Activity of UDP-GlcNAc:alpha-mannoside beta(1,6)N-acetylglucosaminyltransferase (GnT V) in cultured cells using a synthetic trisaccharide acceptor

N-acetylglucosaminyltransferase V activity has been measured under saturating conditions in the extracts of seven cultured cell lines using as substrates, UDP-[3H]-GlcNAc and a synthetic 8-methoxylcarbonyloctyl trisaccharide. The unreacted sugar-nucleotide and its breakdown products were separated from the radiolabeled tetrasaccharide product by reverse-phase chromatography. Enzyme activity was present in six of the cell lines, which were derived originally from either human, mouse, or hamster tissues, with the highest activity in mouse lymphoma BW5147 cells. The PHAR 2.1 variant cell line, derived from the BW5147 line, expressed no detectable activity.     

Synthesis of alpha-D-Manp-(1----3)-[beta-D-GlcpNAc-(1----4)]-[alpha-D-Manp+ ++-(1----6)] - beta-D-Manp-(1----4)-beta-D-GlcpNAc-(1----4)-[alpha-L-Fucp- (1----6)]-D- GlcpNAc, a core glycoheptaose of a "bisected" complex-type glycan of glycoproteins

A synthesis of alpha-D-Manp-(1----3)-[beta-D-GlcpNAc-(1----4)]-[alpha-D-Manp++ +-(1----6)]- beta-D-Manp-(1----4)-beta-D-GlcpNAc-(1----4)-[alpha-L-Fucp-( 1----6)]-D- GlcpNAc was achieved by employing benzyl O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1--- -4)-O- (2-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-(3,6-di-O-benzyl-2-deoxy-2 - phthalimido-beta-D-glucopyranosyl)-(1----4)-3-O-benzyl-2-deoxy-6-O-p- methoxyphenyl-2-phthalimido-beta-D-glucopyranoside as a key glycosyl acceptor. Highly stereoselective mannosylation was performed by taking advantage of the 2-O-acetyl group in the mannosyl donors. The alpha-L-fucopyranosyl residue was also stereoselectively introduced by copper(II)-mediated activation of methyl 2,3,4-tri-O-benzyl-1-thio-beta-L-fucopyranoside.     

2-Bromoethyl glycosides in glycoside synthesis: preparation of glycoproteins containing alpha-L-Fuc-(1----2)-D-Gal and beta-D-Gal-(1----4)-D-GlcNAc

The applicability of 2-bromoethyl glycosides in carbohydrate synthesis is demonstrated by the synthesis of glycosides of alpha-L-Fuc-(1----2)-D-Gal and beta-D-Gal-(1----4)-D-GlcNAc. The bromoethyl aglycon was transformed into the methoxycarbonylethylthioethyl spacer, which allowed coupling of the sugars to proteins (BSA and KLH).     

A practical synthesis of alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-[alpha-D-Glcp-(1-->3)]-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp, an O-specific heterohexasaccharide fragment of Citrobacter braakii O7a, 3b, 1c

An O-specific heterohexasaccharide fragment of Citrobacter braakii O7a, 3b, 1c, alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-[alpha-D-Glcp-(1-->3)]-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp was synthesized as its methyl glycoside. Acetylation of allyl 4,6-O-benzylidene-alpha-D-mannopyranoside, followed by debenzylidenization and benzoylation gave allyl 2,3-di-O-acetyl-4,6-di-O-benzoyl-alpha-D-mannopyranoside (3), and subsequent deacetylation of 3 with CH(3)COCl-MeOH gave the monosaccharide acceptor 4. Condensation of isopropyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-glucopyranoside (6) with 4 selectively afforded the alpha-(1-->3)-linked disaccharide 7. Condensation of 7 with the (1-->3)-linked disaccharide donor 9, followed by deallylation and trichloroacetimidation, afforded the tetrasaccharide donor 12. Coupling of 12 with disaccharide acceptor 13, followed by debenzylation and deacylation, furnished the target heterohexasaccharide 16.     

