid and methyl $\text{7-}\text{deoxy-}\text{D}\text{-glycero}\text{-β-}\text{D}\text{-galacto-heptopyranoside}$ had no measurable affinity; the other sugars showed a lower affinity than lactose. The correlations between these differences and the conformations of the sugars obtained by X-ray analysis are discussed.     

Occurrence of mono- or disaccharides and polysaccharide reserves in mature pollen grains

 Pollen from 13 species of gymnosperms and angiosperms was studied for soluble and insoluble carbohydrates at dispersal. Starch reserves stored during pollen development give rise to carbohydrates at maturity. Combinations of different types of carbohydrates in mature pollen may depend on the extent of starch hydrolysis. An inverse relationship was found between the extent of starch hydrolysis and sucrose content. If the starch was scarcely de-polymerized, the cytoplasm had very low levels of soluble sugars and none of the periodic acid-Schiff (PAS)-positive material as found in pollen not subject to high dehydration (Cucurbita pepo L., Zea mays L.). After total or partial starch hydrolysis, insoluble PAS-positive oligo/polysaccharides were found in the cytoplasm associated with much soluble sugar, and the pollen grains were dehydrated at dispersal as in Typha latifolia L., Chamaerops humilis L., Trachycarpus excelsa Wendl., and other specimens. Intermediate levels of starch and soluble sugars, together with cytoplasmic PAS-positive material, characterized species with dehydrated pollen such as Pinus halepensis Miller. Carbohydrates may be related to pollen longevity, which largely depends on the abundance of sucrose, which is known to protect membrane integrity. The relationship between PAS-positive material and pollen viability is unclear at present. Received: 30 July 1996 / Revision accepted: 18 December 1996     

Screening of lipases for regioselective hydrolysis of peracetylated β-monosaccharides

The monodeacetylation of peracetylated-β-d-galactose (1) and peracetylated N-acetyl-β-d-glucosamine (2) by different lipases is here described. Lipases from different sources in an immobilized form were evaluated to find those that offer the higher activity and regioselectivity in the reactions. In the hydrolysis of 1, the lipase from Aspergillus niger was the most active one, although it hydrolyzed the anomeric position. Using the lipase from Candida rugosa, 30% yield of the corresponding 6-OH isomer was achieved. On the other hand, in the hydrolysis of 2, the lipase from A. niger was the most active and regioselective catalyst, producing more than 75% of the 6-OH derivative product.     

Enzymatic regioselective synthesis of sucrose acrylate esters

Using a protease (at 100 g l^–1) from Bacillus licheniformis, enzymatic acryloylation of sucrose (1 M) with vinyl acrylate (4 M) was carried out in anhydrous pyridine and yielded sucrose acrylate esters with more than 90% of sucrose converted in 24 h. After 5 days of reaction, the ester products consisted of 70% sucrose monoacrylate and 30% sucrose diacrylate. The monoester product was a sucrose 1-acrylate and the diester products consisted of sucrose 6,1-diacrylate and sucrose 6,1-diacrylate in the ratio of 3:2.     

Stimulation of the labellar sugar receptor of the fleshfly by mono- and disaccharides

Responses of the labellar sugar receptor of the fleshfly, Boettcherisca peregrina, were studied over a wide range of concentrations of several sugars (sucrose, maltose, glucose, fructose, and mannose) in single solutions and in mixtures. The results suggest (a) that the receptor sites are not completely differentiated for glucose and for fructose combination, (b) that the receptor site is composed of two subunits. Such suggestions are based on the classical model, where the response is proportional to the number of the sites, two subunits of each site being simultaneously occupied with one molecule of disaccharides or two molecules of monosaccharides. It is shown, however, that an allosteric model gives a somewhat better interpretation of the experimental results.     

Enzymatic synthesis of sulfated disaccharides using beta-D-galactosidase-catalyzed transglycosylation.

