Biological method for protection of 6-position of sucrose and its use in synthesis of disaccharide high-intensity sweetener

A general method for protecting the 6 primary hydroxyl position of sucrose is described. It involves the production of glucose-6-acetate by fermentation of glucose using a strain of Bacillus megaterium followed by conversion to sucrose-6-acetate as a kinetic product using a specially selected fructosyl transferase producted by a newly isolated strain of Bacillus subtilis. The sucrose-6-acetate was found to be more lipophilic than expected, and this property aided its purification by chromatography. Pure sucrose-6-acetate may then be chlorinated and subsequently deacetylated to give the high-intensity sweetener 4,1',6'-trichlo-4,1',6'-trideoxygalactosucrose (sucralose) in high yields. This process involves fewer steps than are required for chemical synthesis using trityl chloride and acetic anhydride. Related intensely sweet molecules which were synthesized by similar methods included 4,1',6'-trichloro, 4,1',6'-trideoxy L-arabinosucrose, and 4,1',6'-trichloro-4,6,1',6'-tetradeoxy-galactosucrose. They were obtained from xylose and 6-deoxyglucose, respectively, via the intermediates xylsucrose and 6-deoxysucrose, formed by the reaction of the fructosyl transferase on the monosaccharide acceptors.     

Selective deprotection of 2',6'-di-O-benzyl-2,3:5,6:3',4'-tri-O-isopropylidenelactose dimethyl acetal

The reactivity order of O-deisopropylidenation of the three isopropylidene protecting groups of 2',6'-di-O-benzyl-2,3:5,6:3',4'-tri-O-isopropylidenelactose dimethyl acetal (2) with various reagents was established. The 5,6-acetal group was, although to a limited extent, more reactive as compared with the 3',4' group, while the 2,3-O-isopropylidene group was definitely less reactive. Conditions were determined for the direct preparation of the 5,6,3',4'-tetraol 5 (60% aqueous acetic acid, room temperature, 48 h, 73% yield) and the 5,6-diol 4 (propylene glycol and p-toluenesulphonic acid in dichloromethane, 46% yield). The diacetonated derivative 3, formally arising from a selective 3',4'-O-deisopropylidenation, was obtained in high yield (90%) through a selective acetonation with 2-methoxypropene of the tetraol 5.     

Synthesis of methyl 2-O-alpha-D-mannopyranosyl-alpha-D-talopyranoside and methyl 2-O-alpha-D-talopyranosyl-alpha-D-talopyranoside

Treatment of methyl 3-O-benzyl-2-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)-alpha-D- mannopyranoside (1) with tert-butyldiphenylsilyl chloride in N,N-dimethylformamide afforded methyl 3-O-benzyl-6-O-tert-butyldiphenylsilyl-2-O-(2,3,4,6-tetra-O-acetyl -alpha-D- mannopyranosyl)-alpha-D-mannopyranoside (2). Oxidation of 2 with pyridinium chlorochromate, followed by reduction of the carbonyl group, and subsequent O-deacetylation afforded methyl 3-O-benzyl-6-O-tert-butyldiphenylsilyl-2-O-alpha-D-mannopyranosyl- alpha-D- talopyranoside (5). Cleavage of the tert-butyldiphenylsilyl group of 5 with tetrabutylammonium fluoride in oxolane, followed by hydrogenolysis, gave methyl 2-O-alpha-D-mannopyranosyl-alpha-D-talopyranoside (7). O-Deacetylation of 1 gave methyl 3-O-benzyl-2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (8). Treatment of 8 with tert-butyldiphenylsilyl chloride afforded a 6,6'-disilyl derivative, which was converted into a 2',3'-O-isopropylidene derivative, and then further oxidized with pyridinium chlorochromate. The resulting diketone was reduced and removal of the protecting groups gave methyl 2-O-alpha-D-talopyranosyl-alpha-D-talopyranoside (15). The structures of both 7 and 15 were established by 13C-n.m.r. spectroscopy.     

