Synthesis of tri- and tetrasaccharide fragments of the Shigella dysenteriae type 1 O-antigen deoxygenated and fluorinated at position 3 of the methyl alpha-D-galactopyranoside terminus

The blockwise synthesis of methyl alpha tri- and tetrasaccharide analogs of the biochemical repeating unit of the Shigella dysenteriae type 1 O-polysaccharide is described. Modifications include deoxygenation and deoxyfluorination at position 3 of the galactopyranoside residue. Methyl 4,6-O-benzylidene-3-deoxy-alpha-D-xylo-hexopyranoside (8) and methyl 4,6-O-benzylidene-3-deoxy-3-fluoro-alpha-D-galactopyranoside (9) were condensed with (2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl)-(1-->3) -2,4-di-O-benzoyl-alpha-L-rhamnopyranosyl chloride to give, after deprotection, the target trisaccharide methyl alpha-L-rhamnopyranosyl-(1-->3)-alpha-L- rhamnopyranosyl-(1-->2)-3-deoxy-alpha-D-xylo-hexopyranoside and the corresponding fluorinated oligosaccharide. For the tetrasaccharide synthesis, the glycosyl acceptors 8 and 9 were condensed with the temporarily protected (2,4-di-O-benzoyl-3-O-chloroacetyl-alpha-L- rhamnopyranosyl)-(1-->3)-2,4-di-O-benzoyl-alpha-L-rhamnopyranosyl chloride. Removal of the chloroacetyl group was followed by condensation of the resulting selectively deblocked trisaccharides with 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-glucopyranosyl chloride. Reduction and deprotection then gave the free methyl 2-acetamido-2-deoxy- alpha-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl- (1-->3)-alpha-L-rhamnopyranosyl-(1-->2)-3-deoxy-alpha-D-xylo-hexopyra noside and the fluorinated analog.     

Facile synthesis of 2-methyl-[4,6-di-O-acetyl-1,2-dideoxy-3-O-(2,3,4,6-tetra-O-acetyl-D-glycopyranosyl)-alpha-D-glucopyrano-[2',1':4,5]-2-oxazolines, key intermediates for the synthesis of oligosaccharides

A simple synthesis of disaccharide oxazolines has been developed. Condensation of methyl 2-acetamido-4,6-O-benzylidene-2-deoxy-alpha-D-glucopyranoside with 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide, followed by removal of the 4,6-O-benzylidene group from the resulting disaccharide derivative, gave crystalline methyl 2-acetamido-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-alpha-D-glucpyranoside which, on acetolysis with acetic anhydride-acetic acid-sulfuric acid, provided 2-methyl-[4,6-di-O-acetyl-1,2-dideoxy-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-alpha-D-glucopyrano]-[2',1':4,5]-2-oxazoline (7). Synthesis of the related alpha-D-mannopyranosyl compound was similarly accomplished. The glycosylating capability of 7 was employed for the synthesis of 6-(benzyloxycarbonylamino)hexyl-2-acetamido-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-beta-D-glucopyranoside (18). An alternative synthesis of compound 18 is also described.     

Chemical synthesis of 6'-alpha-maltosyl-maltotriose, a branched oligosaccharide representing the branch point of starch

