A comprehensive profiling of sulfatides in myelin from mouse brain using liquid chromatography coupled to high-resolution accurate tandem mass spectrometry.

Sulfatides are sulfoglycolipids found in the myelin sheath. The composition ratio of sulfatide molecular species changes with age, and it has also been associated with the pathogenesis of various human central nervous system diseases. However, profiling sulfatides in biological samples is difficult, due to the great variety of molecular species. In this work, a new, easy and reliable liquid chromatography-electrospray tandem mass spectrometry (LC-ESI(+)-MS/MS) method has been developed to profile sulfatide content in biological samples of myelin. The 'wrong-way-round' ionization effect has been described for this type of molecules for the first time, making it possible to correctly identify as many as 37 different sulfatides in mouse brain myelin samples, including molecules with different fatty acid chain lengths and varying degrees of unsaturation and hydroxylation. A chemometric analysis of their relative abundances showed that the main difference among individuals of different ages was the content of sulfatides with odd-numbered fatty acid chains, in addition to hydroxylated species.

Sustratos electrohilados de poli(hidroxibutirato-co-hidroxihexanoato) con glicósidos para reparación de la lesión medular / Glycosides-bearing electrospun poly(hydroxybutyrate-co-hydroxyhexanoate) substrates for the repair of injured spinal cord

Objetivo: Obtención mediante electrohilado de fibras micro- y submicrométricas de poliésteres funcionalizadas con glicósidos que constituyen elementos estructurales de proteoglicanos, para su uso en la reparación del tejido medular. Material y métodos: Las fibras se prepararon a partir de disoluciones de poli(3-hidroxibutirato-co-3-hidroxihexanoato) con glicósidos sintéticos mediante electrohilado variando sistemáticamente las condiciones del proceso. La morfología de las fibras fue analizada mediante microscopía electrónica de barrido. Asimismo, se evaluó la estabilidad de la interacción entre el glicósido y la fibra en medio acuoso, y su toxicidad en cultivos de células neurales. Resultados: La morfología de las fibras obtenidas depende principalmente de los parámetros de la disolución. En medio acuoso, el glicósido sulfatado se liberó de las fibras más lentamente que el que no tenía dicho grupo funcional. La viabilidad de las células neurales no se vio afectada por los glicósidos. Conclusión: La preparación de microfibras alineadas de poliéster funcionalizadas con glicósidos es posible. La mayor parte del glicósido permanece retenido en las fibras sumergidas en agua después de varios días. El electrohilado es una técnica muy accesible y versátil para la fabricación de soportes en estrategias de terapia celular de lesiones medulares (AU)

Objective: Preparation of functionalized micro- and submicrofibers by electrospinning of polyesters with glycosides which are structural elements of proteoglycans, for application to the repair of spinal cord lesions. Material and methods: Solutions of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with synthetic glycosides were prepared varying systematically the processing conditions. Fiber morphology assessed by scanning electron microscopy. The stability of the interaction between the glycoside and the polymer fiber was evaluated in aqueous medium, and their toxicity in cultures of neural cells. Results: The fiber morphology was altered mainly by the solution parameters. In aqueous medium, the glycoside with a sulfate group was released from fibers at slower rate than the non-sulfated glycoside. The viability of neural cells was not affected by the glycosides. Conclusion: It is possible to fabricate aligned polyester micro fibers with glycosides. Most of the glycoside present in the fibers remains in the substrate after extraction in water for several days. Electrospinning is a very accessible and versatile technique for application to strategies of cellular therapy in spinal cord injuries (AU)

A study on partially biodegradable microparticles as carriers of active glycolipids.

This paper describes a study on the preparation and characterisation of partially biodegradable microparticles of poly(epsilon-caprolactone)/poly(ethyl methacrylate) (PCL/PEMA) as carriers of synthetic glycolipids with antimitotic activity against brain tumour cells. Microparticles prepared by suspension polymerisation of methacrylate in the presence of already polymerised PCL showed a predominantly spherical but complex morphology, with segregation of PCL micro/nano-domains towards the surface. Small diameter discs were prepared by compression moulding of blends of microparticles and the active principle under mild conditions. The in vitro behaviour of the discs and release of the glycolipid were studied in different simulated fluid models. Ingress of fluids increased with increasing hydrophobicity of the medium. Release of the glycolipid was sustained in all fluids, the most prolonged profile being in human synovial fluid and phosphate-buffered saline modified with 20 vol.% dioxane. Slow disintegration of the discs and partial degradation of the microparticles was evident in accelerated studies. The antimitotic activity of glycolipid released from the discs was proved against a human glioblastoma line. This activity, along with selectivity against human fibroblasts, could be controlled by the amount of drug charged in the disc.