Synthesis of four novel trisaccharides by induction of loose acceptor specificity in Gal beta 1----4 transferase (EC 2.4.1.22): Galp(beta 1----4)Glcp(X)Glc where X = beta 1----3: beta 1----4: beta 1----6: alpha 1----4.

Development of tandem mass spectral methods for direct linkage determination in oligosaccharides requires sets of trisaccharides differing only in one structural parameter. In this case, we chose the position of linkage to the reducing-end hexose. These sets of compounds would also be useful for the development of high-resolution separation techniques geared to resolve linkage types. Conventional organic synthesis of such a set could take as long as 2-5 months for each member of the set. Each trisaccharide would require 10-20 steps of synthesis. Instead, we utilized low pH to induce a loose acceptor specificity for bovine milk galactosyltransferase (lactose synthase: EC 2.4.1.22) and by this method, within 2 weeks, generated four novel oligosaccharides for NMR and mass spectral studies. The disaccharides cellobiose (beta 1----4), laminaribiose (beta 1----3), gentiobiose (beta 1----6) and maltose (alpha 1----4) acted as acceptors for EC 2.4.1.22 under these conditions. The beta 1----2-linked disaccharide, sophorose, was not commercially available and is not included in this study. The alpha-linked disaccharides were also examined, but except for the alpha 1----4 disaccharide maltose, were very poor acceptors under a variety of conditions. From these four acceptors, the following four novel trisaccharides were synthesized in micromole amounts, suitable for studies of linkage position using low-energy collision-induced-dissociation tandem mass spectrometry (FAB-MS-CID-MS), and for NMR: Galp(beta 1----4)Glcp(beta 1----3)-Glc, Galp(beta 1----4)Glcp(beta 1----4)Glc, Galp(beta 1----4)Glcp(beta 1----6)-Glc and Galp(beta 1----4)Glcp(alpha 1----4)Glc.     

Synthesis of monodeoxy analogues of the trisaccharide alpha-D-Glcp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-ManpOMe recognised by Calreticulin/Calnexin

Six (3,4,4′,6′,3″ or 6″)-monodeoxy analogues of the title trisaccharide (1-6) have been prepared utilising monodeoxy monosaccharide precursors. The reducing end deoxy derivatives were synthesised by N-iodosuccinimide/silver trifluoromethanesulfonate (NIS/AgOTf)-promoted couplings of a common disaccharide thioglycoside donor 10 to suitably protected monodeoxy acceptors 9 and 12, affording trisaccharides, which after deprotection yielded target structures 1 and 2. The non-reducing end deoxy derivatives could similarly be produced by halide-assisted glycosylations of a common disaccharide acceptor 17 with monodeoxy glycosyl bromide donors (obtained from thioglycosides 18 and 20) to yield, after removal of protecting groups, target trisaccharides 3 and 4. The analogues with the deoxy function in the middle mannose residue, were obtained through orthogonal halide-assisted coupling of tetrabenzyl-glucopyranosyl bromide to monodeoxy thioglycoside acceptors to give thioglycoside disaccharides, which subsequently were used as donors in NIS/AgOTf-promoted couplings to a common 2-hydroxy-mannose acceptor 15 to afford trisaccharides; deprotection yielded the final target compounds 5 and 6.     

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 a mucin-type O-glycosylated amino acid, beta-Gal-(1----3)-[alpha-Neu5Ac-(2----6)]-alpha-GalNAc-(1----3)- Ser

Total synthesis of O-beta-D-galactopyranosyl-(1----3)-O-[(5-acetamido-3,5-dideoxy- D-glycero-alpha-D-galacto-2-nonulopyranosylonic acid)-(2----6)]-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-(1----3 )-L- serine was achieved by use of the key glycosyl donor O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1----3)-O- [methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galact o-2- nonulopyranosyl)onate-(2----6)]-4-O-acetyl-2-azido-2-deoxy-a lpha-D- galactopyranosyl trichloroacetimidate and the key glycosyl acceptor N-(benzyloxycarbonyl)-L- serine benzyl ester in a regiocontrolled way.     

