Syntheses of 19-[O-(carboxymethyl)oxime] haptens of epipregnanolone and pregnanolone

O-(Carboxymethyl)oximes 1 and 2 derived from two epimeric 5beta-pregnanolones (3beta-hydroxy-5beta-pregnan-20-one and 3alpha-hydroxy-5beta-pregnan-20-one) in position 19 were prepared. Two synthetic routes were employed, both using protection of the 20-keto group after reduction into the (20R)-alcohol in the form of acetate. In the first route, (20R)-19-hydroxy-5beta-pregnan-3beta,20-diyl diacetate (3) was transformed into the corresponding 19-[O-(carboxymethyl)oxime] methyl ester 6, then deacetylated by acid and partially silylated with tert-butyldimethylsilyl chloride. The desired 3-O-silylated derivative 8 was separated, oxidized to the 20-ketone and protecting groups were sequentially removed to give the first title hapten 1. The second route started from (20R)-19-hydroxy-3-oxopregn-4-en-20-yl acetate (11), which was hydrogenated in the presence of base to the 5beta-pregnan-3-one derivative 12, protected in position 19 with tert-butyldimethylsilyl group and reduced with borohydride. The prevailing 3alpha-alcohol 15 was separated, protected in position 3 with a methoxymethyl group, deprotected in position 19 and transformed into the 19-[O-(carboxymethyl)oxime] 19. After deacetylation, esterification with diazomethane and oxidation in position 20, the pregnanolone skeleton was regenerated. Final deprotection steps gave the second title hapten 2. Both haptens, i.e., (19E)-3beta- and -3alpha-hydroxy-20-oxo-5beta-pregnan-19-al 19-[O-(carboxymethyl)oxime], were designed for the development of immunoassays of the corresponding parent neuroactive steroids.     

The synthesis of racemic purpurosamine B

Starting from methyl 4,6-dichloro-4,6-dideoxy-α-D-galactopyranoside (1), D-chalcose (4,6-dideoxy-3-O-methyl-D-xcylo-hexopyranose) (5) was prepared by dechlorination with tributyltin hydride, selective benzoylation with benzoyl cyanide at O-2, methylation at O-3, and acid hydrolysis. D-Chalcose (5) was obtained as well by direct methylation of 1 with diazomethane at O-3, reduction with tin hydride, and hydrolysis. Chalcosyl bromide prepared from 5 was not very suitable for β-glycoside synthesis under Koenigs-Knorr conditions, and better results were obtained with 2- O-acetyl-4,6-dichloro-4,6-dideoxy-3-O-methyl-α-D-galactopyranosyl bromide, which gave β-glycosides with methanol, cyclohexanol, benzyl alcohol, 1,2:3,4-di-O-isopropylidene-α-D-galactopyranose, and methyl 2,3-di-O-benzyl-6-deoxy-α-D-glucopyranoside. After dechlorination with tributyltin hydride, the corresponding β-glycosides of D-chalcose were obtained in good yield.     

Immunochemical Studies on Bacterial Blood Group Substances

Blood group H-active polysaccharide has been prepared from “smooth” strain Escherichia coli 2B-V by Freeman's method. a-Fucosidase derived from Bacillus fulminans caused the liberation of fucose from this polysaccharide, together with concomitant loss of blood group H activity. The results of quantitative microanalysis, borohydride reduction, the Morgan-Elson reaction and enzymic hydrolysis with β-galactosidase using isolated oligosaccharides obtained by partial acid hydrolysis indicated that the O-specific side chain of the polysaccharide has a pentasaccharide unit which is β-d-Gal-(1→3)-d-GalNAc-(1→3)-d-GalNAc-Fuc with a D -glucose residue bound at some undetermined point on this structure. It was considered that terminal non-reducing fucose of the polysaccharide was liberated by partial acid hydrolysis.     

Immunochemical studies on bacterial blood group substances. VIII. The structure of oligosaccharides from blood group H-active polysaccharide of Escherichia coli 2B-V.

