[Pharmaceutical use of cyclodextrines: perspectives for drug targeting and control of membrane interactions]. / Les cyclodextrines en pharmacie: perspectives pour le ciblage d'actifs thérapeutiques et le contrôle d'interactions membranaires.

Cyclomaltooligosaccharides (cyclodextrins, CDs) comprise a family of biocompatible cage devices which have been developed during the last thirty years in order to improve the solubility, stability and the bioavailability of drugs. Chemical modification usually improves the solubility and solubilisation properties and generally alleviates the renal toxicity of native cyclodextrins. Red cell lysis, which is ascribed to membrane interactions is also monitored. Selective and commercially accessible functionalisation processes are now available which avoid the problems of heterogeneity commonly found with the existing industrial approaches. These allow a convenient access to modular structures which could fit the molecular characteristics of the host ("bouquet" and dimeric CDs). Grafting of saccharide ligands which are recognised by membrane proteins is another promising aspect for the transport and targeting of drugs and the control of cell interactions. Several topological aspects of ligand presentation toward a membrane lectin have been assessed with concanavalin A and mannosyl CD-dendrimers and the results have been extended to molecular targeting to macrophages. Advantage has been taken of the autoassociation properties of amphiphilic derivatives of cyclodextrins for the preparation of stable nanoparticles of interest for the transport and targeting of drugs and macromolecular systems.

Carbohydrate-derived spiroketals. Stereoselective synthesis of di-D-fructose dianhydrides by boron trifluoride promoted glycosylation-spiroketalization of acetal precursors.

[reaction: see text] Di-D-fructose 1,2':2,1'-dianhydrides, dispiro-tricyclic disaccharides widely found in food materials, have been stereoselectively prepared in one-pot reaction from O-protected D-fructose 1,2-acetonide precursors by treatment with boron trifluoride diethyl etherate. The dimerization sequence involves (i) cleavage of the anomeric acetal linkage, (ii) autoglycosylation, and (iii) final spiroketalization, the stereochemical outcome being strongly dependent on the nature of the hydroxyl protecting groups.

Qualitative and quantitative evaluation of mono- and disaccharides in D-fructose, D-glucose and sucrose caramels by gas-liquid chromatography-mass spectrometry. Di-D-fructose dianhydrides as tracers of caramel authenticity.

The monosaccharide (D-fructose, D-glucose, anhydrosugars), disaccharide (glucobioses) and pseudodisaccharide (di-D-fructose dianhydrides) content of D-fructose, D-glucose and sucrose caramels has been determined by gas-liquid chromatography-mass spectrometry (GLC-MS) of their trimethylsilyl (TMS) or TMS-oxime derivatives. The chromatographic profiles revealed significant differences in the disaccharide/pseudodisaccharide distribution depending on the caramel source: a D-fructose caramel contains prominent proportions of di-D-fructose dianhydrides, a D-glucose caramel mainly D-glucobioses, and a sucrose caramel similar proportions of both disaccharide/pseudodisaccharide series. It is noteworthy that di-D-fructose dianhydrides are found in all three types of caramels and might then be used as specific tracers of the authenticity of caramel, i.e., a product resulting from the controlled heat treatment of food-grade carbohydrates for use as food additives.

Hydrogen fluoride-mediated synthesis of 1-thiotrehaloses involving reaction of D-glucose with hydrogen sulfide.

Hydrogen sulfide reacted with D-glucose in hydrogen fluoride solution to yield preponderantly alpha,alpha-1-thiotrehalose, beta,beta-1-thiotrehalose, and the alpha,beta anomer. Conditions were found under which the thiotrehaloses were obtained in the respective proportions of 8:5:5.