Convergent synthesis of a trisaccharide as its 2-(trimethylsilyl)ethyl glycoside related to the flavonoid triglycoside from Gymnema sylvestre

The glycone part of the flavonoid triglycoside, kaempferol 3-O-beta-D-glucopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-galactopyranoside, has been synthesized in good yield and stereoselectivity using N-iodosuccinimide and HClO4-silica promoted glycosylations of thioglycoside donors.     

Synthesis of oligosaccharide fragments of O-specific Shigella flexneri polysaccharides. II. Synthesis of trisaccharide Glc alpha1----3RHa alpha1----2Rha alpha1----OMe and tetrasaccharide GlcNAc beta1----2(Llc alpha1----3)Rha alpha1----2Rh alpha1----OMe

Methyl glycosides of the title linear trisaccharide and branched tetrasaccharide were synthesized by stepwise glycosylation. These oligosaccharides represent the fragments of O-antigenic polysaccharides of Shigella flexneri serotypes 2b, 3a, 5b, and X.     

Purification and characterization of a novel broad-specificity (alpha 1----2, alpha 1----3 and alpha 1----6) mannosidase from rat liver.

We have identified a mannosidase in rat liver that releases alpha 1----2, alpha 1----3 and alpha 1----6 linked manose residues from oligosaccharide substrates, MannGlcNAc where n = 4-9. The end product of the reaction is Man alpha 1----3[Man alpha 1----6]Man beta 1----4GlcNAc. The mannosidase has been purified to homogeneity from a rat liver microsomal fraction, after solubilization into the aqueous phase of Triton X-114, by anion-exchange, hydrophobic and hydroxyapatite chromatography followed by chromatofocusing. The purified enzyme is a dimer of a 110-kDa subunit, has a pH optimum between 6.1 and 6.5 and a Km of 65 microM and 110 microM for the Man5GlcNAc-oligosaccharide or Man9GlcNAc-oligosaccharide substrates, respectively. Enzyme activity is inhibited by EDTA, by Zn2+ and Cu2+, and to lesser extent by Fe2+ and is stabilized by Co2+. The pattern of release of mannose residues from a Man6GlcNAc substrate shows an ordered hydrolysis of the alpha 1----2 linked residue followed by hydrolysis of alpha 1----3 and alpha 1----6 linked residues. The purified enzyme shows no activity against p-nitrophenyl-alpha-mannoside nor the hybrid GlcNAc Man5GlcNAc oligosaccharide. The enzyme activity is inhibited by swainsonine and 1-deoxymannojirimycin at concentrations 50-500-fold higher than required for complete inhibition of Golgi-mannosidase II and mannosidase I, respectively. The data indicate strongly that the enzyme has novel activity and is distinct from previously described mannosidases.     

Use of N-acetyl-2'-O-methyllactosamine as a specific acceptor for the determination of alpha-L-(1----3)-fucosyltransferase in human serum

A synthetic substrate, N-acetyl-2'-O-methyllactosamine, was employed as a specific acceptor for alpha-L-(1----3)-fucosyltransferase from human serum. The fucosyl linkage of the product from this substrate was characterized by hydrolysis with a specific alpha-L-(1----3)/(1----4)-fucosidase. Using this acceptor, the pH optimum for the serum alpha-L-(1----3)-fucosyltransferase was 6.5. The enzyme was activated by Mn2+ or Mg2+ ions and was inhibited by EDTA. The apparent Km for this enzyme using N-acetyl-2'-O-methyllactosamine was 20.4 mM and Vmax was 5.6 pmol/h/ml serum.     