Synthesis of some D-mannosyl-oligosaccharides containing the methyl and 4-nitrophenyl beta-D-mannopyranoside units

Methyl 3,4,6-tri-O-benzyl-beta-D-mannopyranoside (2), methyl 2,3-O-isopropylidene-beta-D-mannopyranoside (11), and 4-nitrophenyl 2,3-O-isopropylidene-beta-D-mannopyranoside (12) were each condensed with 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl bromide (1) in the presence of mercuric cyanide, to give after deprotection, methyl 2-(5) and 6-O-alpha-D-mannopyranosyl-beta-D-mannopyranoside (15), and 4-nitrophenyl 6-O-alpha-D-mannopyranosyl-beta-D-mannopyranoside (20), respectively. A similar condensation of 11 with 3,4,6-tri-O-acetyl-2-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)-a lpha-D- mannopyranosyl bromide (21) and 2,3,4-tri-O-acetyl-6-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)-a lpha D-mannopyranosyl bromide (25), followed by removal of protecting groups, afforded methyl O-alpha-D-mannopyranosyl-(1----2)-O-alpha-D-mannopyranosyl-(1----6)-beta -D- mannopyranoside (24) and methyl O-alpha-D-mannopyranosyl-(1----6)-O-alpha-D-mannopyranosyl-(1----6)-beta -D- mannopyranoside (28), respectively. Bromide 25 was also condensed with 12 to give a trisaccharide derivative which was deprotected to furnish 4-nitrophenyl O-alpha-D-mannopyranosyl-(1----6)-alpha-D-mannopyranosyl-(1----6)-beta-D - mannopyranoside (31). Phosphorylation of methyl 3,4,6-tri-O-benzyl-2-O-alpha-D-mannopyranosyl-beta-D-mannopyranoside and 15 with diphenyl phosphorochloridate in pyridine gave the 6'-phosphates 6 and 16, respectively. Hydrogenolysis of the benzyl and phenyl groups provided methyl 2-O-(disodium alpha-D-mannopyranosyl 6-phosphate)-beta-D-mannopyranoside (7) and methyl 6-O-(disodium alpha-D-mannopyranosyl 6-phosphate)-beta-D-mannopyranoside (17) after treatment with Amberlite IR-120 (Na+) cation-exchange resin. The structures of compounds 5, 7, 15, 17, 20, 24, 28, and 31 were established by 13C-n.m.r. spectroscopy.     

Synthesis and characterization of 6-O-beta-lactosyl-alpha,beta-lactoses, 1-O-(6-O-beta-lactosyl-beta-lactosyl)-(R,S)-glycerols, and 4,6-di-O-beta-D-galactopyranosyl-alpha,beta-D-glucoses.

1,2,3,2',3',4',6'-Hepta-O-acetyl-beta-lactose (4) was coupled with 2,3,6,2',3',4',6'-hepta-O-acetyl-alpha-lactosyl bromide (7) in the presence of Hg(CN)2 to afford 1,2,3,2',3',4',6'-hepta-O-acetyl-6-O-(2,3,6,2',3',4',6'-hepta-O-acetyl-b eta- lactosyl)-beta-lactose (11) which, upon O-deacetylation, gave 6-O-beta-lactosyl-alpha,beta-lactoses (64% from 4). In contrast, the reaction of 7 with benzyl 2,3,2',3',4',6'-hexa-O-acetyl-beta-lactoside in the presence of Hg(CN)2 produced 3,6,2',3',4',6'-hexa-O-acetyl-1,2-O- (2,3,2',3',4',6'-hexa-O-acetyl-1-O-benzyl-beta-lactos-6-yl orthoacetyl)-alpha-lactose (63%) and 3,6,2',3',4',6'-hexa-O-acetyl-1,2-O-(1- cyanoethylidene)-alpha-lactose (27%). The glycosidation of 4 using 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide in the presence of Hg(CN)2 afforded, after deprotection, 4,6-di-O-beta-D-galactopyranosyl-alpha,beta-D-glucoses (66%). The reaction of 11 with 1,2-di-O-benzyl-(R,S)-glycerols and trimethylsilyl trifluoromethanesulfonate yielded, after deprotection, 1-O-(6-O-beta-lactosyl-beta-lactosyl)-(R,S)-glycerols (18%). Under the same coupling conditions 11 reacted with 2-O-benzylglycerol to form 3-O-acetyl-2-O-benzyl-1-O-[2',3',4',6'-hexa-O-acetyl-6-O-(2,3,6,2',3',4' ,6'- hepta-O-acetyl-beta-lactosyl)-beta-lactosyl]-(R,S)-glycerols (16%).     

