Quantitative analysis by various g.l.c. response-factor theories for partially methylated and partially ethylated alditol acetates

Results are presented which demonstrate that the molar flame-responses of partially methylated partially ethylated alditol acetates should be calculated on an effective carbon response (e.c.r.) basis. The relative responses of 2,3,4,6-tetra-O-ethyl-D-glucitol 1,5-diacetate, 2,3,6-tri-O-ethyl-D-glucitol 1,4,5-triacetate, hexa-O-ethyl-D-glucitol, hexa-O-methyl-D-glucitol, α-D-galactopyranose pentaacetate were measured and compared to the predicted values from three theories: equal molar response, equal weight response, effective carbon response. The observed values agree very well (±0?6%) with the e.c.r.-calculated values. The other theories of relative response can result in as much as 100% error in quantitation. The e.c.r-calculated relative response-factors for all commonly found partially methylated partially ethylated alditol acetates are presented, and their use is suggested for accurate quantitation.     

A method of purification of partially methylated alditol acetates in the methylation analysis of glycoproteins and glycopeptides

A method for methylation analysis of intact glycoproteins is described. Starting with intact glycoprotein, the oligosaccharides are methylated, hydrolyzed, reduced, and acetylated. The partially methylated alditol acetates are then separated from noncarbohydrate contaminants on a silica gel G column. Partially methylated hexitol acetates are eluted from the column with petroleum ether:ethyl acetate (1:1, $\text{v}\text{v}$) and partially methylated N-acetylhexosaminitol acetates are subsequently eluted with methanol. Analysis by gas-liquid chromatography/mass spectrometry of the partially methylated alditol acetates shows no interfering contaminants. This method circumvents the need to make pronase glycopeptides and avoids the pitfalls of other methylation procedures.     

Synthesis of a series of fully methylated aldobiouronic acids,and β-elimination reactions of their synthetic precursors

Treatment of methyl 2,3,4-tri-O-acetyl-l-bromo-l-deoxy-α-d-glucopyranuronate severally with 2,4,6-, 2,3,6-, and 2,3,4-tri-O-methyl derivatives of methyl α-d-glucopyranoside and with methyl 4,6-O-benzylidene-3-O-methyl-α-d-glucopyranoside, in the presence of silver carbonate, afforded crystalline aldobiouronic acid derivatives in high yield. Deacetylation followed by methylation gave a series of fully methylated derivatives of laminaribiouronic, cellobiouronic, and gentiobiouronic acids, and the (1 → 2)-linked analogue. Methylation with methyl iodide and silver oxide in N,N-dimethylformamide was invariably accompanied by a small amount ofβ-elimination, with the formation of olefinic disaccharides which were also obtained by β-elimination reactions of the precursor acetates followed by methylation. Methyl 4,5-unsaturated 4-deoxyhexopyranosyluronate derivatives were the main products of the reaction, but these underwent further degradation with cleavage of the interglycosidic linkage and formation of 6-methoxycarbonyl-4-pyrone.     

Methylated cycloamyloses (cyclodextrins) and their inclusion properties

2,3,6-Tri-O-methyl and 2,6-di-O-methyl derivatives of cyclohexa- and cyclo-hepta-amylose (2,3,6-tri-O-methyl- and 2,6-di-O-methyl-α- and -β-cyclodextrin) have been prepared and shown to be versatile complexing agents. Their complexes in aqueous solution are usually more stable than the corresponding complexes of un-substituted cycloamyloses. The methylated cycloamyloses also form crystalline complexes, the stability of which depends on the size and shape of the guest molecule. The decomposition temperature of the crystalline complexes with homologous n-alkanes, which is relatively high increases with increasing chain-length of the hydro-carbon. The shift of the i.r. carbonyl band of oleic acid in its solid complex with methylated α-cyclodextrin probably reflects inclusion of the fatty acid in the mono-meric form. The methylated cycloamyloses, when used as the stationary phases for g.l.c. or dissolved in a conventional stationary phase, affect the retention times of organic compounds in a manner which suggests that inclusion phenomena are operative.     