Polymeric drugs based on bioactive glycosides for the treatment of brain tumours.

Polymeric drugs carrying glycolipids have been designed as target macromolecules for the treatment of brain tumours. A methacrylate derivative of oleyl 2-acetamido-2-deoxy-alpha-D-glucopyranoside (OAGMA) has been prepared and the corresponding glycopolymer obtained by free radical polymerisation. To modulate the hydrophobic character of the polymeric drug, the acrylic glycomonomer was copolymerised with vinyl pyrrolidone (VP). Reactivity ratios obtained by performing copolymerisation reactions inside the NMR apparatus were r(OAGMA)=5.94 and r(VP)=0.01, indicating the much higher reactivity of the glycomonomer. The hydrolytical release of oleyl 2-acetamido-2-deoxy-alpha-D-glucopyranoside (OAG) from the copolymeric drugs was produced in vitro by the ester enzymatic hydrolysis using enzyme/buffered solutions. The cytotoxicity of OAG and OAGMA tested against a human glioblastoma line and normal fibroblasts revealed a concentration dependent selectivity towards tumour cells versus fibroblasts. The antimitotic activity of the copolymeric drugs was also confirmed. The addition of the eluates of the copolymeric systems collected at 1 and 2 days produced a significant decrease in cellular viability of the glioblastoma cells without affecting that of normal fibroblasts. On the contrary, fibroblasts were able to adhere and proliferate onto the copolymeric systems showing normal morphology and revealing a good biocompatibility of the copolymeric drugs against healthy cells.

Heterologous over-expression of alpha-1,6-fucosyltransferase from Rhizobium sp.: application to the synthesis of the trisaccharide beta-D-GlcNAc(1-->4)-[alpha-L-Fuc-(1-->6)]-D-GLcNAc, study of the acceptor specificity and evaluation of polyhydroxylated indolizidines as inhibitors.

An efficient heterologous expression system for overproduction of the enzyme alpha-1,6-Fucosyltransferase (alpha-1,6-FucT) from Rhizobium sp. has been developed. The gene codifying for the alpha-1,6-FucT was amplified by PCR using specific primers. After purification, the gene was cloned in the plasmid pKK223-3. The resulting plasmid, pKK1,6FucT, was transformed into the E. coli strain XL1-Blue MRF'. The protein was expressed both as inclusion bodies and in soluble form. Changing the induction time a five-fold increase of enzyme expressed in soluble form was obtained. In this way five units of enzyme alpha-1,6-FucT can be obtained per liter of culture. A crude preparation of the recombinant enzyme was used for the synthesis of the branched trisaccharide alpha-D-GlcNAc-(1-->4)-[alpha-L-Fuc-(1-->6)]-D-GlcNAc (3), from chitobiose (2) and GDP-Fucose (1). After purification, the trisaccharide 3 was obtained in a 84% overall yield. In order to elucidate the structural requirements for the acceptors, the specificity of the enzyme was studied towards mono-, di- and trisaccharides, which are structurally related to chitobiose. The enzyme uses, among others, the disaccharide N-acetyl lactosamine as a good substrate; the monosaccharide GlcNAc is a weak acceptor. Finally, several racemic polyhydroxylated indolizidines have been tested as potential inhibitors of the enzyme. Indolizidine 21 was the best inhibitor with an IC50 of 4.5 x 10(-5) M. Interestingly, this compound turned out to be the best mimic for the structural features of the fucose moiety in the presumed transition state.

Stereospecific synthesis of alpha- and beta-C-glycosides from glycosyl sulfoxides: scope and limitations.

C-Glycosides derived from alpha-L-fuco-, alpha-D-gluco-, beta-D-gluco-, and alpha-D-mannopyranose have been synthesized from the corresponding glycosyl phenyl sulfoxide through phenylsulfinyl-lithium exchange, to generate an anomeric carbanion, and subsequent reaction with a carbon electrophile. The reactions were stereospecific and proceeded with retention of the configuration at the anomeric center. Improved yields of C-glycosides were obtained by an inverse addition protocol. Trapping of the anomeric carbanion with aldehydes gave best results. Reaction with ketones, chloroformates, nitriles, and alkyl halides was also explored. Mechanistic aspects of the reaction are discussed.

