New and more efficient multivalent glyco-ligands for asialoglycoprotein receptor of mammalian hepatocytes

New multi-valent, carbohydrate ligands that contain terminal N-acetylgalactosamine (GalNAc) or lactose (Lac) were prepared using a nitrilotriacetic acid (NTA) derivative of L-lysine as scaffold. Tri-valent structures were prepared by attaching an ω-amino glycoside of GalNAc or Lac to each of the three carboxyl groups of N(ε)-protected N(α)-dicarboxymethyl-L-lysine. In addition, a hexa-valent lactoside was synthesized by attaching N(ε)-deprotected trivalent lactoside to each of the carboxyl group of N(α)-(trifluoroacetamido)hexanoyl L-aspartic acid. Tri-valent GalNAc glycosides and the hexa-valent lactoside had high affinity (dissociation constants approaching nM) for rat hepatocytes. The hexa-valent lactoside, after de-N(ε)-protection, was modified with a chelator, diethylenetriaminepentaacetic acid (DTPA), through which a fluorescent or radioactive tag, such as europium or indium, can be firmly attached. Intravenous infusion of (111)Indium-tagged hexa-valent lactoside to rats and mice resulted in nearly exclusive accumulation of radioactivity in the liver.     

Synthesis of Lewis X and three Lewis X trisaccharide analogues in which glucose and rhamnose replace N-acetylglucosamine and fucose, respectively

Three analogues of the Le(x) trisaccharide: alpha-L-Fucp-(1-->3)-[beta-D-Galp-(1-->4)]-D-GlcNAcp as well as the Le(x) trisaccharide itself were synthesized as methyl glycosides. In the analogues, either only the fucose residue is replaced by rhamnose or both the N-acetylglucosamine and the fucosyl residues are replaced by glucose and rhamnose, respectively. Our synthetic strategy relied on the use of lactoside and 2-azido lactoside derivatives as disaccharide acceptors, which were submitted to either fucosylation or rhamnosylation. Our results confirm that the reactivity of lactose in protection and glycosylation reactions is greatly affected by (1) the structure of the aglycone and (2) the presence of an azido substituent at C-2 of the glucose moiety. Thus, a methyl lactoside acceptor was easily glycosylated at O-3 with perbenzylated beta-thiophenyl fucoside and rhamnoside to give anomerically pure alpha-fucosylated and alpha-rhamnosylated trisaccharides, respectively. In contrast, the same reactions on a 2-azido methyl lactoside acceptor led to the formation of anomeric mixtures. While the alpha- and beta-fucosylated 2-azido trisaccharides could be separated by RP-HPLC, such separation was not possible for the rhamnosylated anomers. The desired rhamnosylated trisaccharide was finally obtained anomerically pure using an isopropylidene-protected rhamnosyl donor. The deprotection sequences also showed that the presence of a 2-azido substituent at C-2 of the glucose residue conferred stability to the vicinal fucosidic linkage at C-3. To test their relative affinity for anti-Le(x) Abs the Le(x) analogues will be used as competitive inhibitors against methyl Le(x). In addition, their conformational behavior will be studied by NMR spectroscopy and molecular modeling experiments.     

Synthesis of propyl and 2-aminoethyl glycosides of alpha-D-galactosyl-(1-->3')-beta-lactoside.

Propyl and 2-aminoethyl alpha-D-galactopyranosyl-(1-->3')-beta-lactosides (1 and 2) were prepared from the corresponding perbenzylated trisaccharide allyl glycoside 6 which, in turn, was obtained by methyl triflate promoted alpha-galactosylation of benzylated allyl lactoside acceptor 4 with thiogalactoside 3. Transformation of the allyl moiety in compound 6 into 2-azidoethyl one was achieved by cleavage of the double bond followed by reduction into alcohol 9, subsequent mesylation, and mesylate-->azide substitution. Alternatively trisaccharide 2 was synthesized using alpha-galactosylation of selectively benzoylated 2-azidoethyl lactoside 19 with 3 as the key step.     

Synthesis of lactoside glycodendrons using photoaddition and reductive amination methodologies

Carbohydrate-based divalent and tetravalent lactoside glycodendrons were constructed in a convergent manner. The dendrons were synthesized beginning with the photoaddition of hepta-O-acetyl-1-thio-beta-lactose, in an anti-Markovnikov manner, to a bis-allyl AB2 trisaccharide to form a divalent dendron. Following two nearly quantitative deprotection steps, the divalent lactoside was coupled to another AB2 trisaccharide by reductive amination to afford a tetravalent dendron. These paucivalent compounds were characterized by NMR spectroscopy and mass spectrometry.     