Blood group H-active polysaccharide has been prepared from "smooth" strain Escherichia coli 2B-V by Freeman's method, alpha-Fucosidase derived from Bacillus fluminans caused the liberation of fucose from this polysaccharide, together with concomitant loss of blood group H activity. The results of quantitative microanalysis, borohydride reduction, the Morgan-Elson reaction and enzymic hydrolysis with betagalactosidase using isolated oligosaccharides obtained by partial acid hydrolysis indicated that the O-specific side chain of the polysaccharide has a pentassaccharide unit which is beta-D-Gal-(1 leads to 3)-D-GalNAc-(1 leads to 3)-D-GalNAc-Fuc with a D-glucose residue bound at some undetermined point on this structure. It was considered that terminal non-reducing fucose of the polysaccharide was liberated by partial acid hydrolysis.     

Alkynyl polysaccharides: synthesis of propargyl potato starch followed by subsequent derivatizations

Potato starch propargyl ethers (PgS) with degrees of substitution (DS) from 0.1 to 2.2 have been prepared by etherification of starch with sodium hydroxide or Li dimsyl in Me(2)SO and propargyl bromide. DS values and substituent distribution were determined after hydrolysis and acetylation by GC-MS. The order of reactivity was 2>6>3, with O-3 substitution being preferably observed in the trisubstituted units. Repeated analysis of the starch derivatives revealed that propargyl residues were lost during storage, a phenomenon that was not fully understood until now. Selected PgS were further functionalized: (a) O- and C-methylated to O-(2-butynyl)-O-methyl starch (BMS), (b) in a Mannich type reaction with diethylamine and formaldehyde to yield O-(4-diethylamino)-2-butinyl starch (DEABiS), (c) in a 1,3-dipolar cycloaddition with benzyl azide ('click-chemistry') to a N-benzyltriazole derivatized starch (BTrS), and (d) with carbon dioxide to O-(3-carboxy)-2-butinyl starch (CBiS). While the yield of carboxylation was only poor, conversion was high or nearly quantitative for reactions a-c. Thus, it is demonstrated that starch propargyl ethers are valuable intermediates for the preparation of functional polysaccharides.     

Synthesis of analogs of a metabolite of sodium 2-pyridinethiolate N-oxide (pyrithione)

Antitumor activities of two (1 → 6)-branched (1 → 3)-β-d-glucans, isolated from the fruiting body of Auricularia auricula-judae (“kikurage”, an edible mushroom), and other branched polysaccharides containing a backbone chain of (1 → 3)-α-d-glucosidic or (1 → 3)-α-d-mannosidic linkages [and their corresponding (1 → 3)-d-glycans, derived by mild, Smith degradation] were compared. Among these polysaccharides, a water-soluble, branched (1 → 3)-β-d-glucan (glucan I) of A. auriculajudae exhibited potent, inhibitory activity against implanted Sarcoma 180 solid tumor in mice. The alkali-insoluble, branched (1 → 3)-β-d-glucan (glucan II), a major constituent of the fruiting body, showed essentially no inhibitory activity. When the latter glucan, having numerous branches attached, was modified by controlled, periodate oxidation, borohydride reduction, and mild, acid hydrolysis, the resulting, water-soluble, degraded glucan, having covalently linked polyhydroxy groups attached at O-6 of the (1 → 3)-linked d-glucosyl residues, exhibited potent antitumor activity. Further investigations using the glucan-polyalcohol indicated that the attachment of the polyhydroxy groups to the (1 → 3)-β-d-glucan backbone may enhance the antitumor potency of the glucan. On the other hand, partial introduction     

Structure of a highly pyruvylated galactan sulfate from the Pacific green alga Codium yezoense (Bryopsidales, Chlorophyta)