Synthesis of the alpha-L-Araf-(1-->2)-beta-D-Galp-(1-->6)-beta-D-Galp-(1-->6)-[alpha-L-Araf-(1-->2)]-beta-D-Galp-(1-->6)-D-Gal hexasaccharide as a possible repeating unit of the cell-cultured exudates of Echinacea purpurea arabinogalactan.

For the characterization of the supposed epitope of an arabinogalactan, isolated from the extract of the cell-cultured Echinacea purpurea, the title hexasaccharide was synthesized. The whole synthetic route was based on the 6-O-(methoxydimethyl)methyl ether (MIP) protecting group strategy. 2-O-Benzyl-3,4-O-isopropylidene-6-O-(methoxydimethyl)methyl-beta-D-galactopyranosyl-(1-->6)-1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranose was used to prepare the desired glycosyl donor and glycosyl acceptor both carrying a persistent O-benzyl group at position 2'. Reaction of the digalactose donor and the digalactose acceptor resulted in a beta-(1-->6)-linked galactose-containing tetrasaccharide in which OH-2' and OH-2"' were substituted with benzyl groups. Hydrogenolytic removal of the benzyl groups of the tetragalactose compound gave the diol aglycon which was diarabinosylated in one step to furnish the protected target compound, whose deprotection led to the title hexasaccharide. All of the synthesized compounds were characterized by 1H and 13C NMR spectra, as well as by MALDI-TOF mass-spectrometry measurements.     

Synthesis of 5-O-β-D-galactofuranosyl-D-galactofuranose

Conversion of benzyl αβ-D-galactofuranoside into the 5,6-O-[α-(dimethyl-amino)benzylidene] derivative, followed by acetylation of HO-2 and HO-3, and selective ring opening or the acetal, gave benzyl 2,3-di-O-acetyl-6-O-benzoyl-αβ-D-galactofuranoside(4). The title disaccharide was synthesised from4 by reaction with 3,4,6-tri-O-acetyl-α-D-galactofuranose 1,2-(methyl orthoacetate) followed by removal of protecting groups     

Tricyclic furanoid dichloroacetyl orthoesters of D-mannose from 1,2-O-trichloroethylidene-beta-D-mannofuranose

1,2-O-(R)-Trichloroethylidene-beta-D-mannofuranose (1) was obtained from the reaction of D-mannose with chloral. Reaction of 1 with potassium tert-butoxide (3 Mequiv) gave the thermodynamically stable 1,2,5-O-orthodichloroacetyl-beta-D-mannofuranose as the sole product whereas 1.5 Mequiv of reagent gave the kinetically controlled 1,2,3-O-orthodichloroacetyl-beta-D-mannofuranose (10) as the main product. Orthoester 10 gave the 5,6-isopropylidene derivative, which was also obtained from the reaction of 5,6-O-isopropylidene-1,2-O-(R)-trichloroethylidene-beta-D-mannofuranose with potassium tert-butoxide (1.5 Mequiv). These novel orthoesters are expected to prove useful as protecting groups and as building blocks in the formations of new mannofuranisidic units.     