A convenient access to beta-(1-->4)-linked 2-amino-2-deoxy-D-glucopyranosyl fluoride oligosaccharides and beta-(1-->4)-linked 2-amino-2-deoxy-D-glucopyranosyl oligosaccharides by fluorolysis and fluorohydrolysis of chitosan.

beta-(1-->4)-Linked 2-amino-2-deoxy-D-glucopyranosyl oligosaccharides, in the form of their alpha-glucopyranosyl fluorides at the reducing end, were obtained by fluorolysis of chitosan in anhydrous hydrogen fluoride at room temperature. The average dp depended on the reaction time and was conveniently monitored by 13C NMR spectroscopy, using the signal ratios for beta-(1-->4) bonded C-1 at approximately 98.5 ppm and the C-1 doublet for the terminal glycosyl fluoride moiety at approximately 104 ppm. Preparative fractionation of dp 2-11 glycosyl fluoride oligosaccharides, obtained after 18 h of fluorolysis, was achieved by gel-permeation chromatography on Bio-Gel P-4 with aqueous acetic acid-ammonium acetate as eluent. Hydrolysis of the anomeric fluoride, with either aqueous perchloric acid, or by a sequence involving formation of the C-2 N-trifluoroacetate and subsequent simultaneous hydrolysis of the glycosyl fluoride and the amide substituent with aqueous methanol, yielded the free beta-(1-->4)-linked 2-amino-2-deoxy-D-glucopyranosyl oligosaccharides which were separated, for dp 2-11, by the same gel-exclusion technique. Both oligosaccharide series, either free or in the form of their alpha-glycopyranosyl fluorides, were fully characterized.

Selective protonic activation of isomeric glycosylfructoses with pyridinium poly(hydrogen fluoride) and synthesis of spirodioxanyl oligosaccharides.

Selective activation of the ketose unit in the isomeric glycosylfructoses, palatinose, leucrose, maltulose, turanose and lactulose, with pyridinium poly(hydrogen fluoride) resulted in the almost quantitative formation of glycosylated difructose dianhydrides. The reaction preferentially involves a reactive fructofuranosyl oxocarbenium ion and is subject to stereoelectronic control. The relative amounts of isomeric spirodioxanyl oligosaccharides obtained within a series was shown to depend on the reaction conditions, especially on the hydrogen fluoride-pyridine ratio. Using suitable concentrations of hydrogen fluoride in pyridine, the reaction was easily directed to the formation of the kinetic difuranosyl or thermodynamic pyranosyl derivatives. More rigorous conditions resulted in the specific hydrolysis of one glycosidic bond in the tetrasaccharides derived from palatinose, leucrose and turanose, to yield spirodioxanyl trisaccharides.

A convenient synthesis for anomeric 2-thioglucobioses, 2-thiokojibiose and 2-thiosophorose.

2-S-alpha-D-Glucopyranosyl-2-thio-D-glucopyranose (2-thiokojibiose, 8) and 2-S-beta-D-glucopyranosyl-2-thio-D-glucopyranose (2-thiosophorose, 14) were conveniently prepared by SN2 reaction of the corresponding anomers of 2,3,4,6-tetra-O-acetyl-1-thio-D-glucopyranose with 1,3,4,6-tetra-O-acetyl-2-O-tri-flyl-beta-D-mannopyranose, followed by a deprotection sequence for the anomeric acetate involving conversion into the 1-propenyl glycosides. Alkaline O-deacetylation was followed by smooth hydrolysis of the propenyl group at pH approximately 2.

Synthesis of dispirodioxanyl pseudo-oligosaccharides by selective protonic activation of isomeric glycosylfructoses in anhydrous hydrogen fluoride.