A polysaccharide fraction consisting of d-galactose, sulfate, and pyruvate in a molar proportion of 4:2:1 was isolated from the green seaweed Codium yezoense by water extraction followed by ion-exchange chromatography. To elucidate its structure, modified polysaccharides were prepared by desulfation, depyruvylation, and by total removal of non-carbohydrate substituents. Structures of the native polysaccharide and of the products of its chemical modifications were investigated by methylation analysis as well as by 1D and 2D (1)H and (13)C NMR spectroscopy. The polysaccharide devoid of sulfate and pyruvate was subjected to two subsequent Smith degradations to afford a rather low-molecular and essentially linear (1-->3)-beta-d-galactan. A highly ramified structure was suggested for the native polysaccharide, which contains linear backbone segments of 3-linked beta-d-galactopyranose residues connected by (1-->6) linkages, about 40% of 3-linked residues being additionally substituted at C-6, probably by short oligosaccharide residues also containing (1-->3) and (1-->6) linkages. Sulfate groups were found mainly at C-4 and in minor amounts at C-6. Pyruvate was found to form mainly five-membered cyclic ketals with O-3 and O-4 of the non-reducing terminal galactose residues. The minor part of pyruvate forms six-membered cyclic ketals with O-4 and O-6. The absolute configurations of ketals (R for six-membered ketals and S for five-membered ones) were established using NMR spectral data.     

Structural studies of a cell wall polysaccharide of Trichosporon asahii containing antigen II.

The structure of a cell-wall polysaccharide containing antigen II from Trichosporon asahii was investigated. A purified glucuronoxylomannan (GXM) antigen was found to contain O-acetyl groups that contribute to the serological reactivity. The structure of GXM was analyzed by partial acid hydrolysis, methylation analysis, controlled Smith degradation, NMR studies, and fluorophore-assisted carbohydrate electrophoresis. GXM has an alpha-(1-->3)-D-mannan backbone with a beta-D-glucopyranosyluronic acid residue bound to O-2 of a mannopyranosyl residue and the same number of beta-D-xylopyranosyl residues as mannose. Side chains of beta-D-xylopyranosyl-D-xylopyranose, forming a nonreducing terminus, and beta-D-xylopyranosyl residues were attached to O-2, O-4, and O-6 of the mannose residues.     

Synthesis and silica-based immobilization of monofunctionalized cyclomaltoheptaose derivatives for enantioselective HPLC

Heptakis(6-O-tert-butyldimethylsilyl-2,3-di-O-methyl)cyclomaltohep taose (6-TBDMS-2,3-Me-beta-CD) and heptakis(2,3,6-tri-O-methyl)cyclomaltoheptaose (per-Me-beta-CD) were monofunctionalized by introduction of a 5-cyanopentyl group attached to one of the O-2, O-3 or O-6 positions and subsequent reduction with lithium aluminum hydride to give the corresponding mono-O-(omega-aminohexyl) derivatives. Alternatively, after attachment of a 7-octenyl group and further epoxidation the corresponding mono-omega-epoxyoctyl derivatives of 6-TBDMS-2,3-Me-beta-CD were obtained. The mono-O-(omega-aminohexyl) derivatives were immobilized by reaction with glycidoxypropyl and 'aldehyde' silica, whereas aminopropyl silica was used for the immobilization of the monoepoxyoctyl derivatives. The immobilized cyclodextrin derivatives were partially evaluated as chiral stationary phases in high-performance liquid chromatography (HPLC) and micro-HPLC.     

Receptors for glucocorticoids in lung tissue

Gas chromatography-mass spectrometric identification of partially methylated aminosugars has been developed: (a) various kinds of O-methylated 2-deoxy-2-(N-methyl)-acetamidohexitols were prepared from partially O-(1-methoxy)-ethylated 2-deoxy-2-acetamidohexoses, and their gas chromatography-mass spectrometric patterns were determined; (b) permethylated glycolipids gave a satisfactory yield of 2-deoxy-2-N-methyl-amidohexoses by acetolysis with 0.5 n sulfuric acid in 95% acetic acid, followed by aqueous hydrolysis; (c) the resulting partially methylated aminosugars and neutral sugars were analyzed after borohydride reduction and acetylation according to the procedure of Lindberg and associates (Björndal, Lindberg and Svennson, 1967; Björndal, Hellerqvist, Lindberg and Svensson, 1970).This method was successfully applied to analysis of aminosugar linkages in blood group B-active ceramide pentasaccharide from rabbit erythrocytes and in Forssman antigen of equine spleen. The structure of blood group B-active glycolipid of rabbit erythrocyte was found to be Galα1 → 3Galβ1 → 4G1cNAcβ1 → 3Ga11 → 4Glc → Cer, and that of Forssman antigen to be GaNAcα1 → 3GalNAcβ1 → 3Galα1 → 4Ga11 → 4Glc → Cer.     