Synthesis and biological studies of glycosyl dopamine derivatives as potential antiparkinsonian agents.

A new approach to deliver dopamine into the central nervous system, based on the use of D-glucose as transportable agent, has been studied. Glycosyl dopamine derivatives bearing the sugar moiety linked to either the amino group or the catechol ring of dopamine through amide, ester or glycosidic bonds were synthesised as potential antiparkinsonian agents. Studies on the binding to dopamine D2 receptor, in vitro stability, and locomotive effect in mice of the synthetic glycoconjugates are reported.

Purification and structure of neurostatin, an inhibitor of astrocyte division of mammalian brain.

Neurostatin was originally described as an inhibitor of astroblast and astrocytoma division present in rat brain extracts and immunologically related to the sugar moiety of epidermal growth factor receptor and to blood group antigens. It was purified recently from mammalian brain extracts and characterized as a glycosphingolipid, but its precise structure remained unknown. Neurostatin has now been purified to apparent homogeneity from ganglioside extracts of rat, bovine, and porcine brain. It is cytostatic for astroblasts, C6 glioma cells, and various human astrocytomas grades III and IV, with IC(50) values ranging from 250 to 450 nM, but does not affect the division of primary or transformed fibroblasts up to concentrations >4 microM. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of purified pig neurostatin showed a molecular ion of 1, 905 Da and ions of 1,863 and 1,934 Da, compatible with a disialoganglioside. Mono- and bidimensional NMR spectra, together with biochemical studies, suggest that neurostatin may be the 9-O-monoacetyl ester of GD1b.

Novel disaccharide inhibitors of human glioma cell division.

Several alpha-L-Fuc-(1-->3)-alpha-D-GlcNAcOC8H17 disaccharide derivatives bearing different hydroxylated alkyl chains, with or without sulfate groups at C-4 and/or C-6 positions of the GlcNAc unit, have been synthesized and tested as inhibitors of human astrocytoma lines U-373 and U-118. The antimitotic activity was dependent on the structure and position of the hydroxylated chain linked to the disaccharide. The compounds with a pentaerythritol or L-glyceryl chain at the C-6 position showed the best inhibitory properties, with an ID50 value of ca. 200 microM. On the contrary, sulfated disaccharide derivatives were inactive. The antimitotic activities of the compounds tested were essentially independent of the mitogen used to stimulate cell division.

Conformational differences between Fuc(alpha 1-3) GlcNAc and its thioglycoside analogue.

NOE measurements and molecular mechanics calculations have been performed to study the conformational behaviour of Fuc(alpha 1-3)GlcNAc and its thioglycoside analogue in solution. Experimental data show that, in contrast with the natural O-disaccharide, which is basically monoconformational, the S-analogue shows two conformational families, namely syn and anti.

Regioselectivity of the enzymatic transgalactosidation of D- and L-xylose catalysed by beta-galactosidases.

The regioselectivity of enzymatic transgalactosidation depends on the source of the beta-galactosidase used. When the galactosyl acceptor only contains secondary hydroxyl groups, e.g., D- or L-xylose, it is possible to find an enzyme that catalyses preferentially the synthesis of any of the three regioisomers 4-, 3- and 2-O-beta-D-galactopyranosyl-D-xylose (1, 2 and 3, respectively) or 4-, 3- and 2-O-beta-D-galactopyranosyl-L-xylose (4, 5 and 6, respectively). Enriched mixtures in 1, 2 or 3 were obtained using beta-galactosidases from Escherichia coli, bovine testes or Aspergillus oryzae, respectively, by transgalactosidation reaction of O-nitrophenyl-beta-D-galactopyranoside and D-xylose, and enriched mixtures in 4, 5 or 6 were obtained in a similar way using beta-galactosidases from Aspergillus oryzae, lamb small-intestine (intestinal lactase-phloridzin hydrolase) or Saccharomyces fragilis, respectively, using L-xylose as acceptor.

A direct enzymatic synthesis of beta-D-galactopyranosyl-D-xylopyranosides and their use to evaluate rat intestinal lactase activity in vivo.

By enzymatic beta-D-galactosylation of D-xylose a mixture of 4-, 3-, and 2-O-beta-D-galactopyranosyl-D-xyloses (1, 4, and 7, respectively) was obtained in 50% isolated yield. Disaccharides 1, 4, and 7 are substrates of intestinal lactase isolated from lamb small intestine with K(m) values of 250.0, 4.5, and 14.0 mM, respectively. The mixture was used to monitor the normal decline in lactase activity in rats that takes place after weaning. The data obtained by this method correlated with the levels of intestinal lactase activity in the same animals after being sacrificed.

