Enzymatic Synthesis of 2-Chloro-4-nitrophenyl 4,6-O-3-Ketobutylidene β-Maltopentaoside,a Substrate for α-Amylase

A transglycosylation reaction with 2-chloro-4-nitrophenyl β-maltoside as an acceptor was done with 4,6-O-3-ketobutylidene maltopentaose and Bacillus macerans cyclodextrin glucanotransferase in an aqueous solution containing 50% n-propanol, and there were two main transglycosylation products. They were identified as 2-chloro-4-nitrophenyl 4,6-O-3-ketobutylidene β-maltopentaoside and 2-chloro-4-nitrophenyl 4,6-O-3-ketobutylidene β-maltohexaoside, and their yields were 30% and 21%, respectively on the basis of the decrease of 4,6-O-3-ketobutylidene maltopentaose. For the production of 2-chloro-4-nitrophenyl 4,6-O-3-ketobutylidene β-maltopentaoside at high substrates concentrations, the addition of n-propanol in this reaction not only increased the solubility of 2-chloro-4-nitrophenyl β-maltoside sufficiently but also suppressed side reactions.     

Synthesis of some monodeoxyfluorinated methyl and 4-nitrophenyl alpha-D-mannobiosides and a related 4-nitrophenyl alpha-D-mannotrioside

Treatment of methyl 3,4,6-tri-O-benzyl-2-O-(2,3,4-tri-O-acetyl-alpha-D-mannopyranosyl)-alpha -D- mannopyranoside with N,N-diethylaminosulfur trifluoride (Et2NSF3), followed by O-deacetylation and catalytic hydrogenolysis, afforded methyl 2-O-(6-deoxy-6-fluoro-alpha-D-mannopyranosyl)-alpha-D-mannopyranoside (8). Methyl 6-deoxy-6-fluoro-2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (11) was similarly obtained from methyl 3-O-benzyl-2-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-alpha-D- mannopyranoside. 1,2,3,4-Tetra-O-acetyl-6-deoxy-6-fluoro-beta-D-mannopyranose (13), used for the synthesis of the 4-nitrophenyl analogs of 8 and 11, as well as their 3-O-linked isomers, was obtained by treatment of 1,2,3,4-tetra-O-acetyl-beta-D-mannopyranose with Et2NSF3. Treatment of 13 with 4-nitrophenol in the presence of tin(IV) chloride, followed by sequential O-deacetylation, isopropylidenation, acetylation, and cleavage of the acetal group, afforded 4-nitrophenyl 4-O-acetyl-6-deoxy-6-fluoro-alpha-D-mannopyranoside (18). Treatment of 13 with HBr in glacial acetic acid furnished the 6-deoxy-6-fluoro bromide 19. Glycosylation of diol 18 with 20 gave 4-nitrophenyl 4-O-acetyl-6-deoxy-6-fluoro-3-O- (21) and -2-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)-alpha-D- mannopyranoside (23) in the ratio of approximately 2:1, together with a small proportion of a branched trisaccharide. 4-Nitrophenyl 4,6-di-O-acetyl-alpha-D-mannopyranoside was similarly glycosylated with bromide 19 to give 4-nitrophenyl 4,6-di-O-acetyl-3-O- and -2-O-(2,3,4-tri- O-acetyl-6-deoxy-6-fluoro-alpha-D-mannopyranosyl)-alpha-D-mannopyranosid e. The various di- and tri-saccharides were O-deacetylated by Zemplén transesterification.     

Application of selectively acylated glycosides for the α-galactosidase-catalyzed synthesis of disaccharides

4-Nitrophenyl alpha-D-galactopyranosyl-(1-->3)-6-O-acetyl-alpha-D-galactopyranoside was prepared in a transglycosylation reaction catalyzed by alpha-D-galactosidase from Talaromyces flavus using 4-nitrophenyl alpha-D-galactopyranoside as a glycosyl donor and 4-nitrophenyl 6-O-acetyl-alpha-D-galactopyranoside as an acceptor. 4-Nitrophenyl 6-O-acetyl-alpha-D-galactopyranoside and 4-nitrophenyl 6-O-acetyl-beta-D-galactopyranoside were prepared in a regioselective enzymic transesterification in pyridine-acetone catalyzed by the lipase PS from Burkholderia cepacia. A series of water-miscible organic solvents (acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, 2-methoxyethanol, pyridine, 2-methylpropan-2-ol, tetrahydrofuran, propargyl alcohol) were used as co-solvents in this enzymic reaction. Their influence on the activity and stability of the alpha-galactosidase from T. flavus was established. 2-Methylpropan-2-ol and acetone (increasing the solubility of the modified substrate acceptors and displaying the minimum impairment of the activity and stability of the enzyme) were used as co-solvents in transglycosylation reactions.     