The reaction of ammonia with acylated disaccharides : XI. The wohl reaction with octa-O-acytylmelibiononitrile

Octa-O-acetylmelibiononitrile (1) was prepared from melibiose oxime. The reaction of aqueous ammonia with 1 gave 1,1-bis(acetamido)-1-deoxy-5-O-α-D-galactopyranosyl-D-arabinitol (2), N-acetyl-5-O-α-D-galactopyranosyl-α-D-arabinofuranosylamine (3), and the anomeric pair of 5-O-α-D-galactopyranosyl-D-arabinofuranoses (4 and 5). The hepta-O-acetyl derivative of 2 was prepared, and the acyclic structure of the nitrogen-containing moiety was established by oxidation with periodate. The α anomeric configuration of 3 was demonstrated by periodate oxidation and subsequent reduction with sodium borohydride and hydrolysis.     

A short efficient synthesis of 16-oxygenated estratriene 3-sulfates

A novel synthesis of sodium 17-oxo-16 alpha-hydroxy-1,3,5(10)-estratrien-3-yl sulfate (4), sodium 16 alpha, 16 beta-dihydroxy-1,3,5(10)-estratrien-3-yl sulfate (5) and sodium 16-oxo-17 beta-hydroxy-1,3,5(10)-estratrien-3-yl sulfate (6) is described. 16 alpha-Bromo-3-hydroxy-1,3,5(10)-estratrien-17-one (1) was efficiently synthesized in one step with 70-97% yield by bromination of 3-hydroxy-1,3,5(10)-estratrien-17-one with cupric bromide. 3,16 alpha-Dihydroxy-1,3,5(10)-estratrien-17-one (3) was quantitatively obtained by controlled stereospecific hydrolysis of the bromoketone 1 with sodium hydroxide in aqueous pyridine. The bromoketone 1 was converted to the 16 alpha-hydroxy-17-ketone 3-sulfate 4 by sulfation with chlorosulfonic acid in pyridine and a subsequent controlled hydrolysis in a high yield without formation of the other ketols. Treatment of the sulfate 4 with sodium borohydride have the triol sulfate 5. The sulfate 4 was also rearranged to the 17 beta-hydroxy-16-ketone 6 with sodium hydroxide in water in a quantitative yield.     

Synthesis of DL- and D-gluco-hept-3-ulose.

DL-gluco-Hept-3-ulose was synthesised by oxidation of tri-O-isopropylidene-meso-glycero-gulo-heptitol with methyl sulphoxide-phosphorus pentaoxide, and subsequent hydrolysis. D-gluco-Hept-3-ulose (3) was synthesised by oxidation of one of the two isopropylidene derivatives from perseitol (D-glycero-D-galacto-heptitol), which is presumed to have the 2,2:4,5:6,7 structure, followed by hydrolysis. The crude product from the reduction of DL-gluco-hept-3-ulose with sodium borohydride showed two peaks corresponding to meso-glycero-gulo-heptitol and perseitol on g.l.c. of the trimethylsilyl derivatives. Isolation and acetylation of the latter heptitol revealed it to be racemic perseitol. Oxodation of DL-gluco-hept-3-ulose with oxygen in alkali followed by treatment with ferric acetate-hydrogen peroxide gave products with chromatographic behaviour characteristic of arabinonolactone and erythrose. Treatment of DL-gluco-hept-3-ulose with 2,4-dinitrophenylhydrazine gave a 1-deoxy-2,4-dinitrophenylosazone.     

Conversion of allyl 2-acetamido-2-deoxy-β-d-glucopyranoside into 2-methyl-(3,4,6-tri-O-benzoyl-1,2-dideoxy-α-d- galactopyrano)-[2′,1′:4,5]-2-oxazoline

2-Methyl-(3,4,6-tri-O-benzoyl-1,2-dideoxy-α-d-galactopyrano)-[2′,1′:4,5]-2-oxazoline (7) was prepared from 1-propenyl 2-acetamido-3,4,6-tri-O-benzoyl-2- deoxy-β-d-galactopyranoside (6). The latter was prepared from allyl 2-acetamido-2-deoxy-β-d-glucopyranoside (1) through selective benzoylation at O-3 and O-6, conversion into the 4-p-bromobenzenesulfonate 4, inversion of configuration at C-4 to afford allyl 2-acetamido-3,4,6-tri-O-benzoyl-β-d-galactopyranoside (5), and subsequent isomerization with palladium-charcoal to give 6.     