Azido glycoside primer: a versatile building block for the biocombinatorial synthesis of glycosphingolipid analogues

A lactoside primer, 12-azidododecyl beta-lactoside, was synthesized via the Koenigs-Knorr method by glycosylation of 1,12-dodecyldiol with perbenzoylated lactosyl bromide. The presence of the 2-O-acyl substituent in the donor gave the beta-lactoside, and an excess of acceptor ensured monoglycosylation of the diol. Mesylation of the omega-hydroxyl group in the aglycon, followed by displacement of the mesylate with azide and subsequent O-debenzoylation gave the desired omega-azidododecyl beta-lactoside. The azido glycoside primer was examined in mouse B16 melanoma cells for its feasibility as a building block for oligosaccharide biosynthesis. Uptake of the azido glycoside primer by B16 cells resulted in the sialylation of the galactose residue of the primer to give a glycosylated product having the same glycan as in ganglioside GM3. After 24 h incubation of B16 cells with the primers, the amount of sialylated omega-azidododecyl beta-lactoside primer was 75% of the amount of sialylated n-dodecyl beta-lactoside. However, after 48 h incubation, both primers gave equal amounts of the sialylated products. Interestingly, the remaining azido glycoside primer after 48 h incubation was 5.6-fold greater than that of the alkyl primer, indicating degradation of the alkyl primer to a larger extent than the omega-azido glycoside primer. The facile chemical synthesis and the efficient uptake in cells make the azido glycoside primer a versatile building block for the biocombinatorial synthesis of glycolipid oligosaccharides.     

Allylmalonamide as a bivalent linker: Synthesis of biantennary GM3-saccharide—Keyhole limpet hemocyanin glycoconjugate and the immune response in mice

A biantennary GM3-saccharide (sialyllactoside) derivative (4) was constructed using allylmalonic acid as a bivalent linker, both carboxylic acids of which were condensed with 3-aminopropyl lactoside (2) prior to enzymatic sialylation with a fusion enzyme. While ozonolysis of its allyl group generated a saccharide having a terminal aldehyde (6), we were unable to couple 6 directly to protein by reductive amination. However, extension of the spacer by means of introducing a maleimide group to 6 through its aldehyde group to give 7 enabled the latter to be successfully coupled to thiolated proteins. The average ratios of saccharide to protein were observed to be 35 in KLH conjugate (13) and 9-12 in HSA conjugates (14 and 15). The antisera obtained by immunizing mice with the biantennary sialyllactoside-KLH conjugate (13) together with MPL adjuvant were analyzed by ELISA. Using several structurally related saccharide-HSA conjugates as screening antigens, it was concluded that anti-sialyllactoside antibodies, both IgG and IgM, were effectively raised. This was further supported by competitive inhibition experiments using lactoside (1), sialyllactoside (8) and biantennary sialyllactoside (4) as inhibitors.     

Efficient sialylation on azidododecyl lactosides by using B16 melanoma cells

Lactoside primers (dodecyl lactoside derivatives) resemble intermediates in the biosynthetic pathway of glycolipids and, therefore, act as substrates for cellular enzyme-catalyzed glycosylation. To establish the optimal condition for the bioproduction of a large amount of valuable materials containing GM3-type oligosaccharides, two kinds of lactoside primers having the azido group in different positions were synthesized and introduced into B16 melanoma cells. The saccharide chains of both primers were elongated by cells to give GM3-type oligosaccharide derivatives, which were released to the culture medium. The amount of glycosylated product from newly synthesized 2-azidododecyl beta-lactoside (primer II) was almost twice that from 12-azidododecyl beta-lactoside (primer I). The effects of seeded cell number, primer concentration, and length of incubation time on the glycosylation efficiency were also investigated. The results showed that the higher the seeded cell number, the larger the amount of sialylated products obtained. The optimum concentrations of primers I and II were found to be 200 and 100 microM, respectively. Above these concentrations, productivity and cell viability decreased. As regards the length of incubation time, the sialylated products increased linearly until 48 h, but productivity did not advance thereafter. These results represent the optimal conditions that are necessary for the mass production of GM3-type oligosaccharide using azidododecyl lactoside primers and B16 cells.     

