Synthesis of Aminoethyl Glycosides of the Carbohydrate Chains of Glycolipids Gb3, Gb4, and Gb5

4-O-Glycosylation of 2-azidoethyl 2,3,6-tri-O-benzoyl-4-O-(2,3,6-tri-O-benzoyl--D-galactopyranosyl)--D-glucopyranoside with ethyl 2,3,4,6-tetra-O-benzyl- and ethyl 3-O-acetyl-2,4,6-tri-O-benzyl-1-thio--D-galactopyranoside in the presence of methyl trifluoromethanesulfonate led to trisaccharide 2-azidoethyl (2,3,4,6-tetra-O-benzyl--D-galactopyranosyl)-(14)-(2,3,6-tri-O-benzoyl--D-galactopyranosyl)-(14)-2,3,6-tri-O-benzoyl--D-glucopyranoside and its 3"-O-acetylated analogue, 2-azidoethyl (3-O-acetyl-2,4,6-tri-O-benzyl--D-galactopyranosyl)-(14)-(2,3,6-tri-O-benzoyl--D-galactopyranosyl)-(14)-2,3,6-tri-O-benzoyl--D-glucopyranoside in yields of 85 and 83%, respectively. Deacetylation of the latter compound and subsequent glycosylation with 4-trichloroacetamidophenyl 3,4,6-tri-O-acetyl-2-deoxy-1-thio-2-trichloroacetamido--D-galactopyranoside and 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-1-thio-2-trichloroacetamido--D-galactopyranoside in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid resulted in the corresponding selectively protected derivatives of the tetrasaccharide GalNAc(13)Gl(14)Gal(14)Glc-OH₂CH₂N₃ and the pentasaccharide Gal(13)GalNAc(13)Gl(14)Gal(14)Glc-OH₂CH₂N₃ in 88 and 73% yields, respectively. Removal of O-protecting groups, substitution of acetyl group for the N-trichloroacetyl group, and reduction of the aglycone azide group resulted in the target 2-aminoethyl globo-tri-, -tetra-, and -pentasaccharide, respectively.     

Synthesis of four structural elements of xylose-containing carbohydrate chains from N-glycoproteins

The synthesis of the oligosaccharides beta-D-Xylp-(1----2)-beta-D-Manp-OMe (12), beta-D-Xylp-(1----2)-[alpha-D-Manp-(1----6)]-beta-D-Manp+ ++-OMe (17), beta-D-Xylp-(1----2)-[alpha-D-Manp-(1----3)]-beta-D-Manp+ ++-OMe (21), and beta-D-Xylp-(1----2)-[alpha-D-Manp-(1----3)] [alpha-D-Manp-(1----6)]-beta-D-Manp-OMe (25) is described. Methyl 3-O-benzyl-4,6-O-isopropylidene-beta-D-mannopyranoside (6) was prepared from the corresponding glucoepimer (4) by oxidation, followed by stereoselective reduction. Condensation of 6 with 2,3,4-tri-O-acetyl-alpha-D-xylopyranosyl bromide in the presence of mercuric cyanide gave a 1:9 mixture of methyl 3-O-benzyl-4,6-O-isopropylidene-2-O-(2,3,4- tri-O-acetyl-alpha- (7a) and -beta-D-xylopyranosyl)-beta-D-mannopyranoside (7), and then 7 was converted into the acetylated disaccharide-glycoside 11. Regioselective mannosylation, with 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl bromide, at position 6 of deisopropylidenated 7 (8), using mercuric bromide as a promoter, afforded the trisaccharide-glycoside derivative 13, which was transformed into the acetylated trisaccharide-glycoside 16. The disaccharide derivative 10, obtained from 8, and the trisaccharide derivative 15, obtained from 13, were glycosylated at position 3 with O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)trichloroacetimidate (19), using trimethylsilyl triflate as a promoter, giving rise to acetylated tri- (20) and tetra-saccharide (24) derivatives, respectively. O-Deacetylation of 11, 16, 20, and 24 gave 12, 17, 21, and 25, respectively.     