Hydrolysis of phenyl beta-maltoside catalyzed by saccharifying alpha-amylase from Bacillus subtilis.

1. The hydrolytic reaction of phenyl beta-maltoside catalyzed by saccharifying alpha-amylase [EC 3.2.1.1] of Bacillus subtilis was studied at 25 degrees C and pH 5.4, by measuring the total reducing power and the amount of phenol liberated, and by thin layer chromatography. 2. The enzyme hydrolyzed phenyl beta-maltoside at the glucosidic linkage between the two glucose residues to form D-glucose and phenyl beta-D-glucoside. Besides these products, maltose, maltotriose, and phenyl beta-maltotrioside were also observed as reaction products. The identification of phenyl beta-maltotrioside is described in detail. The formation of these products was attributed to the transglycosylation reaction of the enzyme. The time course of reaction as followed by reducing power measurement showed an induction period of several minutes.     

Enzymatic synthesis of p-nitrophenyl alpha-maltopentaoside in an aqueous-methanol solvent system by maltotetraose-forming amylase: a substrate for human amylase in serum

Transglycosylation from maltopentaose to the 4-position of p-nitrophenyl alpha-glucoside was efficiently induced through the use of maltotetraose-forming amylase from Pseudomonas stutzeri in an aqueous solution containing methanol at a high concentration. The enzyme specifically formed p-nitrophenyl alpha-maltopentaoside (12% of the enzyme-catalyzed net decrease of maltopentaose) from maltopentaose as a donor and p-nitrophenyl alpha-glucoside as an acceptor. The rate of the transglycosylation depended on the concentration of methanol solvent, the pH and the temperature. Use of the aqueous methanol system in this reaction not only ensured a sufficient solubility of p-nitrophenyl alpha-glucoside but also resulted in a remarkable increase in the formation of p-nitrophenyl alpha-maltopentaoside, which is a useful substrate for assay of human amylase in serum and urine.     

Chemoenzymatic synthesis of 2-chloro-4-nitrophenyl beta-maltoheptaoside acceptor-products using glycogen phosphorylase b.

In the present work, we aimed at developing a chemoenzymatic procedure for the synthesis of beta-maltooligosaccharide glycosides. The primer in the enzymatic reaction was 2-chloro-4-nitrophenyl beta-maltoheptaoside (G(7)-CNP), synthesised from beta-cyclodextrin using a convenient chemical method. CNP-maltooligosaccharides of longer chain length, in the range of DP 8-11, were obtained by a transglycosylation reaction using alpha-D-glucopyranosyl-phosphate (G-1-P) as a donor. Detailed enzymological studies revealed that the conversion of G(7)-CNP catalysed by rabbit skeletal muscle glycogen phosphorylase b (EC 2.4.1.1) could be controlled by acarbose and was highly dependent on the conditions of transglycosylation. More than 90% conversion of G(7)-CNP was achieved through a 10:1 donor-acceptor ratio. Tranglycosylation at 37 degrees C for 30 min with 10 U enzyme resulted in G(8-->12)-CNP oligomers in the ratio of 22.8, 26.6, 23.2, 16.5, and 6.8%, respectively. The reaction pattern was investigated using an HPLC system. The preparative scale isolation of G(8-->11)-CNP glycosides was achieved on a semipreparative HPLC column. The productivity of the synthesis was improved by yields up to 70-75%. The structures of the oligomers were confirmed by their chromatographic behaviours and MALDI-TOF MS data.     