Dispirodioxanyl pseudotetrasaccharides 6-O-alpha-D-glucopyranosyl-alpha-D-fructofuranose 6-O-alpha-D-glucopyranosyl-beta-D-fructofuranose 1,2':2,1'-dianhydride, 5-O-alpha-D-glucopyranosyl-alpha-D-fructopyranose 5-O-alpha-D-glucopyranosyl-beta-D-fructopyranose 1,2':2,1'-dianhydride, 4-O-alpha-D-glucopyranosyl-alpha-D-fructofuranose 4-O-alpha-D-glucopyranosyl-beta-D-fructopyranose 1,2':2,1'-dianhydride, 4-O-beta-D-galactopyranosyl-alpha-D-fructofuranose 4-O-beta-D-galactopyranosyl-beta-D-fructopyranose 1,2':2,1'-dianhydride, and 3-O-alpha-D-glucopyranosyl-alpha-D-fructofuranose 3-O-alpha-D-glucopyranosyl-beta-D-fructofuranose 1,2':2,1'-dianhydride were respectively obtained, on a preparative scale, by dissolution of the isomeric glycosylfructoses palatinose, leucrose, maltulose, lactulose, and turanose in anhydrous hydrogen fluoride. The reaction, involving selective protonation at the free anomeric position of the fructose unit, was extended to the preparation of the pseudotrisaccharides 6-O-alpha-D-glucopyranosyl-alpha-D-fructofuranose beta-D-fructopyranose 1,2':2',1-dianhydride from palatinose and fructose, and to its 3-O-, 4-O-, and 4'-O-glucosyl analogues using turanose and maltulose as the disaccharide precursor. The cross-reactions of palatinose with maltulose and with leucrose resulted in the preparation of 6-O-alpha-D-glucopyranosyl-alpha-D-fructofuranose 4-O-alpha-D-glucopyranosyl-beta-D-fructopyranose 1,2':2,1'-dianhydride and 6-O-alpha-D-glucopyranosyl-alpha-D-fructofuranose 5-O-alpha-D-glucopyranosyl-beta-D-fructopyranose 1,2':2,1'-dianhydride, respectively.

Protonic reactivity of sucrose in anhydrous hydrogen fluoride.

Sucrose reacts quantitatively, when dissolved at high concentration in anhydrous hydrogen fluoride, to afford a complex mixture of difructose dianhydrides and their glucosylated derivatives. Oligo- and small poly-saccharides up to dp 14 were detected by FABMS. Oligosaccharides up to dp 4, representing approximately 50% of the total mixture, have been isolated and characterized by mass spectrometry, 13C NMR spectroscopy, and comparison with reference oligosaccharides previously obtained by unambiguous synthesis. alpha-D-Fructofuranose beta-D-fructopyranose 1,2':2,1'-dianhydride is the main spirodioxanyl pseudodisaccharide entity found in the mixture, either free or glucosylated at C-6 and to a lesser extent at C-3, C-4, C-4', C-6, and C-5' C-6. Minor spirodioxanyl pseudodisaccharide components are di-beta-D-fructopyranose 1,2':2,1'-dianhydride, which has also been found glucosylated at C-5, alpha-D-fructopyranose beta-D-fructopyranose 1,2':2,1'-dianhydride, beta-D-fructofuranose beta-D-fructopyranose 1,2':2,3'-dianhydride, and the 6,6'-diglucosylated alpha-D-fructofuranose beta-D-fructofuranose 1,2':2,1'-dianhydride. A 13C NMR examination of the higher mass oligomeric fraction suggests that it may involve 6-O-isomaltooligoglycosyl alpha-D-fructofuranose beta-D-fructopyranose 1,2':2,1'-dianhydrides as the main structural components. The reaction of sucrose in anhydrous HF is believed to proceed through initial selective protonic activation of the tertiary anomeric carbon atom of the fructose moiety, resulting in the quantitative formation of difructose dianhydrides, which subsequently suffer electrophilic substitution by glucopyranosyl oxocarbenium ions generated in a second step by action of the HF.

Steric and electronic effects in the formation of dihexulose dianhydrides. Reaction of racemic sorbose in anhydrous hydrogen fluoride and a facile synthesis of D-sorbose.