Synthesis of methyl 3-O-alpha-D-mannopyranosyl-alpha-D-talopyranoside and methyl 3-O-alpha-D-talopyranosyl-alpha-D-talopyranoside

Methyl 2-O-benzyl-3-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)-alpha- D-mannopyranoside (4) and methyl 2-O-benzyl-3-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (6) were prepared from a common intermediate, namely, methyl 2-O-benzyl-4,6-O-benzylidene-3-O-(2,3,4,6-tetra-O-acetyl-alpha-D- mannopyranosyl)-alpha-D-mannopyranoside. On treatment with tert-butylchlorodiphenylsilane, in N,N-dimethylformamide in the presence of imidazole, 4 and 6 afforded methyl 2-O-benzyl-6-O-tert-butyldiphenylsilyl-3-O-(2,3,4,6-tetra-O-acetyl -alpha-D- mannopyranosyl)-alpha-D-mannopyranoside (7), and methyl 2-O-benzyl-6-O-tert-butyldiphenylsilyl-3-O-(6-O-tert- butyldiphenylsilyl-alpha-D-mannopyranosyl)-alpha-D-mannopyranoside (8), respectively. Compound 8 was converted into its 2,3-O-isopropylidene derivative (9), and oxidation of 7 and 9 with pyridinium chlorochromate, and reduction of the resulting carbonyl intermediates gave methyl 2-O-benzyl-6-O-tert-butyldiphenylsilyl-3-O-(2,3,4,6-tetra-O-acetyl -alpha-D- mannopyranosyl)-alpha-D-talopyranoside and methyl 2-O-benzyl-6-O-tert-butyldiphenylsilyl-3-O-(6-O-tert-butyldiphe nylsilyl- 2,3-O-isopropylidene-alpha-D-talopyranosyl)-alpha-D-talopyranoside , respectively. Removal of the protecting groups furnished the title disaccharides.     

Preparation of 6,6,1',1',6',6'-hexadeutero sucrose

The preparation of 6,6,1',1',6',6'-hexadeutero sucrose is reported. The synthesis is based on a triple oxidation of a protected sucrose 6,1',6'-triol to the corresponding 6,1',6'-tricarboxylic acid or ester, followed by reduction with lithium aluminium deuteride. This triple oxidation could be achieved either using cat. TEMPO-NaOCl (to the acid) or PDC-Ac(2)O-t-BuOH (to the t-butyl carboxylic ester).     

Characterization of the genus Pseudozyma by the formation of glycolipid biosurfactants, mannosylerythritol lipids

Pseudozyma antarctica is one of the best producers of the glycolipid biosurfactants known as mannosylerythritol lipids (MELs), which show not only excellent surface-active properties but also versatile biochemical actions. In order to obtain a variety of producers, all the species of the genus were examined for their production of MELs from soybean oil. Pseudozyma fusiformata, P. parantarctica and P. tsukubaensis were newly identified to be MEL producers. Of the strains tested, P. parantarctica gave the best yield of MELs (30 g L(-1)). The obtained yield corresponded to those of P. antarctica, P. aphidis and P. rugulosa, which are known high-level MEL producers. Interestingly, P. parantarctica and P. fusiformata produced mainly 4-O-[(4',6'-di-O-acetyl-2',3'-di-O-alkanoyl)-beta-d-mannopyranosyl]-meso-erythritol (MEL-A), whereas P. tsukubaensis produced mainly 4-O-[(6'-mono-O-acetyl-2',3'-di-O-alkanoyl)-beta-d-mannopyranosyl]-meso-erythritol (MEL-B). Consequently, six of the nine species clearly produced MELs. Based on the MEL production pattern, the nine species seemed to fall into four groups: the first group produces large amounts of MELs; the second produces both MELs and other biosurfactants; the third mainly produces MEL-B; and the fourth is non-MEL-producing. Thus, MEL production may be an important taxonomic index for the Pseudozyma yeasts.     