Synthesis of selected aminodeoxy analogues of galabiose and globotriose

Six aminodeoxy 2-(trimethylsilyl)ethyl (Me3SiEt) glycoside analogues of galabiose (4'- and 6'-aminodeoxy) and globotriose (6"-, 4"-, 2"-, and 6'-aminodeoxy) were synthesized by glycosylation of protected Me3SiEt galactoside and lactoside acceptors with azido-substituted monosaccharide donors, followed by reduction of the azido groups and removal of the protecting groups.     

Requirement for a different hydrophobic moiety and reliable chromogenic substrate for endo-type glycosylceramidases.

A series of synthetic lactosides with aglycones that differed in length and structure were used to determine the substrate specificity of endo-type glycosylceramidases. Endoglycoceramidases (EGCase) from bacteria preferred lactosides with an acylamide structure over simple n-alkyl lactosides. While ceramide glycanase (CGase) from leech did not show preference. N -Acylaminoethyl beta-lactosides and n -alkyl lactosides were substrates for both EGCase and CGase, but N-acylaminobutyl beta-lactosides, whose acylamide residue differs from that in ceramide, were not hydrolyzed by EGCases. Thus, EGCases, but not CGase, appear to require an N-acyl group at the same position as that of intact glycosphingolipid for substrate recognition. A p-nitrophenyl lactoside derivative possessing an N-acyl chain was degraded by both EGCases and CGase and this chromogenic substrate may be an alternative substrate for endo-type glycosylceramidase activity. Km of the chromogenic lactoside for CGase and Rhodococcus EGCase were 28 microM and 2.9 mM, respectively.     

Facile synthesis of 1-thio-beta-lactoside clusters scaffolded onto p-methoxyphenyl,beta-D-galactopyranoside,beta-D-glucopyranoside,and lactoside

The free-radical addition of 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-acetyl-1-thio-beta-D-glucopyranose to the allyl ether functions of p-methoxyphenyl per-O-allyl-D-galactopyranoside, D-glucopyranoside, and lactoside provides a concise and effective route for synthesis of glycoside clusters, of use for exploring anti-metastatic activity.     

Aryl O- and S-galactosides and lactosides as specific inhibitors of human galectins-1 and -3: role of electrostatic potential at O-3

Phase transfer catalyzed reaction was used for the high yielding synthesis of aryl 1-thio-beta-d-galacto- and lacto-pyranosides carrying a panel of substituents on the phenyl groups. Best galectin-1 inhibitors were simple p-nitrophenyl thiogalactoside 5a for the monosaccharide and o-nitrophenyl thiolactoside 6f or napthylsulfonyl lactoside 8c, both being 20 times better relative to natural ligands. Relative inhibitory properties as low as 2500 and 40 microM were observed, respectively. The electronic effects of the lactoside aglycons directly influenced the electrostatic potential at O-3, which was associated with the inhibitory potencies against galectin-1.     

The Chemical Structure of Glycolipids of Bovine Milk

Experiments were executed to elucidate the chemical structure of ceramide monohexoside (CMH) and ceramide dihexoside (CDH) isolated from cow’s milk, especially with regard to the nature of the sugar moiety of the molecules. The results have shown that the structure of CMH and CDH in bovine milk is β-glucosyl-(l→l)-N-acyl-sphingosine, namely ceramide glucoside, and β-galactosyl-(1→4)-β-glucosyl-(1→1)-N-acyl-sphingosine, namely ceramide lactoside, respectively.     

Synthesis of globo- and isoglobotriosides bearing a cinnamoylphenyl tag as novel electrophilic thiol-specific carbohydrate reagents

The galactosyl donor, 4,6-di-O-acetyl-2,3-di-O-benzyl-D-galactopyranosyl trichloroacetimidate, was efficiently coupled with regioselectively benzylated lactoside acceptors under standard conditions to stereoselectively afford the corresponding globotrioside and isoglobotrioside derivatives in very good yields. These glycosides were smoothly functionalized with a 6-(p-cinnamoylphenoxy)-hexyl tether tag as novel electrophilic thiol-specific carbohydrate reagents. Immobilization of the globotrioside conjugate to Thiopropyl Sepharose 6B for purification of B-subunit of Shiga toxin (StxB) and coupling of a model cysteine-containing protein (StxB-Z(n)-Cys) to the isoglobotrioside conjugate were both performed with high efficiency.     