Chemical synthesis of the branched pentasaccharide 6'-alpha-maltosyl-maltotriose (15) is reported, based on the use of one synthon as a glycosyl acceptor and another synthon as a glycosyl donor. The synthon used as glycosyl acceptor was phenyl 2,3,6-tri-O-benzyl-1-thio-beta-D-glucopyranoside (7) and was synthesized from D-glucose with phenyl 2,3-di-O-acetyl-4,6-O-benzylidene-1-thio-beta-D-glucopyranoside and phenyl 2,3-di-O-benzyl-4,6-O-benzylidene-1-thio-beta-D-glucopyranoside as key intermediates. The synthon used as glycosyl donor was O-(2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl)-(1-->4)-O-(2,3,6-tri-O -benzyl - alpha-D-glucopyranosyl)-(1-->6)-O-[(2,3,4,6-tetra-O-benzyl-alpha-D- glucopyranosyl)-(1-->4)]-2,3-di-O-benzyl-alpha,beta-D-glucopyranosyl trichloroacetimidate (12) and was synthesized from phenyl O-2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl)-(1-->4)-O-(2,3,6-tri-O- benzyl- alpha-D-glucopyranosyl)-(1-->6)-O-[(2,3,4,6-tetra-O-acetyl-alpha-D- glucopyranosyl)-(1-->4)]-2,3-di-O-acetyl-1-thio-beta-D-glucopyranoside with O-(2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl)-(1-->4)-O-(2,3,6-tri-O - benzyl-alpha-D-glucopyranosyl)-(1-->4)]-2,3-di-O-benzyl-D-glucopyranose as an intermediate. Condensation of compounds 7 and 12 followed by removal of the phenylthio group and debenzylation provided the branched pentasaccharide 15. Alternatively, the branched pentasaccharide was produced from amylopectin by consecutive alpha- and beta-amylase treatments and purified by chromatography. The identity of the products obtained by chemical synthesis and enzymatic hydrolysis is documented by 1H and 13C NMR spectra.     

Extracellular matrix (mesoglea) of Hydra vulgaris III. Formation and function during morphogenesis of hydra cell aggregates

The cell wall arabinogalactans of strains of Mycobacterium, Rhodococcus, and Nocardia were per-O-methylated, partially hydrolyzed with acid, and the resulting oligosaccharides were reduced and per-O-ethylated to yield per-O-alkylated oligoglycosyl alditol fragments. Analyses of these fragments by gas chromatography-mass spectrometry and of the intact solubilized polysaccharides by 1H and 13C NMR revealed the major structural features of the different arabinogalactans from representatives of the different genera. All of the mycobacterial products contained a homogalactan segment of alternating 5-linked alpha-galactofuranosyl (Galf) and 6-linked beta-Galf residues. The arabinan segment consisted of three major domains, linear 5-linked alpha-arabinofuranosyl (Araf) residues and branched (3-->5)-linked Araf units substituted with either 5-linked Araf or the disaccharide beta-Araf-(1-->2)-alpha-Araf at both branched positions. The recognition of these features in in vivo grown Mycobacterium leprae is an important development. The arabinan from strains of Nocardia contains a nonreducing-end motif composed of the linear trisaccharide, beta-Araf-(1-->2)-alpha-Araf-(1-->5)-Araf, attached to linear 5-linked alpha-Araf units. The galactan segment of the arabinogalactan of Nocardia sp. is composed of linear 5-linked beta-Galf units substituted in part at O-6 with terminal beta-glucosyl units. The two representative strains of Rhodococcus also differed in the composition of the galactan moiety; in addition to the 5-linked Galf, 2- and 3-linked beta-Galf units are present. The reducing end of the galactans, and therefore, apparently, of the entire arabinogalactans from all species from all genera, are apparently composed of the unit, rhamnosyl-(1-->3)-N-acetyl-glucosamine, which, in turn, is apparently attached to peptidoglycan via phosphodiester linkage.     

Characterization of a novel branched tetrasaccharide of 3-deoxy-D-manno-oct-2-ulopyranosonic acid. The structure of the carbohydrate backbone of the lipopolysaccharide from Acinetobacter baumannii strain nctc 10303 (atcc 17904)