Synthesis of a fully protected derivative of O-(N-acetyl-alpha-D-neuraminyl)-(2----3)-O-beta-D-galactopyranosyl-(1- ---3)-O- [(N-acetyl-alpha-D-neuraminyl)-(2----6)]-O-(2-acetamido-2-deoxy-alpha- D-galactopyranosyl)-(1----3)-L-serine

N-(Benzyloxycarbonyl)-O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galact o-2- nonulopyranosyl)onate]-(2----3)-O-(2,4,6-tri-O-acetyl-beta-D - galactopyranosyl)-(1----3)-O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galact o-2- nonulopyranosyl)onate-(2----6)]-O-(2-acetamido-4-O-acetyl-2- deoxy-alpha-D- galactopyranosyl)-(1----3)-L-serine benzyl ester was synthesized by using O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5- di-deoxy-D-glycero-alpha-D-galacto-2-nonulopyranosyl)onate]- (2----3)-O-(2,4,6- tri-O-acetyl-beta-D-galactopyranosyl)-(1----3)-O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galact o-2- nonulopyranosyl)onate-(2----6)]-4-O-acetyl-2-azido-2-deoxy-a lpha- and -beta-D-galactopyranosyl trichloroacetimidate as a key glycotetraosyl donor which, upon reaction with N-(benzyloxycarbonyl)-L-serine benzyl ester, afforded a 44% yield of a mixture of the alpha- and beta-glycosides in the ratio of 2:5.     

Synthesis of spacer-arm, lipid, and ethyl glycosides of the trisaccharide portion [alpha-D-Gal-(1----4)-beta-D-Gal-(1----4)-beta-D-Glc] of the blood-group Pk antigen: preparation of neoglycoproteins

The title compounds were prepared via the acetylated 2-bromoethyl glycoside 11 of alpha-D-Gal-(1----4)-beta-D-Gal-(1----4)-beta-D-Glc by displacement of bromide ion with methyl 3- mercaptopropionate , octadecanethiol , and hydrogen, respectively. Silver triflate -promoted glycosylation of 2-bromoethyl 2,3,6-tri-O-benzyl-beta-D-glucopyranoside with 2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl) -alpha -D-galactopyranosyl bromide gave 11. A tetradeuterated analogue of 11 was prepared by essentially the same route. The spacer-arm glycoside formed from methyl 3- mercaptopropionate was coupled to bovine serum albumin and keyhole limpet haemocyanin.     

A bisubstrate analog inhibitor for alpha(1----2)-fucosyltransferase

Porcine submaxillary beta-galactoside alpha(1----2)-fucosyltransferase is known to transfer a fucosyl residue from guanosine 5'-diphosphofucose (GDP-fucose) to the 2-OH group of beta-D-galactopyranosides with inversion of configuration at the fucopyranosyl anomeric carbon. A bisubstrate analog (1) of the postulated transition-state for this reaction, which has O-2 of phenyl beta-D-galactopyranoside attached to the terminal phosphorous of GDP through a flexible ethylene bridge, has been chemically synthesized and evaluated as an inhibitor of this enzyme. Compound 1 was found to be a competitive inhibitor with respect to both GDP-fucose and phenyl beta-D-galactopyranoside for both the membrane-bound and soluble forms of the fucosyltransferase. It was also a competitive inhibitor with respect to the alternate acceptor beta DGal(1----3)beta DGlcNAcO(CH2)8-COOMe. The Ki values were in the range 2.3-16 microM. Compound 1 is the first example of a bisubstrate analog inhibitor for a glycosyltransferase which binds to both the acceptor and donor recognition sites of the enzyme. The potential of a bisubstrate analog strategy for the production of specific glycosyltransferase inhibitors is discussed.     