Synthesis of O-alpha-D-glucopyranosyl-(1----4)-O-alpha -D-xylopyranosyl-(1----4)-O-alpha -D-xylopyranosyl-(1----4)-D-glucopyranose as a substrate analogue of alpha amylase

The tetrasaccharide a-D-Glcp-(1----4)-a-D-Xylp-(1----4)-a-D-Xylp-(1----4)-D- Glcp (1) has been synthesized, as a substrate analogue of alpha amylase, by silver perchlorate-catalyzed glycosylation of benzyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-benzyl-a-D-xylopyranosyl)-beta-D- glucopyranoside (30) with 2,3-di-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-a-D- glucopyranosyl)-a-D-xylopyranosyl chloride or by methyl triflate-promoted condensation of 30 with methyl 2,3-di-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl)-1-thio- beta-D-xylopyranoside, followed by removal of protecting groups of the resulting tetrasaccharide derivative 40.     

Synthesis of cerebroside, lactosyl ceramide, and ganglioside GM3 analogs containing beta-thioglycosidically linked ceramide

Coupling of the sodium salt of 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranose, -beta-D-galactopyranose, O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1----4)-2,3,6-tri-O- acetyl- 1-thio-beta-D-glucopyranose, or O-(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galacto -2- nonulopyranosylonate)-(2----3)-O-(2,3-di-O-acetyl-6-O-bezoyl -beta-D- galactopyranosyl)-(1----4)-3-O-acetyl-2,6-di-O-benzoyl-1-thio-beta-D- glucopyranose, which were prepared from the corresponding 1-S-acetates, 1, 3, 6, and 9, with (2S,3R,4E)-2-azido-3-O-benzoyl-1-O-(p-tolylsulfonyl)-4-oc tadecene-1,3-diol (12) derived by tosylation of 11, gave the corresponding beta-thioglycosides 13, 17, 21, and 25, respectively in good yield. The beta-thioglycosides obtained were converted, via selective reduction of the azide group, condensation with octadecanoic acid, and removal of the protecting groups, into the title compounds.     

Chemical transformation of lactose into 4-O-beta-D-galactopyranosyl-D-glucuronic acid (pseudolactobiouronic acid) and some derivatives thereof

The selective oxidation of the primary alcoholic function of the reducing unit of lactose was achieved in good overall yield (67%) starting from 2',6'-di-O-benzyl-2,3:3',4'-di-O-isopropylidenelactose dimethyl acetal (1) through a simple multi-step procedure based on the selective acetylation of OH-5 of 1 (methoxyisopropylation, acetylation, de-methoxyisopropylation) followed by a two-step oxidation at C-6 (TPAP-NMO then TEMPO-NaOCl) and finally, complete removal of the protecting groups.     

Biocatalytic synthesis of disaccharide high-intensity sweeterner sucralose via a tetrachlororaffinose intermediate

A second high-yielding bioorganic synthesis of the highintensity sweetener sucralose (4,1',6'-trichloro-4,1',6'-trideoxygalactosucrose) is described. This procedure involves the chemical chlorination of raffinose to form a novel tetrachloroaffinose intermediate (6,4',1',6'-tetrachloro-6,4',1',6'-tetradeoxygalactoraffinose; TCR) followed by the enzymic hydrolysis of the alpha-1-6 glycosidic bond of TCR to give sucralose and 6-chlorogalactose. Commercial enzyme preparations and microorganisms were screened to select alpha-galactosidases which have high catalytic activity on this compound. The most active enzyme was produced by a strain of Mortierella vinacea and had a maximum rate of 118 mumol sucralose/g dry weight cells/h, which was approximately 5% of the activity toward raffinose, and a K(m) of 5.8 mM toward TCR. The enzyme could be used in the form of mycelial pellets in a continuous packed bed column reactor. The reaction was also studied in a water-immiscible hydrophilic organic solvent, such as methyl isobutyl ketone, to overcome the poor aqueous solubility of TCR and to increase volumetric productivity. Synthesis of raffinose was achieved from saturated aqueous solutions of galactose and sucrose using a selected alpha-galactosidase from Aspergillus niger. When raffinose is used as a starting material for sucralose synthesis, this route has fewer steps than either the preceeding method using glucose-6-acetate as an intermediate or the complete chemical synthesis from sucrose. The relative merits of the two bioorganic routes and the utility of such methods to synthesize new sugars are discussed.     