Immobilization of fluorous oligosaccharide recognized by influenza virus on polytetrafluoroethylene filter

The lactoside with PEG-fluorous tag was introduced to BHK-21(C-13) cells to generate a GM3-type oligosaccharide (Siaα2-3Galβ1-4Glc). The GM3-type oligosaccharide obtained was easily immobilized by spotting onto commercially available polytetrafluoroethylene (PTFE) filter through non-covalent fluorous affinity and simply assessed by dot blot method using the interaction of carbohydrate- with proteins which recognize sialic acid such as virus membrane proteins.     

Synthesis of O-galactosyl aldoximes as potent LacNAc-mimetic galectin-3 inhibitors

A panel of anomeric oxime ether derivatives of beta-galactose were synthesized via the reaction of O-beta-D-galactopyranosylhydroxylamine with aldehydes. The oxime ethers were evaluated as inhibitors against galectin-3 in a competitive fluorescence polarization assay. The best inhibitor, [E]-O-(beta-D-galactopyranosyl)-indole-3-carbaldoxime (E-52), had a Kd value of 180 microM, which is 24 times better than methyl beta-D-galactopyranoside (Kd=4400 microM) and in the same range as methyl lactoside (Kd=220 microM).     

Enzymic hydrolysis of sphingolipids: Hydrolysis of ceramide lactoside by an enzyme from rat brain

Ceramide lactoside [1-O-(galactosido-4-β-glucosido)-2-N-acyl-sphingosine] was hydrolysed to ceramide glucoside and galactose by β-galactosidase of rat brain. The reaction was not reversible, required cholate or taurocholate, had optimum pH5·0 and K_m 2·2×10⁻⁵m. It was inhibited by γ-galactonolactone and galactose as well as by ceramide, sphingosine and fatty acid. Ceramide lactoside could be degraded to ceramide, galactose and glucose by mixtures of rat-brain β-galactosidase and ox-brain β-glucosidase.     

Synthesis and galectin-binding activities of mercaptododecyl glycosides containing a terminal β-galactosyl group

Mercaptododecyl glycosides containing a terminal β-galactosyl group were prepared from d-galactose or from d-lactose via hexa-O-acetyl-lactal (10) as a key intermediate. Interactions of these glycolipids (5 kinds) and galectins (β-galactoside binding lectins, 6 species) were evaluated by surface plasmon resonance (SPR) method. High binding responses were observed for the lactoside, 2-deoxy-lactoside, and lactosaminide with some galectins (Gal-3, -4, -8), whereas the galactoside and 2,3-dideoxy-lactoside showed low binding activities.     

Structure-activity relationships of synthetic diosgenyl diglycosides.

The haemolytic and antifungal activities of six synthetic diosgenyl diglycosides and diosgenyl maltotrioside were compared with each other and with those of the parent glucoside. In general, the haemolytic activity of each of these glycosides was higher than, and the antifungal activity as strong as, that of the glucoside. However, both activities of the lactoside were much lower than those of the others.     

'Click chemistry' on polysaccharides: a convenient, general, and monitorable approach to develop (1-->3)-beta-D-glucans with various functional appendages

(1-->3)-beta-D-Glucans having various functional appendages (lactoside, ferrocene, pyrene, and porphyrin) can be prepared in an convenient, quantitative, and regioselective manner through regioselective bromination-azidation of curdlan to afford 6-azido-6-deoxycurdlan followed by chemoselective [3+2]-cycloadditions with various functional modules bearing a terminal alkyne group. The ability to monitor reaction conversions is an additional advantage of this synthetic approach over the conventional direct modifications on polysaccharides; the reaction can be readily monitored based on the intensity of azido peaks in the in situ attenuated total reflection infrared spectra.     

Active-latent glycosylation strategy toward Lewis X pentasaccharide in a form suitable for neoglycoconjugate syntheses.

Glycosylation of 4-nitrophenyl 2-acetamido-6-O-tert-butyldiphenylsilyl-2-deoxy-1-thio-beta-D-gluc opyranoside with phenyl 2,3,4,6-tetra-O-benzoyl-1-thio-beta-D-galactopyranoside in the presence of NIS and TfOH as catalyst gave the lactosamine derivative regiospecifically in high yield. Further 3-O-fucosylation with phenyl 2,3,4-tri-O-benzyl-1-thio-beta-L-fucopyranoside using DMTST as promoter afforded the Lex trisaccharide intermediate. The latent glycosyl donor was transformed into its active form (p-acetamidothiophenyl) by reduction with zinc in acetic acid and N-acetylation. Glycosidation with p-nitrothiophenyl lactoside acceptor in the presence of NIS/TfOH as catalyst gave the Lex pentasaccharide in 71% yield.