A highly convergent and effective synthesis of the phytoalexin elicitor hexasaccharide.

The peracetylated hexasaccharide 1,2,4-tri-O-acetyl-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-6- O- (2,3,4-tri-O-acetyl-6-O-(2,4-di-O-acetyl-3,6-di-O-(2,3,4,6-tetra-O-acety l- beta-D-glucopyranosyl)-beta-D-glucopyranosyl)-beta-D-glucopyranosyl)-alp ha, beta-D-glucopyranose 21 was synthesized in a blockwise manner, employing trisaccharide trichloroacetimidate 2,4-di-O-acetyl-3,6-di-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)- alpha-D-glucopyranosyl trichloroacetimidate 17 as the glycosyl donor, and trisaccharide 4-O-acetyl-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-6-O-(2,3,4 -tri -O-acetyl-beta-D-glucopyranosyl)-1,2-O-(R,S)ethylidene-alpha-D-glucopyra nose 18 as the acceptor. The donor 17 and acceptor 18 were readily prepared from trisaccharides 3-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-6-O-(2,3,4-tri-O-acet yl- 6-O-chloroacetyl-beta-D-glucopyranosyl)-1,2-O-(R,S)ethylidene-alpha-D- glucopyranose 10 and 3,6-di-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-1,2-O-(R,S) ethylidene-alpha-D-glucopyranose 11, respectively, which were obtained from rearrangement of orthoesters 3,4-di-O-acetyl-6-O-chloroacetyl-alpha-D-glucopyranose 1,2-(3-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-1,2-O-(R,S) ethylidene-alpha-D-glucopyranosid-6-yl orthoacetate) 8 and 3,4,6-tri-O-acetyl-alpha-D-glucopyranose 1,2-(3-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-1,2-O-(R,S) ethylidene-alpha-D-glucopyranosid-6-yl orthoacetate) 9, respectively. The orthoesters were prepared from selective coupling of the disaccharide 3-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-1,2-O-(R,S) ethylidene-alpha-D-glucopyranose 4 with 'acetobromoglucose' (tetra-O-acetyl-alpha-D-glucopyranosyl bromide) and 6-O-chloroacetylated 'acetobromoglucose', respectively. To confirm the selectivity of the orthoester formation and rearrangement, the disaccharide 4-O-acetyl-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl)-1,2-O-(R,S ) ethylidene-alpha-D-glucopyranose 7 was prepared from 4 by selective tritylation, acetylation and detritylation. The title compound, an elicitor-active D-glucohexaose 3-O-(beta-D-glucopyranosyl)-6-O-(6-O-(3,6-di-O-(beta-D-glucopyranosyl)-b eta -D-glucopyranosyl)-beta-D-glucopyranosyl)-alpha,beta-D-glucopyranose 1, was finally obtained by Zemplén deacetylation of 21 in quantitative yield.     

Synthesis of neoglycoproteins containing D-glycero-D-talo-oct-2-ulopyranosylonic acid (Ko) ligands corresponding to core units from Burkholderia and Acinetobacter lipopolysaccharide

Glycal esters of Kdo derivatives were converted into 2,3-anhydro intermediates, which were transformed into D-glycero-D-talo-oct-2-ulopyranosylonic acid (Ko), as well as 3-O- and 4-O-p-nitrobenzoyl-Ko derivatives. The exo-allyl orthoester derivative, methyl [5,7,8-tri-O-acetyl-4-O-(4-nitrobenzoyl)-2,3-O-[(1-exo-allyloxy)-ethylidene]-D-glycero-beta-D-talo-oct-2-ulopyranos]onate, prepared from the 4-O-pNBz-protected Ko derivative, was elaborated into the alpha-Ko allyl ketoside, the reducing disaccharide alpha-Kdop-(2-->4)-Ko and the disaccharide alpha-Kdop-(2-->4)-Kop-(2-->OAll). Conversely, methyl[4,5,7,8-tetra-O-acetyl-3-O-(4-nitrobenzoyl)-alpha-D-glycero-D-talo-2-octulopyranosyl bromide]onate [Carbohydr. Res., 244 (1993) 69-84], was coupled with a Kdo acceptor to give the disaccharide alpha-Kop-(2-->4)-Kdop-(2-->OAll) after orthoester rearrangement and deprotection. The allyl glycosides were treated with cysteamine and converted into neoglycoproteins. The ligands correspond to inner core units from Acinetobacter haemolyticus and Burkholderia cepacia lipopolysaccharides.     

The synthesis of mono- and oligosaccharide 1,2-orthoesters by way of a glycosyl nitrate intermediate

Sugar orthoesters with complex alcohols were obtained in high yield in the reaction of acylated 1,2-cis-glycosyl halides with partially protected sugar derivatives in the presence of silver nitrate and 2,4,6-trimethylpyridine in dry acetonitrile. The reaction has been shown to proceed by way of the acylated 1,2-trans-glycosyl nitrate intermediate.     