Synthesis of two phosphate-containing "heptasaccharide" fragments of the capsular polysaccharides of Streptococcus pneumoniae types 6A and 6B.

The "heptasaccharides" O-alpha-D-galactopyranosyl-(1----3)- O-alpha-D-glucopyranosyl-(1----3)-alpha, beta-L-rhamnopyranose 2'-[O-alpha-D-galactopyranosyl-(1----3)-O-alpha-D-glucopyranosyl- (1----3)-O-alpha-L-rhamnopyranosyl-(1----3)-D-ribit-5-yl sodium phosphate] (25) and O-alpha-D-galactopyranosyl- (1----3)-O-alpha-D-glucopyranosyl-(1----3)-alpha, beta-L-rhamnopyranose 2'-[O-alpha-D-galactopyranosyl-(1----3)-O-alpha-D-glucopyranosyl- (1----3)-O-alpha-L-rhamnopyranosyl-(1----4)-D-ribit-5-yl sodium phosphate] (27), which are structural elements of the capsular polysaccharides of Streptococcus pneumoniae types 6A and 6B ([----2)- -alpha-D-Galp-(1----3)-alpha-D-Glcp-(1----3)-alpha-L-Rhap- (1----X)-D-RibOH-(5-P----]n; 6A X = 3, 6B X = 4), respectively, have been synthesized. 2,4-Di-O-acetyl- 3-O-[2,4,6-tri-O-acetyl-3-O-(2,3,4,6-tetra-O-acetyl-alpha-D- galactopyranosyl)-alpha-D-glucopyranosyl]-alpha-L-rhamnopyranosyl trichloroacetimidate (13) was coupled with 5-O-allyloxycarbonyl-1,2,4-tri-O- benzyl-D-ribitol (10), using trimethylsilyl triflate as a promotor (----14), and deallyloxycarbonylation (----15) and conversion into the corresponding triethylammonium phosphonate then gave 16. Condensation of 16 with 4-methoxybenzyl 2,4-di-O-benzyl-3-O-[2,4,6-tri-O-benzyl-3-O-(3,4,6-tri-O-benzyl-alpha-D- galactopyranosyl)-alpha-D-glucopyranosyl]- alpha-L-rhamnopyranoside (22) followed by oxidation and deprotection afforded 25. 5-O-Allyl-1-O-allyloxycarbonyl-2,3-di-O-benzyl-D-ribitol (12) was coupled with 13, using trimethylsilyl triflate as a promoter, the resulting tetrasaccharide-alditol derivative 17 was deallyloxycarbonylated (----18), acetylated (----19), and deallylated (----20), and the product was converted into the triethylammonium phosphonate derivative 21. Condensation of 21 with 22 followed by oxidation and deprotection afforded 27.     

Synthesis and reactions of O-acetylated benzyl alpha-glycosides of 6-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-N-acetylmuramoyl-L- alanyl-D-isoglutamine esters: the base-catalysed isoglutamine in equilibrium glutamine rearrangement in peptidoglycan-related structures