Origin of the different strengths of the (i,i+4) and (i,i+3) leucine pair interactions in helices

Pairs of leucine side chains, spaced either (i,i+3) or (i,i+4), are known to stabilize alanine-based peptide helices, Experiments with new peptide sequences confirm that the (i,i+4) pair interaction is markedly stronger than the (i,i+3) pair interaction. This result is not expected from reported Monte Carlo simulations, which predict that the (i,i+3) interaction is slightly stronger. The interaction strength can be predicted from recently reported measurements of buried non-polar surface area, obtained from structures in the Protein Data Bank: the agreement is reasonable for the (i,i+3) LL interaction but underestimates the (i,i+4) LL interaction. Solvation of peptide groups in the helix backbone may contribute to the different strengths of the two LL pair interactions because different chi(1) leucine rotamers are used and the (i,i+3) pair shields two peptide groups whereas the (i,i+4) pair shields only one. A rough estimate of the backbone solvation effect, based on the difference between the helix propensities of leucine and alanine, agrees with the size of the difference between the (i,i+3) and (i,i+4) leucine pair interactions.     

Synthesis of glycosides of 3-deoxy-4-thiopentopyranosid-2-uloses and their reduction products: 3-deoxy-4-thiopentopyranosides

Michael addition of common thiols to the enone system of (2S)-2-benzyloxy-2H-pyran-3(6H)-one (1) afforded the corresponding 3-deoxy-4-thiopentopyranosid-2-ulose derivatives (2-4). The reaction was highly diastereoselective, and the addition was governed by the quasiaxially disposed 2-benzyloxy substituent of the starting pyranone. As expected from the enantiomeric excess of 1 (ee > 86%) the corresponding thiouloses 2-4 exhibited the same optical purity. However, the enantiomerically pure thioulose 5 was obtained by reaction of 1 with the chiral thiol, N-(tert-butoxycarbonyl)-L-cysteine methyl ester. The thio derivative 7 was also synthesized by reaction of 6 (enantiomer of 1) with the same chiral thiol. Alternatively, 4-thiopent-2-uloses 9-12 were prepared in high optical purity by 1,4-addition of thiols to (2S)-[(S)-2'-octyloxy]dihydropyranone 8. Similarly, reaction of 13 (enantiomer of 8) with benzenemethanethiol afforded 14 (enantiomer of 10). This way, the stereocontrol exerted by the anomeric center on the starting dihydropyranone led to 4-thiopentuloses of the D and L series. Sodium borohydride reduction of the carbonyl function of uloses 10 and 12 gave the corresponding 3-deoxy-4-thiopentopyranosid-2-uloses (16-19). The diastereomers having the beta-D-threo configuration (16, 18) slightly predominated over the beta-D-erythro (17, 19) analogues. However, the reduction of the enantiomeric pyranones 10 and 14 with K-Selectride was highly diastereofacial selective in favor of the beta-D- and beta-L-threo isomers 16 and 20, respectively.     

Analysis of N-acetyl-4-O-acetylneuraminic-acid-containing N-linked carbohydrate chains released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Application to the structure determination of the carbohydrate chains of equine fibrinogen

The carbohydrate chains of equine fibrinogen were enzymatically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The oligosaccharides obtained were fractionated by a combination of FPLC and HPLC and analyzed by 500-MHz 1H-NMR spectroscopy. Four monosialo and four disialo diantennary N-acetyllactosamine type of carbohydrate chains occur: (formula; see text)     

Cell surface glycoconjugates as possible target structures for human natural killer cells: evidence against the involvement of glycolipids and N-linked carbohydrate chains

Natural killer (NK) cells can spontaneously kill various malignantcells, but the susceptibility towards NK cells differs greatlyamong different types of tumour cells. The molecules, whichare recognized by NK cells, have not yet been identified, butthere is ample evidence that target cell surface glycoconjugatesare involved in the interaction with NK cells. In this report,we show that the recognition of K562 target cells by human NKcells depends on the presence of protein-bound determinants,implying that glycolipids are not the primary target structureson K562 cells. The NK susceptibility of K562 cells was not alteredby enzymic removal of various cell surface carbohydrates oroligosaccharides, mostly related to N-linked carbohydrate chains.Treatment of K562 cells with 1-deoxynojirimycin and 1-deoxymannojirimycin,inhibitors of N-glycan processing, resulted in drastic alterationsin the carbohydrate phenotype of the cell surface, as couldbe shown by flow cytometric analysis of the lectin-binding propertiesof the cells. Despite these clear changes in N-glycosylation,the NK susceptibility of K562 cells remained unaffected. Summarizing,the results described in this report show that potential targetstructures for NK cells are protein bound, but the involvementof a specific (N-linked) carbohydrate determinant in the interactionbetween NK cells and target cells could not be established. cell adhesion molecules cell—cell interaction cell surface glycoconjugates natural killer cells target structures     

Synthesis of 5'-C-methyl-1',3'-dioxolan-4'-yl nucleosides

Novel racemic 5'-C-methyl-1',3'-dioxolan-4'-yl nucleosides were synthesized from the key intermediate, 2-benzoyloxymethyl-4-oxo-5-C-methyl-1,3-dioxolane, which was prepared from racemic lactic acid.     