We have established a unique enzymatic approach for obtaining sulfated disaccharides using Bacillus circulans beta-D-galactosidase-catalyzed 6-sulfo galactosylation. When 4-methyl umbelliferyl 6-sulfo beta-D-galactopyranoside (S6Gal beta-4MU) was used as a donor, the enzyme induced transfer of 6-sulfo galactosyl residue to GlcNAc acceptor. As a result, the desired compound 6'-sulfo N-acetyllactosamine (S6Gal beta1-4GlcNAc) and its positional isomer 6'-sulfo N-acetylisolactosamine (S6Gal beta1-6GlcNAc) were observed by HPAEC-PAD, in 49% total yield based on the donor added, and in a molar ratio of 1:3.5. With a glucose acceptor, the regioselectivity was substantially changed and S6Gal beta1-2Glc was mainly produced along with beta-(1-1)alpha, beta-(1-3), beta-(1-6) isomers in 74% total yield. When methyl alpha-D-glucopyranoside (Glc alpha-OMe) was an acceptor, the enzyme also formed mainly S6Gal beta1-2Glc alpha-OMe with its beta-(1-6)-linked isomer in 41% total yield based on the donor added. In both cases, it led to the predominant formation of beta-(1-2)-linked disaccharides. In contrast, with the corresponding methyl beta-D-glucopyranoside (Glc beta-OMe) acceptor, S6Gal beta1-3Glc beta-OMe and S6Gal beta1-6Glc beta-OMe were formed in a low total yield of 12%. These results indicate that the regioselectivity and efficiency on the beta-D-galactosidase-mediated transfer reaction significantly depend on the anomeric configuration in the glucosyl acceptors.     

A convenient preparation of 1,2,3-tri-O-acetyl-beta-D-ribofuranose by enzymatic regioselective 5-O-deacetylation of the peracetylated ribofuranose

The lipase from Candida rugosa (Sigma) was used to catalyze the regioselective deacetylation at the 5-position of 1,2,3,5-tetra-O-acetyl-beta-D-ribofuranose on a preparative scale. Enzymatic deacetylation provides a convenient one-step preparation of 1,2,3-tri-O-acetyl-beta-D-ribofuranose.     

Desulfobulbus mediterraneus sp. nov., a sulfate-reducing bacterium growing on mono- and disaccharides

A new sulfate-reducing bacterium, strain 86FS1, was isolated from a deep-sea sediment in the western Mediterranean Sea with sodium lactate as electron and carbon source. Cells were ovoid, gram-negative and motile. Strain 86FS1 contained b- and c-type cytochromes. The organism was able to utilize propionate, pyruvate, lactate, succinate, fumarate, malate, alanine, primary alcohols (C(2)-C(5)), and mono- and disaccharides (glucose, fructose, galactose, ribose, sucrose, cellobiose, lactose) as electron donors for the reduction of sulfate, sulfite or thiosulfate. The major products of carbon metabolism were acetate and CO(2), with exception of n-butanol and n-pentanol, which were oxidized only to the corresponding fatty acids. The growth yield with sulfate and glucose or lactate was 8.3 and 15 g dry mass, respectively, per mol sulfate. The temperature limits for growth were 10 degrees C and 30 degrees C with an optimum at 25 degrees C. Growth was observed at salinities ranging from 10 to 70 g NaCl l(-1). Sulfide concentrations above 4 mmol l(-1) inhibited growth. The fatty acid pattern of strain 86FS1 resembled that of Desulfobulbus propionicus with n-14:0, n-16:1omega7, n-16:1 omega5, n-17:1 omega6 and n-18:1 omega7 as dominant fatty acids. On the basis of its phylogenetic position and its phenotypic properties, strain 86FS1 affiliates with the genus Desulfobulbus and is described as a new species, Desulfobulbus mediterraneus sp. nov.     

Immobilisation on polystyrene of diazirine derivatives of mono- and disaccharides: biological activities of modified surfaces

The potential of surface glycoengineering for biomaterials and biosensors originates from the importance of carbohydrate-protein interactions in biological systems. The strategy employed here utilises carbene generated by illumination of diazirine to achieve covalent bonding of carbohydrates. Here, we describe the synthesis of an aryl diazirine containing a disaccharide (lactose). Surface analysis techniques [X-ray photoelectron spectroscopy (XPS) and time of flight secondary ion mass spectroscopy (ToF-SIMS)] demonstrate its successful surface immobilisation on polystyrene (PS). Results are compared to those previously obtained with an aryl diazirine containing a monosaccharide (galactose). The biological activity of galactose- or lactose-modified PS samples is studied using rat hepatocytes, Allo A lectin and solid-phase semi-synthesis with alpha-2,6-sialyltransferase. Allo A shows some binding to galactose-modified PS but none to lactose-modified surfaces. Similar results are obtained with rat hepatocytes. In contrast, sialylation of lactose-modified PS is achieved but not with galactose-modified surfaces. The different responses indicate that the biological activity depends not only on the carbohydrate per se but also on the structure and length of the spacer.     