Condensation of benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl-2- deoxy-3-O-[(R)-1-carboxyethyl]-alpha-D-glucopyranoside (2) and its 4-acetate (4) with L-alanyl-D-isoglutamine benzyl ester via the mixed anhydride method yielded N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D- glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyranosid-3-yl]-(R)-lacto yl)-L- alanyl-D-isoglutamine benzyl ester (5) and its 4-acetate (6), respectively. Condensation by the dicyclohexylcarbodi-imide-N-hydroxysuccinimide method converted 2 into benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl- 2-deoxy-beta-D-glucopyranosyl)-3-O-[(R)-1-carboxyethyl]-2-deoxy-alpha-D- glucopyranoside 1',4-lactone (7). In the presence of activating agents, 7 underwent aminolysis with the dipeptide ester to give 5. Zemplén O-deacetylation of 5 and 6 led to transesterification and alpha----gamma transamidation of the isoglutaminyl residue to give N-(2-O-[benzyl 2-acetamido-6-O-(2- acetamido-2-deoxy-beta-D-glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyr anosid-3- yl]-(R)-lactoyl)-L-alanyl-D-isoglutamine methyl ester (8) and -glutamine methyl ester (9). Treatment of 6 with MgO-methanol caused deacetylation at the GlcNAc residue to give a mixture of N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-2- deoxy-beta-D-glucopyranosyl)-4-O-acetyl-2,3-dideoxy-alpha-D-glucopyra nosid-3- yl]-(R)-lactoyl)-L-alanyl-D-isoglutamine methyl ester (11) and -glutamine methyl ester (12). Benzyl or methyl ester-protection of peptidoglycan-related structures is not compatible with any of the reactions requiring alkaline media. Condensation of 2 with L-alanyl-D-isoglutamine tert-butyl ester gave N-(2-O-[benzyl 2-acetamido- 6-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-2,3-d ideoxy- alpha-D-glucopyranosid-3-yl]-(R)-lactoyl-L-alanyl-D-isoglutamine tert-butyl ester (16), deacetylation of which, under Zemplén conditions, proceeded without side-reactions to afford N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyranosid-3-yl]-(R)-la cotyl)-L- alanyl-D-isoglutamine tert-butyl ester (17).     

Synthesis of a spacer-containing repeating unit of the capsular polysaccharide of Streptococcus pneumoniae type 23F.

The synthesis is reported of 3-aminopropyl 4-O-(4-O-beta-D-glucopyranosyl-2-O-alpha-L-rhamnopyranosyl-beta-D- galactopyranosyl)-beta-L-rhamnopyranoside 3'-(glycer-2-yl sodium phosphate) (25 beta), which represents the repeating unit of the capsular polysaccharide of Streptococcus pneumoniae type 23F (American type 23) [(----4)-beta-D-Glcp-(1----4)-[Glycerol-(2-P----3)] [alpha-L- Rhap-(1----2)]-beta-D-Galp-(1----4)-beta-L-Rhap-(1----)n). 2,4,6-Tri-O-acetyl-3-O-allyl-alpha-D-galactopyranosyl trichloroacetimidate (5) was coupled with ethyl 2,3-di-O-benzyl-1-thio-alpha-L-rhamnopyranoside (6). Deacetylation of the resulting disaccharide derivative, followed by benzylidenation, and condensation with 2,3,4-trio-O-acetyl-alpha-L-rhamnopyranosyl trichloroacetimidate (10) afforded ethyl 4-O-[3-O-allyl-4,6-O-benzylidene-2-O-(2,3,4-trio-O-acetyl- alpha-L-rhamnopyranosyl)-beta-D-galactopyranosyl]-2,3-di-O-benzyl-1-thio - alpha-L-rhamnopyranoside (11). Deacetylation of 11, followed by benzylation, selective benzylidene ring-opening, and coupling with 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate (15) gave ethyl 4-O-[3-O-allyl-6-O-benzyl-4-O-(2,3,4,6- tetra-O-acetyl-beta-D-glucopyranosyl)-2-O-(2,3,4-tri-O-benzyl-alpha-L- rhamnopyranosyl)-beta-D-galactopyranosyl]-2,3-di-O-benzyl-1-thio-alpha-L - rhamnopyranoside (16). Deacetylation of 16 followed by benzylation, deallylation, and acetylation yielded ethyl 4-O-[3-O-acetyl-6-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-beta-D-glucopy ran osyl)- 2-O-(2,3,4-tri-O-benzyl-alpha-L-rhamnopyranosyl)-beta-D-galactopyranosyl ]-2,3- di-O-benzyl-1-thio-alpha-L-rhamnopyranoside (20). The glycosyl bromide derived from 20, when coupled with 3-benzyloxycarbonylamino-1-propanol, gave the beta-glycoside (21 beta) as the major product. Deacetylation of 21 beta followed by condensation with 1,3-di-O-benzylglycerol 2-(triethylammonium phosphonate) (27), oxidation, and deprotection, afforded 25 beta.     