A regio- and stereo-controlled synthesis of beta-D-Glcp NAc6SO3-(1----3)-beta-D-Galp6SO3-(1----4)-beta-D-GlcpNAc 6SO3- (1----3)-D-Galp, a linear acidic glycan fragment of keratan sulfate I

A stereocontrolled synthesis of beta-D-GlcpNAc6SO3-(1----3)-beta-D-Galp6SO3-(1----4)-beta-D- GlcpNAc6SO3- (1----3)-D-Galp, was achieved by use of benzyl O-(2-acetamido-3,4 di-O-benzyl-2-deoxy-6-O-p-methoxyphenyl-beta-D- glucopyranosyl)-(1----3)-O-(2,4-di-O-tert-butyldiphenylsilyl-beta- D- galactopyranosyl-(1----4)-O-(2-acetamido-3-O-benzyl-2-deoxy-6-O-p-methox yphenyl - beta-D-glucopyranosyl)-(1----3)-2,4,6-tri-O-benzyl-beta-D-galactopyranos ide as a key intermediate, which was in turn prepared by employing two glycosyl donors, 3,4-di-O-benzyl-2-deoxy-6-O-p-methoxyphenyl-2-phthalimido-beta-D- glucopyranosyl trichloroacetimidate and O-(3,6-di-O-acetyl-2,4-di-O-benzyl-beta-D-galactopyranosyl)-(1----4)-3-O - benzyl-2-deoxy-6-O-p-methoxyphenyl-2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate, and a glycosyl acceptor, benzyl 2,4,6-tri-O-benzyl-beta-D-galactopyranoside.     

Synthesis from pullulan of spacer-arm, lipid, and ethyl glycosides of a tetrasaccharide [alpha-D-Glc-(1----6)-alpha-D-Glc-(1----4)-alpha-D-Glc-(1----4)-D-Glc] found in human urine; preparation of neoglycoproteins

Enzymic hydrolysis of pullulan, followed by acetylation and chromatography, gave acetylated alpha-D-Glcp-(1----6)-alpha-D-Glcp-(1----4)-alpha-D-Glcp-(1----4)-D-Glcp which, with 2-bromoethanol and boron trifluoride etherate in dichloromethane, gave the 2-bromoethyl glycoside. The reactions of the glycoside with methyl 3- mercaptopropionate , methyl 11- mercaptoundecanoate , and octadecanethiol are described, and also its hydrogenolysis to give an ethyl glycoside. The mercaptopropionate -derived, spacer-arm glycoside has been coupled to bovine serum albumin and keyhole limpet haemocyanin.     

6-keto-prostaglandin E1 is not equipotent to prostacyclin (PGI2) as an antiaggregatory agent

A direct comparison of the relative potencies of the two antiaggregatory prostaglandins PGI2 and 6-keto-PGE1 showed PGI2 was at least 20 times more potent than 6-keto-PGE1 when tested against ADP-induced human platelet aggregation. This marked difference in potency was even more evident when the ability of PGI2 and 6-keto-PGI2 to stimulate platelet cyclic AMP levels was determined. When cyclic AMP levels were measured direct comparisons were difficult because the respective dose response curves were not parallel, but 10 ng of PGI2 was equivalent to 300 ng of 6-keto-PGE1. PGI2 was also more potent (10-20 times) than 6-keto-PGE1 as a disaggregatory agent, and the disaggregatory activity of both prostaglandins was enhanced by the phosphodiesterase inhibitor 1-methyl-3-isobutylmethylxanthine. PGI2 was also more active than 6-keto-PGE1 as an inhibitor of thrombus formation in dog coronary arteries in vivo. In vivo, 6-keto-PGE1 was at least 10 times less potent thatn PGI2, the exact difference could not be determined because 6-keto-PGE1 caused significant falls in blood pressure before anti-platelet activity could be detected. PGI2 is an intrinsically more potent anti-aggregatory molecule than 6-keto-PGE1, but these data do not rule out the possibility that some of the activities attributed to PGI2 could be the result of the conversin of PGI2 and/or 6-keto-PGF1 alpha to 6-keto-PGE1.