Synthesis of 3, 5-Di-sec-butylphenol

Synthesis of 3, 5-di-sec-butylphenol, an intermediate in the synthesis of auxin b lactone, was described. Hydrogenation of 3, 5-di-sec-butylphenol afforded 1, 3-di-sec-butylcyclo-hexane.     

Synthesis and distribution of carbohydrate chains in cleavage-stage mouse embryos carrying the t12 lethal mutation

Carbohydrate chains on the t12 mutant embryos were analyzed. No abnormalities of the synthesis of the carbohydrate chains were observed in the t12 homozygotes until the late morula stage, when radiolabeled carbohydrate chains were analyzed by Sephadex G-50 column chromatography. Furthermore, polarization of the Con A receptor occurred normally at the eight-cell stage. Therefore, the possible defects of the carbohydrate chains on the t12 embryos were suggested to be neither gross abnormality of the synthesis nor abnormal localization on the surface, but rather minor structural changes on a specific molecule.     

Synthesis and biological evaluation of 5-fatty-acylamido-1, 3, 4-thiadiazole-2-thioglycosides

In the present study, the synthesis of 1, 3, 4-thiadiazole-based thioglycosides were accomplished in good yields with employing a convergent synthetic route. The starting material 5-amino-1, 3, 4-thiadiazole-2-thiol and followed by a series of 5-fatty-acylamido-1, 3, 4-thiadiazole-2-thiols (4a–4j) were synthesized with different fatty acid chlorides. The glycosylation of compounds 4a–4j were achieved with trichloroacetimidate methodology. Antimicrobial and cytotoxicity activities of title compounds were evaluated. Among the entire compounds lauric acid and myristic acid derivatives showed good and moderate antimicrobial activity. In case of cytotoxicity results of compounds 8a–8j and 9a–9j, the acetate protected short chain (C6:0, C8:0, C10:0) compounds and the free hydroxyl long chain saturated (C16:0, C18:0) and unsaturated (C18:1, C22:1) compounds exhibited good activity against different cancer cell lines. Further, the free hydroxyl compounds 9a, 9c–9j did not show any toxicity towards normal CHO-K1 cell line whereas acylated compounds 8a–8j exhibited toxicity.     

Structural studies of the carbohydrate chains of human gamma-interferon

Human gamma-interferon (IFN-gamma) was prepared biotechnologically using Chinese hamster ovary cells. These cells were shown to be able to produce glycosylated IFN-gamma. Sugar analysis revealed the presence of Man, Gal, GlcNAc, NeuAc and Fuc residues in a molar ratio of 3.8:2.0:3.5:0.6:0.4 suggesting the occurrence of N-glycosidically linked N-acetyllactosamine type of carbohydrate chains. For structure determination of these chains, the glycoprotein was subjected to the hydrazinolysis procedure, yielding oligosaccharide-alditols. The latter compounds were analysed by 500-MHz 1H-NMR spectroscopy. The carbohydrate material was found to consist of biantennary structures, exhibiting microheterogeneity as to the terminal sialic acids and the core Fuc residue: (Formula: see text). As similar carbohydrates are present on several human secreted proteins, this glycosyl group is not expected to be immunogenic in man. It remains to be established to what extent the carbohydrate chains of this biotechnologically produced IFN-gamma are identical to those of naturally occurring human IFN-gamma.     

Synthesis of Neu5Ac- and Neu5Gc-alpha-(2-->6')-lactosamine 3-aminopropyl glycosides

In order to prepare 3-aminopropyl glycosides of Neu5Ac-alpha-(2-->6')-lactosamine trisaccharide 1, and its N-glycolyl containing analogue Neu5Gc-alpha-(2-->6')-lactosamine 2, a series of lactosamine acceptors with two, three, and four free OH groups in the galactose residue was studied in glycosylations with a conventional sialyl donor phenyl [methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-alpha- and beta-D-galacto-2-nonulopyranosid]onates (3) and a new donor phenyl [methyl 4,7,8,9-tetra-O-acetyl-5-(N-tert-butoxycarbonylacetamido)-3,5-dideoxy-2-thio-D-glycero-alpha- and beta-D-galacto-2-nonulopyranosid]onates (4), respectively. The lactosamine 4',6'-diol acceptor was found to be the most efficient in glycosylation with both 3 and 4, while imide-type donor 4 gave slightly higher yields with all acceptors, and isolation of the reaction products was more convenient. In the trisaccharides, obtained by glycosylation with donor 4, the 5-(N-tert-butoxycarbonylacetamido) moiety in the neuraminic acid could be efficiently transformed into the desired N-glycolyl fragment, indicating that such protected oligosaccharide derivatives are valuable precursors of sialo-oligosaccharides containing N-modified analogues of Neu5Ac.     