For the first time, the tetrasaccharide Kdoalpha2-->5Kdoalpha2-->5(Kdoalpha2-->4)Kdo (Kdo is 3-deoxy-D-manno-oct-2-ulopyranosonic acid) has been identified in a bacterial lipopolysaccharide (LPS), i.e. in the core region of LPS from Acinetobacter baumannii NCTC 10303. The LPS was analyzed using compositional analysis, mass spectrometry, and NMR spectroscopy. The disaccharide D-GlcpNbeta1-->6D-GlcpN, phosphorylated at O-1 and O-4', was identified as the carbohydrate backbone of the lipid A. The Kdo tetrasaccharide is attached to O-6' of this disaccharide and is further substituted by short L-rhamnoglycans of varying length and by the disaccharide D-GlcpNAcalpha1-->4D-GlcpNA (GlcpNA, 2-amino-2-deoxy-glucopyranosuronic acid). The core region is not substituted by phosphate residues and represents a novel core type of bacterial LPS. The complete carbohydrate backbone of the LPS is shown in Structure I as follows: where Rha is rhamnose. Except were indicated, monosaccharides possess the D-configuration. Sugars marked with an asterisk are present in non-stoichiometric amounts.     

An economic synthesis of 1,2,3,4-tetra-O-acetyl-5-thio-D-xylopyranose and its transformation into 4-substituted-phenyl 1,5-dithio-D- xylopyranosides possessing antithrombotic activity

D-Xylose was converted via 1,2-O-isopropylidene-alpha-D-xylofuranose (4) into 3-O-benzoyl-5-S-benzoyl-1,2-O-isopropylidene-alpha-D-xylofuranose which, after methanolysis, acetylation and subsequent acetolysis afforded 1,2,3,4-tetra-O-acetyl-5-thio-alpha-D-xylopyranose (14) in an overall yield of 36%. Reaction of 4 with thionyl chloride gave a mixture of the diastereomeric cyclic sulfites, the structures of which were established by X-ray crystallography. Their oxidation with sodium periodate afforded the corresponding cyclic sulfate 23. Treatment of 23 with potassium thioacetate gave the potassium salt of 5-S-acetyl-1,2-O-isopropylidene-alpha-D-xylofuranose 3-O-sulfonic acid (26) which, after methanolysis, acetylation and subsequent acetolysis afforded 14 in an overall yield of 56%. Treatment of 4 with sulfuryl chloride gave a mixture containing 5-chloro-3-O-chlorosulfonyl-5-deoxy-1,2-O-isopropylidene-alpha-D- xylofuranose, 3,7,9,11-tetraoxa-4-thia-10-dimethyl-tricyclo[6,3,0, 0(2,6)]undecane S-dioxide and 23 in a 2:3:7 ratio. Tetraacetate 14 was converted into the alpha-1-bromide 18 as well as into the alpha-1-O-trichloroacetimidate 17. These three compounds were used as donors for the glycosylation with 4-cyanothiophenol, affording the 4-cyanophenyl 2,3,4-tri-O-acetyl-1,5-dithio-alpha- (29) and beta-D-xylopyranoside (30) in different ratios, depending on the reaction conditions. When donor 18 was used in the presence of potassium carbonate, besides 29 and 30 two aryl C-glycosylated-thioglycosides, i.e. 4-cyano-2-(2,3,4-tri-O-acetyl-5-thio-beta-D-xylopyranosyl)phenyl 2,3,4-tri-O-acetyl-1,5-dithio-alpha- and beta-D-xylopyranoside (32 and 33) as well as 4-cyano-2-(2,3,4-tri-O-acetyl-5-thio-beta-D-xylopyranosyl)phenyl disulfide 34 could be isolated as byproducts. Deacetylation of 30 with sodium methoxide in methanol afforded, besides 4-cyano-phenyl 1,5-dithio-beta-D-xylopyranoside (1), the corresponding 4-[(methoxy)(imino)methyl]phenyl glycoside 2. The 4-cyano group of 1 was converted into the 4-aminothiocarbonyl, the 4-(methyl-thio)(imino)methyl, the 4-amidino and the 4-(imino)(hydrazino)methyl group. All of these glycosides showed a significant antithrombotic activity on rats.     