Synthesis and biological evaluation of 2',3'-didehydro-2',3'-dideoxy-9-deazaguanosine, a monophosphate prodrug and two analogues, 2',3'-dideoxy-9-deazaguanosine and 2',3'-didehydro-2',3'-dideoxy-9-deazainosine

2',3'-Didehydro-2',3'-dideoxy-9-deazaguanosine (1), its monophosphate prodrug (2), and two analogues, 2',3'-dideoxy-9-deazaguanosine (3) and 2',3'-didehydro-2',3'-dideoxy-9-deazainosine (4), have been synthesized from benzoylated 9-deazaguanosine (5). Basic hydrolysis of 5, selective protection of the 2-amino and 5'-hydroxy functions with isobutyryl and silyl groups, respectively, followed by reaction with thiocarbonyldiimidazole gave the cyclic thiocarbonate, which, upon reaction with triethyl phosphite, followed by deprotection, afforded 1. Treatment of 1 with phenyl methoxyalaninylphosphochloridate and N-methylimidazole gave 2. Catalytic hydrogenation of 1 gave 3. Hydrodediazoniation of 1 with tert-butyl nitrite and tris(trimethylsilyl)silane gave 4. Compounds 1-4 were found to be inactive against the human immunodeficiency virus and exhibited minimal to no cytotoxic activity against the L1210 leukemia, CCRF-CEM lymphoblastic leukemia, and B16F10 melanoma in vitro.     

Synthetic mucin fragments: methyl 3-O-(2-acetamido-2-deoxy-beta-D-galactopyranosyl-beta-D- 3-O-(2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl- beta-D-glucopyranosyl)-beta-D-galactoyranoside. pyranosyl)-beta-D-galactopyranoside

Methyl 2,4,6-tri-O-benzyl-beta-D-galactopyranoside (5) was obtained crystalline by way of its 3-O-allyl derivative, which was in turn obtained by ring-opening of a presumed 3,4-O-stannylene derivative of methyl beta-D-galactopyranoside, followed by benzylation. Condensation of 5 with 2-methyl-(2-acetamido-3,4,6-tri-O-acetyl-1,2-dideoxy-beta-D-glucopyra no)-[2,1-d]-2-oxazoline in 1,2-dichloroethane in the presence of p-toluenesulfonic acid afforded the disaccharide derivative methyl 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-2, 4,6-tri-O-benzyl-beta-D-galactopyranoside (6) Deacetylation of 6 in methanolic sodium methoxide afforded the disaccharide derivative 7, which was acetalated with alpha, alpha-dimethoxytoluene to afford the 4',6'-O-benzylidene acetal (10). Catalytic hydrogenolysis of the benzyl groups of 7 afforded the title disaccharide 8. Glycosylation of 10 with 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide in 1:1 benzene-nitromethane in the presence of mercuric cyanide gave the fully protected trisaccharide derivative 12. Systematic removal of the protecting groups of 12 then furnished the title trisaccharide 14. The structures of 5, 8, and 14 were all confirmed by 13C-n.m.r. spectroscopy. The 13C-n.m.r. chemical shifts for methyl alpha- and beta-D-galactopyranoside, and also those of their 3-O-allyl derivatives, are recorded, for the sake of comparison, in conjunction with those of compound 5.