Neutralizing activity of polyvalent Gb3, Gb2 and galacto-trehalose models against Shiga toxins

Shiga toxin (Stx) is one of the most critical factors in the development of hemolytic uremic syndrome and other systemic complications following enterohemorrhagic Escherichia coli (EHEC) infection. Substances neutralizing Stx by interfering with toxin-receptor binding have been explored as therapeutic candidates for EHEC infection. In this study, we examined globotriaosyl (Gb3), galabiosyl (Gb2) and galacto-trehalose, each of which was synthetically conjugated with a polyacrylamide backbone, for Stxneutralizing activity. Galacto-trehalose was designed as a Gb2 mimicking, unnatural Stx-ligand that was expected to show tolerance to enzymatic degradation in vivo. Galacto-trehalose copolymer showed neutralizing activity against Stx-1 but not Stx-2 in a HeLa cell cytotoxicity assay. It was thought that galactotrehalose copolymer could be a lead compound for the treatment of Stx-mediated diseases, although it requires modification to show neutralizing activity to Stx-2. The Gb3 copolymer with high sugar unit density showed stronger neutralizing activity against Stx-2 than those with lower density. However, the density-dependency of the neutralizing activity was less obvious against Stx-1. Intravenous administration of the Gb3 copolymer prevented death in mice lethally infected with Stx-1- and Stx-2-producing E. coli O157:H7. Thus, we demonstrated that the artificial Gb3 copolymer could neutralize Stx-1 and the more clinically relevant Stx-2 in vitro and effectively inhibit Stx toxicity in vivo.     

Synthesis of 5-Chloro-l-(2, 3-Orisopropylidene-4-keto Rhamnopyranosyl)-uracil

Abstract

Syntheses of 5-chloro-l-C2, 3, 4-tri-0-acetyl-αa-L-rhamnopyrano-syl)-uracil (1), 5-chloro-l-(a-L-rhamnopyranosyl)-uracil (2), 5-chloro-l (2, 3-Oisopropylidene-α-L-rhamnopyranosyl)-uracil (3), and 5-chloro-l-(2, 3-O-isopropylidene-4-Tceto-rhamnopyranosyl)-uracil (4 are reported. Oxidation of 3 to 4 was effected using pyridinium chlorochromate.     

Syntheses, structures and properties of 3d/5d-4f metal complexes with novel polycationic chains

Two 3d/5d-4f metal complexes [DyL₃(H₂O)₂]_n(1.5nHgCl₄) · 2nH₂O (1) and (1.5nZnCl₄) · nH₂O (2), where L and L′ are isonicotinic acid and nicotinic acid, respectively, have been synthesized via hydrothermal reaction and structurally characterized by single-crystal X-ray diffractions. Both complexes are characteristic of a one-dimensional polycationic chain-like structure. Photoluminescent investigation reveals that complex 1 displays emissions in violet, blue and yellow regions, and the violet emission is stronger than the blue and yellow ones. Complex 2 displays emissions in orange and red regions, and the emission are attributed to the characteristic emissions of ⁵D₀ → ⁷F_J (J = 0, 1, 2, 3, 4) of Eu³⁺ ions. Optical absorption spectra reveal the presence of an optical gap of 3.31 and 3.86 eV for 1 and 2, respectively. The magnetic properties show that complex 1 exhibits antiferromagnetic-like interactions.     

Synthesis of 3-(2-aminoethylthio)propyl glycosides

Anomeric pairs of 3-(2-aminoethylthio)propyl d-galactopyranoside (4, 4a), d-glucopyranoside (5, 5a), and 2-acetamido-2-deoxy-d-glucopyranoside (6, 6a) were prepared by addition of 2-aminoethanethiol to the corresponding, anomeric, allyl glycosides. The allyl α-glycosides were prepared by refluxing the sugars with allyl alcohol in the presence of an acid catalyst; the allyl β-glycosides were prepared by the reaction of acetylated glycosyl bromides with allyl alcohol in the presence of mercuric cyanide, followed by O-deacetylation. The rate of thiol addition to the allylic group was found to be different for each glycoside.