Synthesis of allyl O-[sodium(alpha-D-glycero-D-talo-2- octulopyranosyl)onate]-(2-->6)-2-acetamido-2-deoxy-beta-D-glucopyranosi de, a core constituent of the lipopolysaccharide from Acinetobacter calcoaceticus NCTC 10305

Reaction of methyl 2,6-anhydro-2,3-dideoxy-D-manno-2-octenoate 1 with 3-chloroperoxybenzoic acid gave the 2,3-anhydro derivative 2, which was converted into the per-O-acetylated anomeric methyl glycosides of D-glycero-D-galacto-2-octulopyranosylonic acid in good yield. Subsequent inversion of the configuration at C-3 and deprotection afforded sodium (methyl beta-D-glycero-D-talo-2-octulopyranosid)onate. Alternatively, 2 was transformed into methyl (alpha-D-glycero-D-talo-2- octulopyranosyl bromide(onate derivatives. Reaction with methanol or allyl 2-acetamido-2-deoxy- 3,4-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-beta-D-g lycopyranoside, promoted by silver triflate, gave good yields of the corresponding orthoester derivatives. Me3Si triflate-catalyzed orthoester rearrangement and removal of the protecting groups afforded sodium O-(methyl alpha-D-glycero- D-talo-2-octulopyranosid)onate and the disacchanide, allyl O-[sodium(alpha-D-glycero-D-talo-2- octulopyranosyl)onate]-(2-->6)-2-acetamido-2-deoxy-beta-D-gl ucopyranoside in high yield.     

Gum heteropolysaccharide and free reducing mono- and oligosaccharides of Anadenanthera colubrina

The gum from Anadenanthera colubrina consists mainly of a complex high-arabinose heteropolysaccharide with a (1-->3)-linked beta-D-Galp main-chain and many different side-chains. These contain beta-D-Galp-[(1-->6)-beta-D-Galp]m-(1-->6)-, substituted in turn at O-3 by alpha-L-Araf-[(1-->3)-alpha-L-Araf-]0-2. Also present are (1) main-chain units substituted at O-4 and O-6 by alpha-L-Araf units, (2) side-chains of Rhap-(1-->4)-beta-D-GlcpA-(1-->6)-beta-Galp-groups, (3) alpha-L-Arap non-reducing end-units linked (1-->6) to D-Galp, and (4) beta-Araf and beta-Arap structures. For the first time, a plant gum exudate was found to contain in the natural state, reducing low M(r) carbohydrates. These were rhamnose (0.6%), arabinose (4.7%), mannose (0.1%), galactose (0.8%) and many oligosaccharides (0.6%; 11 with different RFs, with the majority containing arabinose). They were all mixtures with the exception of alpha-Rhap-(1-->4)-beta-D-GlcpA-(1-->6)-alpha beta-Gal and an incompletely identified hexasaccharide, probably having alpha-L-Araf-(1-->4)-beta-D-Galp- and -alpha-L-Araf-(1-->3)-beta-D-Galp- structures. The mono- and oligosaccharides do not appear to arise via in situ autohydrolysis of the gum.     

Microsurgical pterional approach to aneurysms of the basilar bifurcation

The ultrastructure and component polysaccharides of the cell wall of Pythium debaryanum IFO-5919 were investigated. From results obtained by means of acid, alkali, Schweitzer reagent and beta-1, 3-glucanase treatments and electron microscopy, it was concluded that 1) the acid-extracted fraction was a 1,3-linked branched glucan, 2) the alkali-extranded fraction was a mixture of 1,3-, 1,6-, and 1,3,6-linked highly branched two glucans, 3) the Schweitzer reagent-extracted fraction was a beta-1, 4-linked glucan, 4) the cell wall was constructed from two types of cullulosic microfibrils, as a frame and as a finer network, and amorphous beta-1, 3-glucan including beta-1, 6-linkage, 5) cellulosic microfibrils were covered by matrix material consisting of a mixture of amorphous beta-1, 3-linked and beta-1, 6-linked branching glucans.     