Transfer Action of Cyclodextrin Glycosyltransferase on Starch

The transglycosylation reaction of the cyclodextrin glycosyltransferase from Bacillus megaterium (No. 5 enzyme) and Bacillus macerans (BMA) were examined. No. 5 enzyme was more efficient in transglycosylation reaction than BMA in the every acceptor employed in the present study. The order of the efficient acceptors for No. 5 enzyme was maltose (G2), glucose (Gl), maltotriose (G3) and sucrose (GF). On the other hand, that found for BMA was Gl, G2, GF and G3. The transglycosylation products to glucose formed by the action of No. 5 enzyme on starch were G2, G3, maltotetraose (G4), maltopentaose (G5), maltohexaose (G6) and maltoheptaose (G7) in the order of their quantities, while, in the case of BMA, they were G2, G3, G5, G7=G4 and G6. The larger transglycosylation products to sucrose formed by the action of No. 5 enzyme on starch were maltosylfructose. On the other hand, that formed by the action of BMA was maltoheptaosylfructose.

It was suggested that cyclodextrin glycosyltransferase could transfer the glucosyl residues to an acceptor directly from starch, as well as through cyclodextrin.     

A concise synthesis of 4-nitrophenyl 2-azido-2-deoxy- and 2-acetamido-2-deoxy-D-mannopyranosides

4-nitrophenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha- and beta-D-mannopyranosides were prepared from methyl 4,6-O-benzylidene-alpha-D-glucopyranoside and 1,3,4,6-tetra-O-acetyl-alpha-D-glucopyranose, respectively. Chemoselective reduction of both azides with hydrogen sulfide readily afforded 4-nitrophenyl 2-acetamido-4,6-di-O-acetyl-2-deoxy-alpha-D- and -beta-D-mannopyranosides in higher yields than reduction with triphenylphosphine or a polymer-supported triarylphosphine. Subsequent de-O-acetylation yielded 4-nitrophenyl 2-acetamido-2-deoxy-alpha-D-mannopyranoside and 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-mannopyranoside in 20% and 44% overall yields, respectively.     

A facile synthesis of nitrophenyl oligosaccharides containing the O-alpha-L-fucopyranosyl-(1----3)-2-acetamido-2-deoxy-beta-D-glucopyrano syl unit at their nonreducing end.

A facile approach towards the synthesis of 4-nitrophenyl O-alpha-L-fucopyranosyl-(1----3)-2-acetamido-2-deoxy-beta-D-glucopyra nos ide, 2-nitrophenyl O-alpha-L-fucopyranosyl-(1----3)-O-(2-acetamido-2-deoxy-beta-D-glucop yra nosyl)- (1----6)-2-acetamido-2-deoxy-alpha-D-galactopyranoside, 4-nitrophenyl O-alpha-L-fucopyranosyl-(1----3)-O-(2-acetamido-2-deoxy-beta-D-glucop yra nosyl)- (1----6)-alpha-D-mannopyranoside, and 4-nitrophenyl O-alpha-L-fucopyranosyl-(1----3)-O-(2-acetamido-2-deoxy-beta-D-glucop yra nosyl)-(1----6)-beta-D-galactopyranoside has been accomplished through the development and use of methyl 3,4-O-isopropylidene-2-O-(4-methoxybenzyl)-1-thio-beta-L-fucopyranoside as the glycosyl donor.     

Synthesis of the isomeric trisaccharides, methyl O-alpha-L-fucopyranosyl- (1----3, 4, and 6)-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----3)-beta- D-galactopyranoside

Benzylation of methyl 3-O-(2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D- glucopyranosyl)-2,4,6-tri-O-benzyl-beta-D-galactopyranoside with benzyl bromide in N,N-dimethylformamide in the presence of sodium hydride afforded methyl 3-O- (2-acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-beta-D-glucopyranosyl) -2,4,6- tri-O-benzyl-beta-D-galactopyranoside (3). Reductive ring-opening of the benzylidene group of 3 gave methyl 3-O-(2-acetamido-3,6-di-O-benzyl-2-deoxy-beta-D- glucopyranosyl)- 2,4,6-tri-O-benzyl-beta-D-galactopyranoside (4). Cleavage of the 4,6-acetal group of 3 with hot, 80% aqueous acetic acid afforded the diol (5). Compounds 3, 4, and 5 were each subjected to halide ion-catalyzed glycosylation with 2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl bromide to produce the corresponding trisaccharide derivatives, which, on catalytic hydrogenation, furnished the title trisaccharides, respectively.     