Treatment of DL-sorbose with anhydrous hydrogen fluoride gave a high yield of alpha-D-sorbopyranose alpha-L-sorbopyranose 1,2':2,1'-dianhydride. Similarly a mixture of D-fructose and D-sorbose gave a good yield of beta-D-fructopyranose alpha-D-sorbopyranose 1,2':2,1'-dianhydride. The formation of these products compared to the more complicated mixtures of compounds obtained by treatment of L-sorbose or D-fructose with hydrogen fluoride, is discussed in terms of conformations, and steric and electronic factors.

The structure of the sialic acid-containing Escherichia coli O104 O-specific polysaccharide and its linkage to the core region in lipopolysaccharide.

Mild acid hydrolysis of Escherichia coli O104 lipopolysaccharide released an O-specific polysaccharide, a tetrasaccharide repeating unit, the corresponding dimer, and a disaccharide fragment of the repeating unit. Complete and incomplete cores, and oligosaccharides comprising fragments of the repeating unit and the core region, were also obtained. On the basis of sugar and methylation analysis, FAB-mass spectrometry and NMR spectroscopy of the hydrolysis products, the repeating unit of the O-specific polysaccharide was shown to be the tetrasaccharide:-->4)-alpha-D-Galp-(1-->4)-alpha-Neup5,7,9Ac3++ +-(2-->3)-beta-D- Galp-(1-->3)-beta-D-GalpNAc (1-->. The linkage between the O-specific polysaccharide chain and the core region, which appeared to be of the R2 type, was established. These results indicate that N-acetylneuraminic acid, located in the O-specific polysaccharide, is an inherent lipopolysaccharide component.

Difructose dianhydrides from sucrose and fructo-oligosaccharides and their use as building blocks for the preparation of amphiphiles, liquid crystals, and polymers.

Controlled selective protonic activation of the fructosyl moiety in sucrose and fructo-oligosaccharides, with pyridinium poly (hydrogen fluoride) at 20 degrees C, yielded either the kinetic product alpha-D-fructofuranose beta-D-fructofuranose 1,2':2,1'-dianhydride (1), or its thermodynamically more stable isomer alpha-D-fructofuranose beta-D-fructopyranose 1,2':2,1'-dianhydride (2), depending on the hydrogen fluoride-pyridine ratio. A similar reaction was performed with 6,6'-dichloro-6,6'-dideoxysucrose, or 6,6'-dideoxy-6,6'-diiodosucrose, using a slightly higher ratio of HF, resulting in the corresponding 6-deoxy-6-halo-alpha-D-fructofuranose 6'-deoxy-6'-halo-beta-D-fructofuranose 1,2':2,1'-dianhydride derivatives. Both 6,6'-dihalides were converted, upon action of the appropriate nucleophile, into the difructofuranose dianhydride derivatives bearing the 6,6'-di-S-heptyl-6,6'-dithio, 6,6'-diazido-6,6'-dideoxy and then 6,6'-diamino-6,6'-dideoxy functionalities. 6-Chloro-6-deoxy and 6-deoxy-6-iodo derivatives of 2 were also prepared by direct halogenation, and further converted into the 6-S-heptyl-6-thio, 6-azido-6-deoxy and then 6-amino-6-deoxy derivatives of 2. Reaction of chloromethyloxirane with 1 or 2 yielded hydrophilic polymers. The 6,6'-di-S-heptyl-6,6'-dithio derivative of 1 displayed liquid crystal properties. The 6,6'-dideoxy-6,6'-diiodosucrose precursor was prepared by the reaction of Garegg's iodine-imidazole-triphenylphosphine reagent with sucrose in N,N-dimethylformamide solution.

Synthesis of 6-deoxymaltooligosaccharides and a study of their lipid-binding properties.