Synthesis of 4-cyanophenyl and 4-nitrophenyl 1,5-dithio-L- and -D-arabinopyranosides possessing antithrombotic activity

5-S-Benzoyl-2,3-O-isopropylidene-5-thio-L-arabinose, prepared from L-arabinose diethyl dithioacetal gave, on treatment with sodium methoxide in methanol, 4-O-benzoyl-2,3-O-isopropylidene-5-thio-L-arabinopyranose 12 which was converted into its 1-O-acetate 14. Hydrolysis of 12 in acetic acid-water afforded, after acetylation, 1,2,3-tri-O-acetyl-4-O-benzoyl-5-thio-L-arabinopyranose 17 which was transformed into 2,3-di-O-acetyl-4-O-benzoyl-5-thio-L-arabinopyranosyl bromide 20. Zemplén deacylation of 17 gave 5-thio-L-arabinopyranose which was converted via 1,2,3,4-tetra-O-acetyl-5-thio-beta-L-arabinopyranose 5 into 2,3,4-tri-O-acetyl-5-thio-beta-L-arabinopyranosyl bromide 6 and into O-(2,3,4-tri-O-acetyl-5-thio-L-arabinopyranosyl) trichloro-acetimidate 7. Glycosidation of 4-nitrophenol with 12 under the Mitsunobu conditions afforded 4-nitrophenyl 4-O-benzoyl-2,3-O-isopropylidene-5-thio-alpha- and beta-L-arabinopyranoside in a approximately 1:2 ratio. Condensation of the glycosyl donors 6, 7, 17, and 20 with 4-cyano- and 4-nitrobenzenethiol yielded, after deacylation, 4-cyano- and 4-nitrophenyl 1,5-dithio-alpha- and beta-L-arabinopyranosides 28 alpha, 28 beta, 29 alpha and 29 beta in different ratios and yields, depending on the reaction conditions applied. In a similar manner the corresponding D-isomers 30 alpha, 30 beta, 31 alpha and 31 beta were also prepared. All of these glycosides, except 28 alpha, showed a stronger oral antithrombotic effect in rats as compared to beciparcil, used as reference.     

Synthesis of a neoglycoprotein containing the Lewis X analogous trisaccharide beta-D-GalpNAc-(1-->4)[alpha-L-Fucp-(1-->3)]-beta-D-GlcpNAc

The trisaccharide allyl glycoside 36 and related disaccharide part structures have been prepared using the 2-trichloroacetamido-2-deoxy-alpha-D-galactopyranosyl trichloroacetimidate derivative 9 as glycosyl donor under promotion with TMSOTf or Sn(OTf)2, respectively, to produce the beta-(1-->4) linkage to suitably protected glucosamine derivatives in fair yields. Fucosylation was effected employing the ethyl 1-thio glycosyl donor 20 in the presence of IDCP. Deprotection of the intermediates afforded the disaccharide allyl glycosides beta-D-GalpNAc-(1-->4)- beta-D-GlcpNAc 13, beta-D-GalpNClAc-(1-->4)-beta-D-GlcpNAc 14, alpha-L-Fucp-(1-->3)-beta-D-GlcpNAc 24, alpha-L-Fucp-(1-->4)-beta-D- GlcpNAc 31 and the branched trisaccharide allyl glycoside beta-D-GalpNAc-(1-->4)[alpha-L-Fucp-(1-->3)]-beta-D-GlcpNAc 36. The trisaccharide which corresponds to a structural motif occurring in N-glycoprotein glycans from human urokinase, human recombinant protein C, phospholipase A2 as well as O-glycans, was converted into a neoglycoprotein following introduction of a cysteamine-derived spacer group and subsequent activation with thiophosgene.     