Synthesis of disaccharide fragments of dermatan sulfate

Condensation of crystalline methyl 2-azido-4,6-O-benzylidene-2-deoxy-beta-D-galactopyranoside with methyl (2,3,4-tri-O-acetyl-alpha-L-idopyranosyl bromide)uronate in dichloromethane, in the presence of silver triflate and molecular sieve, provided 54% of methyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O-(methyl 2,3,4-tri-O-acetyl-alpha-L-idopyranosyluronate)-beta-D-galactopyranoside . The use of methyl (2,3,4-tri-O-acetyl-alpha-L-idopyranosyl trichloroacetimidate)uronate as glycosyl donor, in the presence of trimethylsilyl triflate, improved the yield to 68%. Regioselective opening of the benzylidene group with sodium cyanoborohydride followed successively by O-sulfation with the sulfur trioxide-trimethylamine complex, saponification, catalytic hydrogenolysis and selective N-acetylation gave the disodium salt of methyl 2-acetamido-2-deoxy-3-O-(alpha-L-idopyranosyluronic acid)-4-O-sulfo-beta-D-galactopyranoside. Condensation of methyl 2-azido-4,6-O-benzylidene-2-deoxy-beta-D-galactopyranoside with methyl (2,3,4-tri-O-acetyl-alpha-D-glucopyranosyl bromide)uronate in dichloromethane, in the presence of silver triflate and molecular sieve, gave methyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O-(methyl 2,3,4-tri-O-acetyl-beta-D-glucopyranosyluronate)-beta-D-galactopryano side in 85% yield. The sequence already described then gave the disodium salt of methyl 2-acetamido-2-deoxy-3-O-(beta-D-glucopyranosyluronic acid)-4-O-sulfo-beta-D-galactopyranoside.     

Synthesis of alpha- and beta-glycosyl donors with a disaccharide beta-D-Gal-(1-->3)-D-GalNAc backbone

The synthesis of thioglycosyl donors with a disaccharide beta-D-Gal-(1-->3)-D-GalNAc backbone was studied using the glycosylation of a series of suitably protected 3-monohydroxy- and 3,4-dihydroxyderivatives of phenyl 2-azido-2-deoxy-1-thio-alpha- and 1-thio-beta-D-galactopyranosides by galactosyl bromide, fluoride, and trichloroacetimidate. In the reaction with the monohydroxylated glycosyl acceptor, the process of intermolecular transfer of thiophenyl group from the glycosyl acceptor onto the cation formed from the molecule of glycosyl donor dominated. When glycosylating 3,4-diol under the same conditions, the product of the thiophenyl group transfer dominated or the undesired (1-->4), rather than (1-->3)-linked, disaccharide product formed. The aglycone transfer was excluded when 4-nitrophenylthio group was substituted for phenylthio group in the galactosyl acceptor molecule. This led to the target disaccharide, 4-nitrophenyl 2-azido-4,5-O-benzylidene-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-beta-D- galactopyranosyl)-1-thio-beta-D-galactopyranoside, in 57% yield. This disaccharide product bears nonparticipating azide group in position 2 of galactosamine and can hence be used to form alpha-glycoside bond. 2-Azide group and the aglycone nitro group were simultaneously reduced in this product and then trichloroacetylated, which led to the beta-glycosyl donor, 4-trichloroacetamidophenyl 4,6-O-diacetyl-2-deoxy-3-O-(2,3,4,6-tetra- O-acetyl-beta-D-galactopyranosyl)-1-thio-2-trichloroacetamido-beta-D- galactopyranoside, in 62% yield. The resulting glycosyl donor was used in the synthesis of tetrasaccharide asialo-GM1.     

Transglycosylating and hydrolytic activities of the β-mannosidase from Trichoderma reesei

A purified β-mannosidase (EC 3.2.1.25) from the fungus Trichoderma reesei has been identified as a member of glycoside hydrolase family 2 through mass spectrometry analysis of tryptic peptides. In addition to hydrolysis, the enzyme catalyzes substrate transglycosylation with p-nitrophenyl β-mannopyranoside. Structures of the major and minor products of this reaction were identified by NMR analysis as p-nitrophenyl mannobiosides and p-nitrophenyl mannotriosides containing β-(1 → 4) and β-(1 → 3) linkages. The rate of donor substrate hydrolysis increased in presence of acetonitrile and dimethylformamide, while transglycosylation was weakly suppressed by these organic solvents. Differential ultraviolet spectra of the protein indicate that a rearrangement of the hydrophobic environment of the active site following the addition of the organic solvents may be responsible for this hydrolytic activation.     