Capillary gas chromatography of methylhexitol acetates obtalned upon methylation of N-glycosidically linked glycoprotein oligosaccharides

The 30 O-methylated hexitol and 2-deoxy-2-(N-methyl)acetamidohexitol acetates which commonly may be obtained during methylation analysis of N-glycosidically linked glycoprotein oligosaccharides or their biosynthetic precursors, were subjected to chromatography through glass capillary columns, wall-coated with either Silar 9CP, Dexsil 410, SE-30, or OV 101. All 22 methylhexitol acetates (e.g., 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol and -mannitol) were optimally resolved on the (most polar) Silar column, whereas the 8 aminohexitol derivatives (as well as the corresponding unmethylated hexitol and aminohexitol acetates) were best separated on either Dexsil 410 or OV 101.     

The preparation of carbohydrate-protein conjugates: cyanuric trichloride coupling of 2-aminoethyl glycosides,and mixed-anhydride coupling of 8-carboxyoctyl glycosides to bovine serum albumin

Preparation of the following glycosides is described: 2-aminoethyl β-d-glycosides of (A) 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-d-glucopyranose, (B) 2-acetamido-4-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-3,6-di-O-acetyl-2-deoxy-β-d-glucopyranose (N,N′-diacetylchitobiose pentaacetate), (C) 4-O-(2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl)-2,3,6-tri-O-acetyl-β-d-glucopyranose (cellobiose heptaacetate); 8-carboxyoctyl glycosides of (D) cellobiose, and (E) N,N′-diacetylchitobiose. Conjugates were prepared from (A), (B), and (C) by coupling to bovine serum albumin by cyanuric trichloride and subsequent deacetylation; (D) and (E) were coupled to bovine serum albumin by the mixed-anhydride reaction. Conjugates (A) and (B) were insoluble; conjugates (C), (D), and (E) functioned as artificial antigens and gave rise to precipitating antibodies in rabbits. Specificities of the antisera were determined by inhibition studies.     

An assay for iduronate sulfatase (Hunter corrective factor)

Acetylation of benzyl α-D-mannopyranoside with acetic anhydride-sodium acetate at room temperature gave crystalline benzyl 2,3,6-tri-O-acetyl-α-D-manno-pyranoside (25%) and benzyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (≈65%). Similar esterification of benzyl β-D-glucopyranoside yielded the crystalline benzyl 2,4,6-triacetate (66%), whereas the corresponding galactopyranoside gave the crystalline 3,4,6-, 2,3,6-, and 2,4,6-triacetates (3, 25, and 9%. respectively). The structures of these compounds were established by methylation with diazomethane-boron trifluoride etherate and were confirmed by n.m.r. studies.     

Synthesis of glycosyl esters of oleanolic acid

The trisaccharide, O-(2,3,4-tri-O-benzoyl-β-L-rhamnopyranosyl)-(1→4)-O-(2,3,6-tri-O-benzoyl-β-D-glucopyranosyl)-(1→6)-1,2,3,4-tetra-O-acetyl-β-D-glucopyranose has been prepared by two different routes. Condensation of this trisaccharide with oleanolic acid afforded the corresponding 1,2-trans glycosyl ester. Some other glycosyl esters of oleanolic acid were also prepared by the same method.     

Synthesis of an intensely sweet chlorodeoxysucrose: Mechanism of 4′-chlorination of sucrose by sulphuryl chloride

Reaction of 6-O-acetylsucrose¹ with sulphuryl chloride in chloroform-pyridine affords, after dechlorosulphation and acetylation, a mixture of two isomeric 2,3,6-tri-O-acetyl-4-chloro-4-deoxy-α-d-galactopyranosyl 3-O-acetyl-1,4,6-trichloro-1,4,6-trideoxy-β-d-hexulofuranosides (6 and 7) and 2,3,6-tri-O-acetyl-4-chloro-4-deoxy-α-d-galactopyranosyl 3,4-di-O-acetyl-1,6-dichloro-1,6-dideoxy-β-d-fructofuranoside (4). Chlorination of C-4, C-1′, and C-6′ occurs by direct displacement of the initially formed chlorosulphonyloxy groups by chloride ions, but displacement of the 4′-chlorosulphate is sterically hindered. The introduction of a 4′-chloro substituent involves ring opening of intermediate 3′,4′-epoxides by chloride ions, the ribo-epoxide producing the sorbo-isomer 6 and the lyxo-epoxide giving the fructo-isomer 7. The proposed mechanism is supported by the formation of 4-chloro-4-deoxyfructofuranosides when 3′,4′-lyxo-hexulofuranosides are treated with sulphuryl chloride under the same conditions.     