Amylose reacted in N,N-dimethylformamide with crystalline bromomethylenedimethylammonium bromide (Vilsmeier bromide) to give 6-bromo-6-deoxyamylose with a high degree of substitution. Reduction of the bromoamylose with sodium borohydride yielded 6-deoxyamylose that, following controlled acetolysis, gave 6-deoxymaltooligosaccharides. Such oligosaccharides, of appropriate chain-length, formed stable complexes with long-chain fatty acids that have a dissociation constant of about 2 microM in their interaction with parinaric acid (trans-9,11,13,15-octadecatetraenoic acid). Treatment of 6-bromoamylose with sodium methoxide in dimethyl sulfoxide produced a 3,6-anhydroamylose that contains few unaltered D-glucose units. This anhydroamylose also bound to a palmitoyl-resin column, suggesting that the polymer may be partially helical and somewhat lipophylic. Finally, reaction of 6-bromoamylose with methylvinyl ether led to the 2,3-di(methoxyethyl) ether that, on reaction with sodium methoxide, gave the 6-methyl ether from which 6-O-methylamylose was obtained by mild acid hydrolysis.

Positional isomers of thioxylobiose, their synthesis and inducing ability for D-xylan-degrading enzymes in the yeast Cryptococcus albidus.

Isomeric S-linked 2-thioxylobiose 10, 3-thioxylobiose 17, and 4-thioxylobiose 19 were conveniently prepared by SN2 displacement of suitable triflylglycoses with the sodium salt of 2,3,4-tri-O-acetyl-1-thio-beta-D-glucopyranose, either in N,N-dimethylformamide, or in oxolan in the presence of a sodium complexing agent. Allyl 3,5-O-isopropylidene-2-O-trifluoromethanesulfonyl-beta-D-lyxofu ranoside was a convenient electrophilic precursor for 10, which was smoothly obtained after a short sequence of deprotection involving conversion to the 1-propenyl glycoside. 1,2:5,6-Di-O-isopropylidene-3-O-trifluoromethylsulfonyl-alpha-D-++ +allofuranose and 1,2,3-tri-O-benzoyl-4-O-trifluoromethylsulfonyl-beta-L-arabinop yranose were the respective precursors for 17 and 19. 4-Thioxylobiose has a highly stimulatory effect on the synthesis of enzymes of the xylanolytic system in the yeast Cryptococcus albidus when applied to the cells in the presence of the natural disaccharide inducer (1----4)-beta-D-xylobiose.

4-Thiocellooligosaccharides. Their synthesis and use as ligands for the separation of cellobiohydrolases of Trichoderma reesei by affinity chromatography.

4-Aminophenyl 1,4-dithio-beta-cellobioside (6) was obtained by treatment of methyl 2,3,6-tri-O-benzoyl-4-O-triflyl-alpha-D-galactopyranoside with the sodium salt of 1-thio-beta-D-glucopyranose, followed by acetolysis and glycosylation of the corresponding bromide with 4-aminobenzenethiol and subsequent deacylation. A similar synthesis starting with the 1-thiolate of 1,4-dithio-beta-cellobiose led to the trisaccharide 4-aminophenyl 1,4,4'-trithiocellotrioside (16). The 4-acetamidophenyl di- and tri-thiocellooligosaccharides were found to be excellent competitive inhibitors of the hydrolysis of 4-methylumbelliferyl beta-lactoside with respective Ki values of 25 and 6.5 mM. The two 4-aminophenyl oligosaccharides 6 and 16 were coupled to CH-Sepharose 4B, and the affinity gels were used for the purification of cellobiohydrolases from a crude commercial cellulolytic extract of T. reesei. Cellobiohydrolases I or II were selectively desorbed from gels bearing ligands 6 and 16.

Synthesis of the HSA-conjugate of the S-linked thiomimetic of the branched tetrasaccharide repeating unit of the immunostimulant polysaccharide, schizophyllan. Evaluation as potential immunomodulator.

The conjugate with human serum albumin (HSA) of the S-linked thioanalogue of the branched tetrasaccharide repeating unit of the polysaccharide, schizophyllan, was synthesized from 1,2,4,6-tetra-O-acetyl-3-S-[2,4-di-O-acetyl-3,6-di-S-(2,3,4,6-tetra-O-ac etyl-beta-D-glucopyranosyl)-3,6-dithio-beta-D-glucopyranosyl]-3-thio-bet a-D-glucopyranose [M.O. Contour-Galcera et al., Carbohydr. Res., 281 (1996) 119-128] in five steps, and its potential immunomodulatory activity was evaluated in human blood mononuclear cells. The protein glycoconjugate did not effect proliferation or production of IL-4, IL-5 and IFN-g in a significant way.