Synthesis of beta-D-GlcA-(1----3)-beta-D-Gal disaccharides with 4- and 6-sulfate groups and 4,6-disulfate groups.

The sodium salts of the 6-sulfate 7, the 4-sulfate 10, and the 4,6-disulfate 12 of benzyl 3-O-(beta-D-glucopyranosyl uronate)-beta-D-galactopyranoside (5) have been synthesized. Methyl (2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-d-glucopyran)uronate (1) was coupled with benzyl 2-O-benzoyl-4,6-O-benzylidene-beta-D-galactopyranoside (2) to yield 3. The benzylidene acetal of 3 was hydrolyzed to give benzyl 2-O-benzoyl-3-O-[methyl (2,3,4-tri-O-acetyl-beta-D-glucopyranosyl)uronate]-beta-D-galactopyra noside (4). Compound 4 was utilized as a key intermediate to prepare the sulfated disaccharides 7,10, and 12. Direct sulfation of 4 with sulfur trioxide-trimethylamine for 2 days yielded the 6-sulfate 6. The 4,6-disulfate 11 was accessible by running the reaction under the same conditions for 14 days. The 4-sulfate 9 was obtained after protecting the 6-OH group of 4 by reaction with benzoyl imidazole to give the 6-benzoate 8, followed by sulfation under vigorous conditions. Treatment of the protected compounds 4, 6, 9, and 11 with aqueous sodium hydroxide in tetrahydrofuran gave the unprotected 5, 7, 10, and 12, respectively.     

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%).     

Glycosylation of daidzein by the Eucalyptus cell cultures

The sequential glycosylation of a soybean isoflavone, daidzein, with cultured suspension cells of Eucalyptus perriniana and cyclodextrin glucanotransferase was studied. Daidzein was converted into two glycosylation products, daidzein 7-O-beta-d-glucopyranoside (39%) and daidzein 7-O-[6-O-(beta-d-glucopyranosyl)]-beta-d-glucopyranoside (beta-gentiobioside, 6%), by cultured E. perriniana cells. Further glycosylation of daidzein 7-O-beta-glucoside with cyclodextrin glucanotransferase gave daidzein 7-O-[4-O-(alpha-d-glucopyranosyl)]-beta-d-glucopyranoside (beta-maltoside, 26%), daidzein 7-O-beta-maltotrioside (15%), and daidzein 7-O-beta-maltotetraoside (7%).     

Synthesis of triazole-linked pseudo-starch fragments

Rapid assembly of starch fragment analogues was achieved using 'click chemistry'. Specifically, a pentadecasaccharide and two hexadecasaccharide mimics containing two parallel maltoheptaosyl chains linked via [1,2,3]-triazoles to glucose or maltose core were synthesised using Cu(I)-catalyzed [3+2] dipolar cycloaddition of azidosaccharides and 4,6-di-O-propargylated methyl alpha-d-glucopyranoside and 6,6'- and 4',6'-di-O-propargylated p-methoxyphenyl beta-maltoside.     

Synthesis of alpha-galactosylated fragments related to the core-structure of the GPI anchor of Trypanosoma brucei