Structure of a highly substituted beta-xylan of the gum exudate of the palm Livistona chinensis (Chinese fan)

Structural features of the acidic, highly substituted glycanoxylan (LCP; 87% yield) from the gum exudate of the palm, Livistona chinensis, family Arecaceae, were determined. It had [alpha]D -30 degrees, Mw 1.9x10(5) and a polydispersity ratio Mw/Mn of approximately 1.0. Acid hydrolysis gave rise to Rha, Fuc, Ara, Xyl, and Gal, in a 1:6:46:44:3 molar ratio, and 12% of uronic acid was present. LCP had a highly branched structure with side-chains containing nonreducing end-units (% values are approximate) of Araf (15%), Fucp (4%), Xylp (7%), GlcpA, and 4-Me-GlcpA, and internal 2-O- (5%) and 3-O-substituted Araf (8%), and 2-O-substituted Xylp (14%) units. The (1-->4)-linked beta-Xylp main-chain units of LCP were substituted at O-3 (4%), O-2 (17%), and O-2,3 (16%). Partial acid hydrolysis gave 4-Me-alpha-GlcpA-(1-->2)-[beta-Xylp-(1-->4)](0-2)-Xyl, identified by showing that the uronic acids were single-unit side-chain substituents on O-2. Milder hydrolysis conditions removed from O-3 other side-chains containing Fucp and Araf nonreducing end-units and internal Arap, and 2-O- and 3-O-substituted Araf units. Carboxyl-reduced LCP contained 4-O-methylglucose and glucose in a 3.2:1 molar ratio, arising from GlcpA and 4-OMe-GlcpA nonreducing end-units, respectively. The gum contained small amounts of free alpha-Fucp-(1-->2)-Ara, which corresponds to structures in the polysaccharide. Free myo- and D- or L-chiro-inositol were present in a 9:1 ratio.     

Synthesis of polydeoxygenated and iodinated disaccharides.]

3-Amino-polydeoxy disaccharides have been prepared by condensation of a glycal with methyl 2,3,6-trideoxy-alpha-L-erythro-(or threo)-hex-2-enopyranoside in the presence of N-iodosuccinimide. After acid hydrolysis of the glycoside, 1,4-addition of hydrazoic acid to the corresponding hex-2-enopyranose led to 3-azido-disaccharides which were acetylated. Reduction of the azido group gave 2,2'-dideoxy- or 2,2'-dideoxy-2'-iodo compounds. Condensation of O-(3,4-di-O-acetyl-2,6-dideoxy-2-iodo-alpha-L-manno-hexopy-rano syl)-(1----4)-1- O-acetyl-2,3,6-trideoxy-3-trifluoroacetamido-alpha-L-arabino-he xopyranose with daunomycinone, followed by 3',4'-O-deacetylation produced the new anthracycline, 7-O-[O-(2,6-dideoxy-2-iodo-alpha-L-manno-hexopyranosyl)-(1----4)-2,3,6- trideoxy-3-trifluoroacetamido-alpha-L-arabino-hexopyranosyl]-da uno-mycinone.     

Structural studies on O- and N-glycosidically linked carbohydrate chains on Collocalia mucin