Synthesis of methyl O-(3-deoxy-3-fluoro-β-d-galactopyranosyl)-(1→6)-β-d-galactopyranoside and methyl O-(3-deoxy-3-fluoro-β-d-galactopyranosyl)-(1→6)-O-β-d-galactopyranosyl-(1→6)-β-d-galactopyranoside

Condensation of 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-α-d-galactopyranosyl bromide (3) with methyl 2,3,4-tri-O-acetyl-β-d-galactopyranoside (4) gave a fully acetylated (1→6)-β-d-galactobiose fluorinated at the 3′-position which was deacetylated to give the title disaccharide. The corresponding trisaccharide was obtained by reaction of 4 with 2,3,4-tri-O-acetyl-6-O-chloroacetyl-α-d-galactopyranosyl bromide (5), dechloroacetylation of the formed methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β-d-galactopyranosyl)-(1→6)- 2,3,4-tri-O-acetyl-β-d-galactopyranoside to give methyl O-(2,3,4-tri-O-acetyl-β-d-galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β-d-galactopyranoside (14), condensation with 3, and deacetylation. Dechloroacetylation of methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β-d-galactopyranosyl)-(1→6)-O-(2,3,4-tri-O-acetyl- β-d-galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β-d-galactopyranoside, obtained by condensation of disaccharide 14 with bromide 5, was accompanied by extensive acetyl migration giving a mixture of products. These were deacetylated to give, crystalline for the first time, the methyl β-glycoside of (1→6)-β-d-galactotriose in high yield. The structures of the target compounds were confirmed by 500-MHz, 2D, ¹H- and conventional ¹³C- and ¹⁹F-n.m.r. spectroscopy.     

Selective benzylation of some D-galactopyranosides. Unusual relative reactivity of the hydroxyl group at C-4

Partial benzylation of methyl 2,3-di-O-benzyl-α-D-galactopyranoside gave methyl 2,3,6-tri-O-benzyl-α-D-galactopyranoside as the major product, whereas the isomeric 2,6-di-O-benzyl ether gave a mixture of products in which the ratio of methyl 2,4,6- to methyl 2,3,6-tri-O-benzyl-α-D-galactopyranoside was ≈4:1. The proportion of unreacted starting-material was low in both cases, whereas after a similar reaction of methyl 2,6-di-O-benzyl-β-D-galactopyranoside more than 50% of the dibenzyl ether was recovered unchanged. In this case also, considerably higher reactivity was exhibited by the hydroxyl group at C-4 than that at C-3. Acid hydrolysis of the methyl glycosides of the tribenzyl ethers afforded crystalline 2,4,6-tri-O-benzyl-α-D-galactose and syrupy 2,3,6-tri-O-benzyl-D-galactose. Structures of intermediates were established by acetylation, examination of their n.m.r. spectra, and conversion into the known 3-O and 4-O-methyl-D-galactose.     

Synthèses et réductions de sucres fonctionnels mono et diinsaturés

Attempts to prepare 1,2:5,6 and 2,3:5,6 di-unsaturated sugars starting from 3,4,6-tri-O-acetyl-1,5-anhydro-1,2-dideo xy-d-arabino-hex-1-enitol or from ethyl 4,6-di-O-acetyl-1,5-anhydro-2,3-dideoxy-α-d-erythro-hex-2-enopyranoside led to 1,5-anhydro-1,2,6-trideoxy-l-threo-hex-5-enitol and its 3,4-diacetate. Hydrogenation and hydrogenolysis of the unsaturated chloro and fluoro derivatives afforded 1,5-anhydro-1,2,6-trideoxy-d-arabino-hexitol and ethyl 4-O-acetyl-2,3,6-trideoxy-α-d-erythro-hexopyranoside.     