Nonreducing end structures of chondroitin sulfate chains on aggrecan isolated from Swarm rat chondrosarcoma cultures

Chondrocyte cultures derived from the Swarm rat chondrosarcoma were metabolically labeled with [35S]sulfate or [6-3H]GlcN. Radiolabeled aggrecan was purified from the cell layer and exhaustively digested with chondroitin ABC lyase. Digestion products were resolved into disaccharide and monosaccharide residues using Toyopearl HW40S chromatography. The separated saccharide pools were reduced with NaBH4 and applied onto a CarboPac PA1 column to resolve all of the internal disaccharide alditols (unsaturated) from the nonreducing end disaccharide (saturated) and monosaccharide alditols. Mercuric acetate treatment was used prior to carbohydrate analysis to identify unambiguously the saturated from the unsaturated disaccharides. The chondroitin sulfate (CS) chains from these aggrecan preparations contained: (a) an internal disaccharide composition of unsulfated (3-4 per chain), 4-sulfated (approximately 32 per chain), 6-sulfated (approximately 1 per 14 chains), and 4,6-sulfated disaccharides (approximately 1 per 6 chains) and (b) a nonreducing terminal composition of 4-sulfated GalNAc (approximately 4 out of every 7 chains), 4,6-disulfated GalNAc (approximately 2 out of every 7 chains), and GlcUA adjacent to a 4-sulfated GalNAc residue (approximately 1 out of every 7 chains). Thus, the vast majority of these CS chains terminated with a sulfated GalNAc residue. The presence of 4,6-disulfated GalNAc at nonreducing termini is 60-fold more abundant than 4,6-disulfated GalNAc in interior disaccharides. This observation is consistent with the suggestion that disulfation of terminal GalNAc residues is involved in chain termination.     

Synthesis of methyl alpha-glycosides of some higher oligosaccharide fragments of the O-antigen of Vibrio cholerae O1, serotype Inaba and Ogawa

The title oligosaccharides, the tri- through the hexasaccharide in the Inaba series and the penta- and the hexasaccharide in the Ogawa series, have been synthesized using 1-thioglycosides of precursors to 3-O-benzyl-perosamine (4-amino-4,6-dideoxy-D-mannose) as building blocks and N-iodosuccinimide/silver triflate as a promoter. The azido groups in the assembled oligosaccharides were reduced to amino groups, which were then acylated using 2,4-O-benzylidene-3-deoxy-L-glycero-tetronic acid as the derivatizing reagent. Catalytic hydrogenolysis, simultaneously of the benzyl and benzylidene groups, gave the desired products that were characterized by 1H and 13C NMR spectroscopy.     

Cells of origin, course, and termination patterns of the ventral, uncrossed component of the mature rat corticospinal tract

A concise practical, large scale-adaptable six-step sequence has been developed for the transformation of diacetone-glucose into 4,6-di-O-benzoyl-3-O-benzyl-alpha-D-arabino-hexopyranos-2-ulosy l bromide (7), a most useful indirect beta-D-mannosyl donor as its blocking group pattern allows the construction of biologically relevant beta-D-mannosides branched at O-3 and O-6. The broad utility of this new ulosyl bromide 7 resides in its high anomeric reactivity, and in the ease and uniformity with which beta-stereocontrol can be achieved over both, glycosidations and carbonyl reduction of the beta-ulosides formed: Koenigs-Knorr conditions exclusively provide beta-glycosiduloses, hydride reduction of their carbonyl functions proceeds with high stereoselectivities (> 20:1) in favor of the beta-D-mannosides. These preparatively auspicious properties are materialized in an efficient, straightforward synthesis of alpha-D-Manp-(1-->6)-[alpha-D-Manp-(1-->3)]-beta-D-Manp++ +-(1-->O)-Octyl, the 3,6-O-branched core-mannotrioside carrying an octyl spacer instead of the chitobiosyl unit.     