A series of octyl glycosides di- to tetrasaccharides related to the GPI anchor of Trypanosoma brucei was prepared. Treatment of octyl 2-O-benzoyl-4,6-O-(1,1,3,3-tetraisopropyl-1,3-disiloxane-1,3 -diyl)-alpha-D-mannopyranoside with ethyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-galactopyranoside under activation with bromine and silver trifluoromethanesulfonate afforded the alpha-linked disaccharide octyl 2-O-benzoyl-3-O-(2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-4,6-O- (1,1,3,3-tetraisopropyl-1,3-disiloxane-1,3-diyl)-alpha -D-mannospyranoside, the siloxane ring of which was regioselectively opened with a HF-pyridine complex to give the disaccharide acceptor octyl 3-O-(2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-2-O-benzoyl-4-O-(3 -fluoro-1,1,3,3-tetraisopropyl-1,3-disiloxane-3-yl)-alpha-D- mannopyranoside (4). Mannosylation of 4 with benzobromomannose (7), followed by fluoride catalyzed desilylation gave the trisaccharide octyl 2-O-benzoyl-6-O-(2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl)-3-O-(2, 3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-alpha-D-mannospyranosi de, which was deblocked via the deacylated intermediate octyl 3-O-(2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-6-O-(alpha-D-manno pyranosyl)-alpha-D-mannospyranoside to afford the octyl glycoside trisaccharide octyl 3-O-(alpha-D-galactopyranosyl)-6-O-(alpha-D-mannopyranosyl)-alpha-D-m annospyranoside. Glycosylation of 4 with 3,4,6-tri-O-acetyl-2-O-(2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl)- alpha-D-mannopyranosyl trichloroacetimidate resulted in the tetrasaccharide octyl 2-O-benzoyl-4-O-(1-fluoro-1,1,3,3-tetraisopropyl-1,3-disiloxane -3-yl)-3-O-(2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-6-O-[2-O -(2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl)-3,4,6-tri-O-acetyl-alp ha-D-mannopyranosyl]-alpha-D-mannospyranoside, sequential desilylation, deacylation and debenzylation, respectively, of which via the intermediate octyl 2-O-benzoyl-3-O-(2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-6-O-[2 -O-(2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl)-3,4,6-tri-O-acetyl-a lpha-D-mannopyranosyl]-alpha-D-mannospyranoside afforded the octyl glycoside tetrasaccharide octyl 3-O-(alpha-D-galactopyranosyl)-6-O-[2-O-(alpha-D-mannopyranosyl)-alpha-D -mannopyranosyl]-alpha-D-mannospyranoside.     

Synthesis of twisted intercalating nucleic acids possessing acridine derivatives. Thermal stability studies

Twisted intercalating nucleic acids (TINA) possessing acridine derivatives have been synthesized via the postsynthetic modifications of oligonucleotides possessing insertions of (R)-1-O-(4-iodobenzyl)glycerol (8) or (R)-1-O-(4-ethynylbenzyl)glycerol (9) at the 5'-end or in the middle as a bulge. In the first postsynthetic step, oligonucleotides 8 and 9 on the CPG support were treated with a Sonogashira coupling reaction mixture containing 9-chloro-2-ethynylacridine or 9-chloro-2-iodoacridine, respectively. After the postsynthetic step, treatment of the oligonucleotides with 32% aq ammonia or 50% ethanolic solution of tris(2-aminoethyl)amine led to the substitution of chloride on acridine concurrent with deprotection of the bases and cleavage of the oligonucleotides from CPG. Molecular modeling of the parallel triplex with a bulged insertion of the monomer (R)-3-O-[4-(9-aminoacridin-2-ylethynyl)benzyl]glycerol in the triplex-forming oligonucleotide (TFO) showed that the acridine moiety was stacking between the bases of the duplex, while phenyl was placed between the bases of the TFO. Thermal denaturation studies and fluorescence properties of TINA-acridine oligonucleotide duplexes and triplexes are discussed.     

Synthesis of a novel asparagine-linked heptasaccharide structure via p-methoxybenzyl-assisted beta-mannosylation

Synthesis of a core heptasaccharide asparagine N4-[alpha-D-mannopyranosyl-(1 --> 6)-[(alpha-D-mannopyranosyl)-(1 --> 3)]-[(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1 --> 2)]-(beta-D-mannopyranosyl)-(1 --> 4)-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1 --> 4)-[(alpha-L-fucopyranosyl)-(1 --> 6)]-2-acetamido-2-deoxy-beta-D-glucopyranosyl]-L-asparagine (1a) found from CHO glycosylation mutant cell LEC 14 is described. The structure of 1a is highly novel in terms of the presence of an extra GlcNAc residue linked to the 2-position of beta-linked mannose. The synthesis was performed using p-methoxybenzyl-assisted intramolecular aglycon delivery as the key transformation. 4,6-O-TIDPS-protected thiomannoside methyl 2-O-p-methoxybenzyl-4,6-O-(1,1,3,3-tetraisopropyl)disiloxanylid ene-3-O-trimethylsilyl-1-thio-alpha-D-mannopyranoside was adopted for this particular purpose, which afforded beta-mannoside p-methoxyphenyl 2,3-O-(p-methoxybenzylidene)-4,6-O-(1,1,3,3-tetraisopropyl)+ ++disiloxanylidene-beta-D-mannopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranoside stereoselectively in 75% yield.