Changes in the hemicellulosic polysaccharides of rye-grass with increasing maturity

Reaction of the C-2 mercurated methyl hexopyranoside acetates 1–3 with an excess of iodine resulted in nearly quantitative replacement of mercury by iodine with retention and inversion of configuration at C-2. Similar replacement was observed with 2-acetoxymercuri-3,4,6-tri-O-acetyl-2-deoxy-α-d-glucopyranose (4). In the iodinolysis of 2-acetoxymercuri-1,3,4,6-tetra-O-acetyl-2-deoxy-α-d-glucopyranose (5) in methanol, however, replacement at C-2 was accompanied to a considerable extent by solvolysis of the 1-acetoxyl group, and a mixture of 1,2-trans isomers of methyl 3,4,6-tri-O-acetyl-2-deoxy-2-iodo-hexopyranosides having the d-gluco and d-manno configurations was obtained, together with 1,3,4,6-tetra-O-acetyl-2-deoxy-2-iodo-α-d-mannopyranose.     

Synthetic routes to higher-carbon sugars. 1-C-Formylation of a 2-deoxyaldose via the anion of its diethyl dithioacetal

Diphenylmethylation of carbohydrate hydroxyl groups may be effected by the thermal reaction with diazo(diphenyl)methane in the absence of catalysts. Migration of the labile ester groups of methyl 2,3,4-tri-O-acetyl-α-d-glucopyranoside and 3-O-benzoyl-1,2-O-isopropylidene-α-d-glucofuranose does not occur during diphenylmethylation by this procedure. The diphenylmethyl group may be readily removed by catalytic hydrogenolysis, and is sufficiently acid-stable to enable the selective hydrolysis of acetal groups. Its use as an O-4 protecting-group and as a non-participating O-2 protecting-group in α-glycoside synthesis has been demonstrated in syntheses of methyl 2,3,6-tri-O-methyl-α-d-glucopyranoside and kojibiose octa-acetate, respectively.     

Synthesis of 5-thio-D-galactose

A synthesis of 5-thio-D-galactose, in the form of its crystalline, anomeric methyl glycopyranosides, is described. Compounds prepared as intermediates included ethyl 2,3-di-O-(tert-butyldimethylsilyl)-5,6-O-carbonyl-β-D-galactofuranoside, the corresponding 5,6-dideoxy-5,6-epithio derivative, and ethyl 2,3,6-tri-O-acetyl-5-S-acetyl-5-thio-β-D-galactofuranoside. On methanolysis, the latter afforded methyl 5-thio-α-D-galactopyranoside which, in turn, was transformed into methyl 5-thio-β-D-galactopyranoside. Acetolysis proved to be less satisfactory for incorporation of the sulfur atom into a pyranose ring-form. Characteristics of the ¹³C-n.m.r. spectra of derivatives of 5-thio-D-galactose are described, including the fact that ¹J_C,H values for the anomeric pyranosides differ by only 1–3 Hz, as compared with ≈ 10 Hz for their oxygen analogs.     

Synthesis of 2,5,6- and 3,5,6-tri-O-methyl-D-galactose

Starting from methyl β-D-galactofuranoside, 3,5,6-tri-O-methyl-D-galactose (9) and 2,5,6-tri-O-methyl-D-galactose (16) were synthesized. The alditol acetates were prepared from 9 and 16, and their behavior in g.l.c. was compared. Mass spectra of the alditol acetates from 9 and 16 showed that these compounds gave fragmentations as expected. The alditol acetate from 16 was also prepared by an alternative route.     

A synthesis of psi-cytidine

2-O-Benzoyl-3,6-di-O-benzyl-4-O-(chloroacetyl)-, 4-O-acetyl-2-O-benzoyl-3,6-di-O-benzyl-, and 2-O-benzoyl-3,4,6-tri-O-benzyl-α-d-galactopyranosyl chloride were converted into the corresponding 2,2,2-trifluoroethanesulfonates, and these were treated with allyl 2-O-benzoyl-3,6-di-O-benzyl-α-d-galactopyranoside, to give allyl 2-O-benzoyl-4-O-[2-O-benzoyl-3,6-di-O-benzyl-4-O-(chloroacetyl)-β-d-galactopyranosyl]-3,6-di-O-benzyl- α-d-galactopyranoside (26; 41% yield), allyl 4-O-(4-O-acetyl-2-O-benzoyl-3,6-di-O-benzyl-β-d-galactopyranosyl)-2-O-benzoyl-3,6-di-O-benzyl- α-d-galactopyranoside (27; 62% yield), and allyl 2-O-benzoyl-4-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-3,6-di-O-benzyl-α-d-galactopyranoside (28; 65% yield). All disaccharides were free from their α anomers. Disaccharides 26 and 27 were found to be base-sensitive, and were de-esterified by KCN in aqueous ethanol, and debenzylated with H₂-Pd. Attempts to produce (1→4)-β-d-galactopyranosides from the coupling of a number of fully esterified d-galactopyranosyl sulfonates to allyl 2,3,6-tri-O-benzoyl-α-d-galactopyranoside were unsuccessful.     