Medicinal foodstuffs. X. Structures of new triterpene glycosides, gymnemosides-c, -d, -e, and -f, from the leaves of Gymnema sylvestre R. Br.: influence of gymnema glycosides on glucose uptake in rat small intestinal fragments

Following the characterization of gymnemosides-a and -b, new triterpene glycosides, gymnemosides-c, -d, -e, and -f, were isolated from the leaves of Gymnema (G.) sylvestre R. BR. Their chemical structures were elucidated on the basis of chemical and physicochemical evidence as follows: 21-O-benzoyl-28-O-acetylgymnemagenin 3-O-beta-D-glucopyranosiduronic acid (gymnemoside-c), 23-O-[beta-D-xylopyranosyl (1-->6)-beta-D-glucopyranosyl (1-->6)-beta-D-glucopyranosyl] gymnestrogenin (gymnemoside-d), 23-O-[beta-D-xylopyranosyl (1-->6)-beta-D-glucopyranosyl (1-->6)-beta-D- glucopyranosyl]-28-O-[beta-D-glucopyranosyl (1-->6)-beta-D-glucopyranosyl] 23-hydroxylongispinogenin (gymnemoside-e), 23-O-[beta-D-xylopyranosyl (1-->6)-beta-D-glucopyranosyl (1-->6)-beta-D-glucopyranosyl]-28-O-[beta-O-glucopyranosyl (1-->6)-beta-D-glucopyranosyl] 3 beta,16 beta,23,28-tetrahydroxyolean-18-ene (gymnemoside-f). The inhibitory effects of gymnemosides-c, -d, -e, and -f and principal triterpene glycosides from G. sylvestre on glucose uptake in rat small intestinal fragments were examined, and gymnemic acids II, III, and IV, gymnemasaponin V, and gymnemoside-f were found to exhibit the inhibitory activity.     

Appearance of ovomucoid in coagulated egg yolk passing through from soluble fraction of coagulated egg white in boiled egg

We examined the in vitro bactericidal effects and efficacy on handwashing of water containing electrolytic products of sodium chloride (electrolytic water). The electrolytic water, whose pH and concentration of free residual chlorine were 6.7-6.9 and 20-22 ppm, respectively, showed equal reduction of both Staphylococcus aureus and Escherichia coli to dilution of commercially available sodium hypochlorite containing 60 ppm of free residual chlorine. This bactericidal effect was calculated to be due to hypochlorous acid, based on the pH and the amount of chlorine in solution. Handwashing with the electrolytic water reduced the numbers of S. aureus on hands by 1/10(2), while running water and 0.2% benzalkonium chloride with 80% ethanol gave a 1/10 and 1/10(5) reduction, respectively. We conclude that electrolytic water might be applicable for handwashing in place of running water.     

Chemical sulfonation and anticoagulant activity of acharan sulfate

Acharan sulfate is a glycosaminoglycan prepared from the giant African snail, Achatina fulica. This polysaccharide has a repeating disaccharide structure of -->4)-2-deoxy-2-acetamido-alpha-D-glucopyranose (1-->4)-2-sulfo-alpha-L-idopyranosyluronic acid (1-->). Its structure is related to heparin and heparan sulfate but is distinctly different from all known members of these classes of glycosaminoglycans. Because of its structural similarities to heparin, chemically modified acharan sulfate was studied to understand the chemical structure effected its anticoagulant activity. After de-N-acetylation, acharan sulfate was N-sulfonated using either chlorosulfonic acid-pyridine or sulfur trioxide-trimethylamine complex. The sulfate level in these products ranged from 22 to 24%(w/w), significantly less than that of heparin at 36%. The molecular weight of both N-sulfoacharan sulfates were comparable with that of heparin. In vitro anticoagulant activity assays showed that N-sulfoacharan sulfate derivatives were moderately active for the inhibition of thrombin and neither product showed any measurable anti-factor Xa activity. The differences in the activities of N-sulfoacharan sulfates produced by these two methods are probably ascribable to a small level of concomitant O-sulfonation obtained when using chlorosulfonic acid-pyridine.     