Oligosaccharides, which are O- and N-glycosidically linked on salivary glycoproteins from the edible bird's nest of chinese swallows, were released by alkaline borohydride treatment of the asialoglycoproteins and fractionated by gel chromatography. Fract. VN1 (oligosaccharides greater than 2 000 dalton) apparently represented a mixture of saccharides derived from complex, N-glycosidically linked glycans (molar ratio Man/GlcNAc/Gal 3:4:8), while fractions VN2 (tetra- to hexasaccharides), VN3 (trisaccharide) and VN4 (disaccharide) were free of mannose, but did contain all the N-acetylgalactosamine released from the protein as its alditol. Oligosaccharides in Fract. VN2 and VN4 were purified by high-performance liquid chromatography, paper chromatography and thin-layer chromatography, methylated and analysed after total or partial acid hydrolysis by gas-liquid chromatography-mass spectrometry. The structures of a hexasaccharide in Fract. VN2/6 and of a tetrasaccharide in fraction VN2/4 were finally established after methylation through direct-probe mass spectrometry: Gal(1----4)GlcNAc(1----3)Gal(1----4)GlcNAc(1----3)Gal(1----3)GalNAc- ol and Gal(1----4)GlcNAc(1----6)[Gal(1----3)]GalNAc-ol. Mass spectrometrical and gas-chromatographical data obtained for a disaccharide in Fract. VN4 were identical with those for Gal(beta 1----3)GalNAc-ol.     

Synthesis of p-nitrophenyl 6(5)-O-benzyl-alpha-maltopentaoside, a substrate for alpha amylases

p-Nitrophenyl alpha-maltopentaoside, having a benzyl group on O-6 of the terminal (nonreducing) D-glucosyl group was prepared by use of a reductive ring-opening reaction. Highly regioselective reduction of p-nitrophenyl O-(2,3-di-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl)-(1----4)- tris[O-(2,3,6-tri-O-benzoyl-alpha-D-glucopyranosyl)-(1----4)]-2,3,6-tri- O- benzoyl-alpha-D-glucopyranoside by dimethylamine-borane and p-toluenesulfonic acid, followed by debenzoylation, gave p-nitrophenyl O-(6-O-benzyl-alpha-D-glucopyranosyl)-(1----4)-tris[O-alpha-D-glucopyran osyl- (1----4)]-alpha-D-glucopyranoside. An experiment was done on the mode of action of human pancreatic and salivary alpha amylases on this derivative. The compound is suitable as a substrate for the assay of alpha amylase when used with glucoamylase and alpha-D-glucosidase as coupling enzymes.     

Potential bile acid metabolites. 14. Hyocholic and muricholic acid stereoisomers

The complete set of the eight theoretically possible stereoisomeric 3,6,7-trihydroxy-5 beta-cholanic acids, four of which are new, related to hyocholic and muricholic acids were prepared from chenodeoxycholic acid. The principal reactions used were 1) cis-dihydroxylation of delta 6-compounds with osmium tetroxide/N-methylmorpholine N-oxide; 2) trans-dihydroxylation of 6 alpha, 7 alpha-epoxy compounds with boron trifluoride etherate in N,N-dimethyl-formamide; 3) inversion of equatorial 3 alpha-hydroxylated compounds to the corresponding 3 beta-epimers with diethyl azodicarboxylate/triphenylphosphine/formic acid; and 4) stereoselective reduction of 7-keto derivatives with zinc borohydride (or sodium borohydride) and by metallic potassium/tert-amyl alcohol.     

Synthesis of 3-amino-2,3,6-trideoxy-D-ribo-hexose hydrochloride.

The title sugar, the 5-epimer of daunosamine, was prepared in a sequence of high-yielding steps from methyl alpha-D-mannopyranoside (1). Conversion of 1 into methyl 3-acetamido-4-O-benzoyl-6-bromo-2,3,6-trideoxy-alpha-D-ribo-hexopyranoside (2), followed by reduction with hydrogen and Raney nickel, gave the 4-benzoate (3) of methyl 3-acetamido-2,3,6-trideoxy-alpha-D-ribo-hexopyranoside (4). Saponification of 3 gave 4 as an oil that gave a crystalline 4-acetate (8). N-Deacetylation of 4 was effected with barium hydroxide, and the resultant glycoside was hydrolyzed to give 3-amino-2,3,6-trideoxy-D-ribo-hexose hydrochloride (7). The 3-benzamido analogue (5) of 4 was prepared from 4 by N-deacetylation and subsequent benzoylation, and hydrolysis of 5 gave crystalline 3-benzamido-2,3,6-trideoxy-D-ribo-hexose (6). The crystalline 3-acetamido analogue (9) of 6 was obtained by acid hydrolysis of the glycoside 4.