Determination of the linkages in some methylated,sialic acid-containing,meningococcal polysaccharides by mass spectrometry

The application of gas-liquid chromatography-mass spectrometric (g.l.c.-m.s.) analysis to a number of sialic acid-containing polysaccharides of meningococcal origin has been studied. Methylation of these polysaccharides by the Hakomori conditions resulted in both O- and N-methylation. Methanolysis of the methylated polysaccharides from serogroup C [(2→9)-linked], colominic acid [(2→8)-linked], and serogroups Y and W-135 [both (1→4)-linked], yielded the respective 4,7,8,4,7,9-, and 7,8,9-tri-O-methyl derivatives of methyl N-acetyl-N-methyl-β-D-neuraminate methyl glycoside. As model compounds, methyl N-acetyl-4,7,8,9-tetra-O-methyl-α-D-neuraminate methyl glycoside and its N-methyl derivative were also synthesized. All of the methylated derivatives could be identified on the basis of their typical fragmentation-patterns, indicating that this method is applicable to the determination of the position of linkages to sialic acid residues in biopolymers.     

The synthesis and cytotoxic activity of some haloacetamidoalkyl β-D-xylopyranosides

2-Hydroxyethyl 2,3,4-tri-O-acetyl-β-D-xylopyranoside was prepared from 2,3,4-tri-O-acetyl-α-D-xylopyranosyl chloride by the action of 1,2-ethanediol and mercuric acetate. Subsequent mesylation and azide displacement gave 2-azidoethyl 2,3,4-tri-O-acetyl-β-D-xylopyranoside, which was hydrogenated over palladiumon-charcoal and the amine acylated with various haloacetyl halides, to afford 2-(haloacetamido)ethyl 2,3,4-tri-O-acetyl-β-D-xylopyranosides. Deprotection to obtain the free sugars was carried out with 5mM ethanolic sodium ethoxide. 2-(Chloroacetamido)ethyl 2,3,4-tri-O-acetyl-β-D-xylopyranoside was further modified by sequential azide displacement, hydrogenation, and subsequent acylation with various haloacetyl halides to afford 2-[(haloacetamido)acetylamino]ethyl 2,3,4-tri-O-acetyl-β-D-xylopyranosides, which were also deprotected to give the corresponding free sugars. The effects of these haloacetamido analogs on the growth of the melanoma cells in tissue culture was evaluated.     

Synthèse du 4-O-[(s)-1-carboxyéthyl]-d-glucose et de son isomère (R)

Alkylation of benzyl 2,3,6-tri-O-benzyl-β-D-glucopyranoside in N,Ndimethyl formamide with (R)-2-chloropropionic acid gave crystalline benzyl 2,3,6-tri-O-benzyl-4-O-[(S)-carboxyethyl]-β-D-glucopyranoside. After hydrogenolysis of the benzyl group 4-O-[(S)-D-carboxyethyl]-D-glucose was obtained which lactonized very easily. Treatment of benzyl 2,3,6-tri-O-benzyl-4-O-[(S)-1-carboxyethyl]-β-D-glucopyranoside with diazomethane gave cristalline benzyl 2,3,6-tri-O-benzyl-4-O-[(S)-1-(methoxycarbonyl)ethyl]-β-D-glucopyranoside, which was reduced with lithium aluminium hydride to crystalline benzyl 2,3,6-tri-O-benzyl-4-O-[(S)-1-(hydroxymethyl)ethyl]-β-D-glucopyranoside After hydrogenolysis of the benzyl groups 4-O-[(S)-1-(hydroxymethyl)ethyl]-D-glucose was obtained. A similar sequence of reactions was performed with (S)-2-chloropropionic acid.