Recombinant lipase from Candida rugosa for regioselective hydrolysis of peracetylated nucleosides. A comparison with commercial non-recombinant lipases

Abstract

Commercial lipases from the yeast Candida rugosa have been compared with two recombinant C. rugosa lipases, rCRL1 and rCRL1lid3, with respect to their immobilization and exploitation in biotransformations aimed at the synthesis of pyrimidine nucleosides. Immobilization on octyl-agarose and decaoctyl-Sepabeads but not on Eupergit^® C gave comparable results to commercial lipases for rCRL1, while only a low percentage (12%) of rCRL1lid3 was efficiently immobilized. When immobilized on decaoctyl-Sepabeads, rCRL1 showed a markedly higher stability to chemical inactivation, since it could maintain 100% activity after 180 h incubation in 30% (v/v) acetonitrile. Hydrolysis of peracylated uridine and cytidine and their fluorinated counterparts proceeded with high regioselectivity and good yield, and even improved when rCRL1 was immobilized on decaoctyl-Sepabeads.     

Enzymatic preparation of heparin disaccharides as building blocks in glycosaminoglycan synthesis.

Pharmaceutical heparin and heparan sulfate, isolated from a side-stream of a commercial heparin manufacturing process, have been enzymatically depolymerzed with heparin lyases obtained from Flavobacterium heparinun. Heparin afforded a trisulfated disaccharide product that was recovered from the reaction mixture using gel permeation chromatography. Heparan sulfate afforded unsulfated disaccharide that was conveniently recovered from the product mixture by ion exchange chromatography. Both disaccharides were obtained in gram amounts at 90% or higher purity. Both enzymatically prepared disaccharides were chemically protected to prepare building blocks required for the future chemical synthesis of therapeutically valuable heparin oligosaccharides.     

Lipase-catalyzed regioselective protection/deprotection of hydroxyl groups of the isoflavone irilone isolated from Iris germanica

The regioselective acylation of irilone, isolated from Iris germanica, with vinylacetate and propenylacetate and deacylation of irilone diacetate with n-butanol were studied using lipases from Aspergillus niger, Mucor miehei, Pseudomonas cepacia, Candida cylindracea, porcine pancreas and Candida antarctica. Significant conversion of irilone to 4′-O-acetylirilone was achieved using P. cepacia lipase, while irilone diacetate was converted to 5-O-acetylirilone by the enzymatic action of lipases from M. miechei, P. cepacia and porcine pancreas under different experimental conditions. This preferential protection/deprotection furnishes an opportunity to modify the structure of irilone by selective derivatization that may help to change its biological activities by modifying its amphiphilic/lipophilic balance.     

Synthesis of peracetylated chacotriose

Steroidal glycoalkaloids of many Solanum species have recognized biological activities, especially those containing the glycosyl moiety alpha-L-rhamnopyranosyl-(1-->2)-[alpha-L-rhamnopyranosyl-(1-->4)]-D-glucopyranose (chacotriose) whose peracetate is here synthesized and characterized by complete 1H and 13C NMR assignment.     

Enzymatic synthesis of fucose-containing disaccharides employing the partially purified alpha-L-fucosidase from Penicillium multicolor

The alpha-L-Fucp-(1 --> 3)-D-GlcpNAc disaccharide structure is a vital core unit of the oligosaccharide components of glycoconjugates isolated from human milk and blood group substances. Alpha-L-Fucosidase from Penicillium multicolor catalyses the transfer of L-fucose from donor structures such as alpha-L-FucpOpNP and alpha-L-FucpF to various GlcpNAc derivatives and Glcp, forming alpha-(1 --> 3) linkages. The synthesis of several biologically relevant disaccharides including alpha-L-Fucp-(1 --> 3)-alpha-D-GlcpNAcOMe, alpha-L-Fucp-(1 --> 3)-alpha-D-GlcpNAcOAll, alpha-L-Fucp-(1 --> 3)-beta-D-GlcpNAcOAll, alpha-L-Fucp-(1 --> 3)-D-GlcpNAc and alpha-L-Fucp-(1 --> 3)-D-Glcp has been achieved in up to 34% yields by application of this enzyme.