Experimental brain glioma: growth arrest and destruction by a blood-group-related tetrasaccharide.

A synthetic tetrasaccharide (TS4), structurally related to blood groups, inhibited the proliferation of the C6 glioma cells in culture and the growth of tumors formed after intracerebral transplantation of C6 cells. TS4-treated tumors were substantially smaller than controls, as expected from TS4 cytostatic action on C6 glioma cells in culture. However, in vivo treatment also caused extensive tumor destruction. This effect appeared to be caused by indirectly, either by activation of natural killer cells, cytotoxic lymphocytes, or by inhibition of tumor vascularization. Enhanced antigenicity of TS4-treated glioma may be related to the increased expression of connexin 43 observed in glioma cell cultures treated with the oligosaccharide. Because concentrations of up to 20 mg/ml of TS4 were not toxic for normal neuronal or glial cells, specific oligosaccharides such as TS4 offer the possibility of selective tumor treatment.

Inhibition of proliferation of normal and transformed neural cells by blood group-related oligosaccharides.

A synthetic tetrasaccharide structurally related to blood groups and selectin ligands inhibited division of astrocytes, gliomas, and neuroblastomas at micromolar concentrations. The compound was cytostatic for primary astrocytes in culture, but cytotoxic for fast proliferating cell lines.

4-O-beta-D-galactopyranosyl-D-xylose: a new synthesis and application to the evaluation of intestinal lactase.

4-O-beta-D-Galactopyranosyl-D-xylose (2) was prepared from benzyl 2,3-O-isopropylidene-beta-D-xylopyranoside by glycosylation with 2,3,4,6-tetra-O-benzoyl-alpha-D-galactopyranosyl bromide and subsequent deprotection. Compound 2 was hydrolyzed in vitro by intestinal lactase; the Vmax was 25% of that with lactose and the Km was 370mM (cf. 27mM for lactose). Oral administration of 2 suckling rats led to urinary excretion of D-xylose which could be estimated colorimetrically.

The synthesis and resolution of (+/-)-1,5,6-tri-O-benzyl-myo-inositol.

Racemic 1,5,6-tri-O-benzyl-myo-inositol was prepared by five routes and converted into 1,5,6-tri-O-benzyl-2,3-O-isopropylidene-myo-inositol, the camphanates of which were readily separated by chromatography. The absolute configurations of the chiral derivatives were established by their conversion into the known chiral 1,4,5,6-tetra-O-benzyl-myo-inositols. 1D-1,5,6-Tri-O-benzyl-2,3-O-isopropylidene-myo-inositol was converted into 1D-1,3,5,6-tetra-O-benzyl-myo-inositol and thence into 1D-2,4-di-O-methyl-myo-inositol. 1D-1,5,6-Tri-O-benzyl-myo-inositol was converted into 1D-1,2,5,6-tetra-O-benzyl-myo-inositol, the diacetate of which is a chiral analogue of "thermosalient crystals". The potential of the above compounds for the synthesis of natural products is surveyed.

4-O-beta-D-Galactopyranosyl-3-O-methyl-D-glucose: a new synthesis and application to the evaluation of intestinal lactase.

4-O-beta-D-Galactopyranosyl-3-O-methyl-D-glucose (1, 3-O-methyl-lactose) has been prepared from benzyl 2,6-di-O-benzyl-4-O-(2,6-di-O-benzyl-3, 4-O-isopropylidene-beta-D-galactopyranosyl)-beta-D-glucopyranoside (5) and from benzyl 2,6-di-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-beta-D-galactopyranosyl)-bet a-D-glucopyranoside (15). Partial benzylation of benzyl 3',4'-O-isopropylidene-beta-lactoside (4) gave 5 and partial benzylation of either benzyl beta-lactoside (13) or benzyl hepta-O-acetyl-beta-lactoside (24) gave 15. All other products from the partial benzylation of 4, 13, and 24 were also isolated and characterised. The hydrolysis of 1 in vitro by intestinal lactase was linear during 20 h; the Vmax was 5% of that with lactose and the Km was 120mM (cf. 30mM for lactose). Oral administration of 1 to suckling rats led to urinary excretion of 3-O-methyl-D-glucose.