Stereocontrolled synthesis of sulfur-linked analogues of the branched tetrasaccharide repeating-unit of the immunostimulant polysaccharide schizophyllan and of its beta-(1-->3)-branched, beta-(1-->6)-linked isomer.

The branched, sulfur-linked tetrasaccharide S-(beta-D-glucopyranosyl)-(1-->3)-S-[(6-S-beta-D-glucopyranosyl)-3,6-dit hio- beta-D-glucopyranosyl]-(1-->3)-S-3-thio-D-glucopyranose (9) has been conveniently prepared by SN2 displacement of the triflate group in 1,2:5,6-di-O-isopropylidene-3-O-trifluoromethylsulfonyl-alpha-D-++ +allofuranose with the sodium salt of 2,4-di-O-acetyl-3,6-di-S-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)- 1,3,6- trithio-beta-D-glucopyranose (5). Conversely, reaction of the sodium salt of 5 with 1,2,3,4-tetra-O-acetyl-6-deoxy-6-iodo-beta-D-glucopyranose afforded the positional isomer S-(beta-D-glucopyranosyl)-(1-->6)-S-[(3-S-beta-D-glucopyranosyl)-3,6-dit hio- beta-D-glucopyranosyl]-(1-->6)-S-6-thio-D-glucopyranose (12).

Synthesis and evaluation of diastereoisomeric alkylating pseudo-disaccharides as potential affinity reagents for trehalase.

Reaction of (+/-)-(3/4,5,6)-4-bromo-5,6-epoxy-3-hydroxycyclohexene with 2,3,4,6-tetra-O-acetyl-1-thio-alpha-D-glucopyranose, followed by treatment of the resulting isolated diastereoisomeric 4-bromo-3,5-dihydroxycyclohexene 1-thioglycoside derivatives with base under phase-transfer conditions, gave (R)- and (S)-(3,4,6/5)-3,4-epoxy-6-S-(1-thio-alpha-D-glucopyranosyl)-5- hydroxycyclohexene. None of them was substrate or inhibitor for cockchafer trehalase.

Structural studies of the major glycolipid from Saccharopolyspora genus.

A major glycolipid was isolated from the well characterized Saccharopolyspora species, S. hirsuta, S. rectivirgula, S. erythraea and one not completely identified strain (Saccharopolyspora sp.). On the basis of sugar and methylation analysis, specific enzymatic and chemical degradations of the carbohydrate moiety, its FAB mass spectrometry and NMR spectroscopy characterizations, the carbohydrate part was shown to be the glycerol linked dimannoside alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->1/3)Gro. The internal mannose residue is esterified at C-6 by one fatty acid residue, whereas another fatty acyl chain substitutes the primary methylene position of glycerol. The main fatty acyl residues are anteiso-branched heptadecanoic acid and the iso-branched fatty acids iso-17:0, iso-16:0, and iso-18:0, with the former species being predominant. The major glycolipid has potential value for taxonomic and diagnostic purposes, especially in the specific diagnosis of farmer's lung disease.

Structure of the Hafnia alvei strain PCM 1188 O-specific polysaccharide.

The lipopolysaccharide was extracted from cells of Hafnia alvei PCM 1188 strain and, after mild acid hydrolysis, the O-specific polysaccharide isolated and characterized. On the basis of sugar and methylation analysis, FAB mass spectrometry and NMR spectroscopy of the polysaccharide and oligosaccharides obtained after Smith degradation, or solvolysis with anhydrous hydrogen fluoride, the repeating unit of the O-specific polysaccharide was shown to be the pentasaccharide: [formula: see text]