Purification and characterization of a feruloyl esterase from the fungus Penicillium expansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and rho-coumaric acid from methyl esters of the acids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-alpha-L-arabinofuranosyl]-(1-->3)-O-beta- D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX) and O-[5-O-((E)-rho-coumaroyl)-alpha-L-arabinofuranosyl]- (1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (PAXX). The esterase was purified 360-fold in successive steps involving ultrafiltration and column chromatography by gel filtration, anion exchange and hydrophobic interaction. These chromatographic methods separated the phenolic acid esterase from alpha-L-arabinofuranosidase, pectate and pectin lyase, polygalacturonase, xylanase and beta-D-xylosidase activities. The phenolic acid esterase had an apparent mass of 65 kDa under non-denaturing conditions and a mass of 57.5 kDa under denaturing conditions. Optimal pH and temperature were 5.6 and 37 degrees C, respectively and the metal ions Cu2+ and Fe3+ at concentrations of 5 mmol 1-1 inhibited feruloyl esterase activity by 95% and 44%, respectively, at the optimum pH and temperature. The apparent Km and Vmax of the purified feruloyl esterase for methyl ferulate at pH 5.6 and 37 degrees C were 2.6 mmol 1-1 and 27.1 mumol min-1 mg-1. The corresponding constants of rho-coumaroyl esterase for methyl coumarate were 2.9 mmol 1-1 and 18.6 mumol min-1 mg-1.     

Specificity of rabbit antisera against the rough lipopolysaccharide of Salmonella minnesota strain R7 (chemotype Rd1P-)

Rabbit polyclonal antibodies against the lipopolysaccharide (LPS) of the Rd1P- mutant strain R7 of Salmonella minnesota were serologically characterized using R7 LPS, dephosphorylated LPS, deacylated LPS, deacylated, dephosphorylated and reduced LPS, and synthetic partial structures. The latter comprised partial structures of the core region of Rd1P- LPS bound to the beta 1-->6-linked glucosamine dissacharide with two amide-linked 3-hydroxytetradecanoic acid residues or artificial glycoconjugates comprised of the synthetic oligosaccharides coupled to bovine serum albumin. Using a passive hemolysis and an enzyme immunoassay, absorption and inhibition experiments, the antibody specificites present could be determined. One group of antibodies required components of the core region and the phosphorylated glucosamine disaccharide of the lipid A moiety for binding. The majority of phosphate-independent antibodies was directed against the trisaccharide L-glycero-alpha-D-manno-heptopyranose(1-->3)- L-glycero-alpha-D-manno-heptopyranose(1-->5)3-deoxy-D-manno-oct ulo sonic acid. Antibodies against the 1-->3- and 1-->7-linked heptose disaccharides and against a single heptose were also detected, however, with low titers. No antibodies were found which required the presence of fatty acids.     

Mitogenic activity of particulate yeast beta-(1-->3)-D-glucan and its water-soluble derivatives

Particulate beta-D-glucan was isolated from baker's yeast using autolysis and delipidization of the cells, followed by alkaline and acid treatment. The residual water-insoluble glucan termed cerevan has a beta-(1-->3)-linked backbone with beta-(1-->6)-linked short side chains. In order to achieve water solubility of the glucan, various derivatives were prepared (carboxymethyl-,carboxyethyl-,hydroxyethyl-,sulfoethyl-), and the beta-glucan was oxidized to glucuronoglucan. Their solubility, degree of substitution (DS), and molecular weight distribution (Mw) were compared. The immunomodulatory activity of these preparations was investigated in mitogenic and co-mitogenic tests on rat thymocytes. Cerevan showed higher stimulation indices compared with the known immunomodulator zymosan. Of the water-soluble derivatives, sulfoethylglucan was found to be the most active. Of the carboxymethyl derivatives of various DS, the preparation with DS = 0.75 exhibited the highest activity. Water-soluble carboxymethyl preparations with DS > 1.0 and low-molecular-weight glucuronoglucan were inactive.