Remote control of alpha- or beta-stereoselectivity in (1-->3)-glucosylations in the presence of a C-2 ester capable of neighboring-group participation

In (1-->3)-glucosylation the glycosyl bond originally present in either donor or acceptor is shown to control the stereoselectivity of the forthcoming bond, i.e., the newly formed glycosidic linkage has the opposite anomeric configuration of that of either the donor or acceptor. Therefore, with alpha-(1-->3)-linked disaccharides with nonreducing ends that have the 3-OH free as the acceptor and an acetylated glucosyl trichloroacetimidate as the donor, or with an alpha-(1-->3)-linked acetylated disaccharide trichloroacetimidate as the donor and a glucoside with 3-OH free as the acceptor, beta-linked trisaccharides were obtained. Meanwhile, with beta-(1-->3)-linked disaccharides that have nonreducing ends with the 3-OH free as the acceptor and an acetylated glucosyl trichloroacetimidate as the donor, or with a beta-(1-->3)-linked acetylated disaccharide trichloroacetimidate as the donor and a glucoside with the 3-OH free as the acceptor, alpha-linked trisaccharides were obtained in spite of the C-2 neighboring group participation.     

Use of a phenyl 1-selenogalactofuranoside as a glycosyl donor for the synthesis of galactofuranosyl-containing disaccharides.

The use of acetylated phenyl 1-seleno-beta-D-galactofuranoside as a glycosyl donor for the synthesis of protected D-Galf-beta-(1-->3)-alpha-D-Manp as its methyl or ethylthio glycoside has been demonstrated. Activation of the selenoglycoside over a thioglycoside acceptor by NIS/TfOH is extremely selective and gives the ethylthio disaccharide in 91% yield. The parent disaccharide is found as a terminal and branched unit in the lipopeptidophosphoglycan oligosaccharides of the protozoan Trypanosoma cruzi, the causative agent of Chagas' disease.     

Natural and synthetic cage compounds incorporating the 2,9,10-trioxatricyclo[4.3.1.03,8]decane type moiety

The 2,9,10-trioxatricyclo[4.3.1.0(3,8)]decane moiety is a tetracyclic cage-like orthoester incorporated in the structure of a series of daphnane derivatives such as resiniferatoxin (RTX), kirkinine, synaptolepis factors, huratoxin etc., exhibiting various biological activities. The approaches to the preparation of cage-like orthoesters starting from partially acylated or unmodified 1,2,4-trihydroxycyclohexane moieties built onto natural as well as synthetic compounds are discussed. Orthoester derivatives of RTX analogs, Ceverathrum alkaloids, myoinositol and pyranoses are included. Stereochemical requirements to the formation of the orthoester unit are discussed. The biological activity of different compounds containing the cage-like orthoester structural fragment is given. The literature is reviewed till 2004.     

Synthesis of a heptasaccharide fragment of the O-deacetylated GXM of C. neoformans serotype C

Beta-D-Xylp-(1-->2)-alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->2)][beta-D-Xylp-(1-->4)]-alpha-D-Manp-(1-->3)-[beta-D-Xylp-(1-->4)]-alpha-D-Manp, the fragment of the exopolysaccharide from Cryptococcus neoformans serovar C, was synthesized as its methyl glycoside. Thus, chloroacetylation of allyl 3-O-acetyl-4,6-O-benzylidene-alpha-D-mannopyranoside (1) followed by debenzylidenation and selective 6-O-benzoylation afforded allyl 2-O-chloroacetyl-3-O-acetyl-6-O-benzoyl-alpha-D-mannopyranoside (4). Glycosylation of 4 with 2,3,4-tri-O-benzoyl-D-xylopyranosyl trichloroacetimidate (5) furnished the beta-(1-->4)-linked disaccharide 6. Dechloroacetylation gave the disaccharide acceptor 7 and subsequent coupling with 5 produced the trisaccharide 8. Deacetylation of 8 gave the trisaccharide acceptor 9 and subsequent coupling with a disaccharide 10 produced the pentasaccharide 11. Reiteration of deallylation and trichloroacetimidate formation from 11 yielded the pentasaccharide donor 12. Coupling of a disaccharide acceptor 13 with 12 afforded the heptasaccharide 14. Subsequent deprotection gave the heptaoside 16, while selective 2-O-deacetylation of 14 gave the heptasaccharide acceptor 15. Condensation of 15 with glucopyranosyluronate imidate 17 did not yield the expected octaoside, instead, an orthoester product 18 was obtained. Rearrangement of 18 did not give the target octaoside; but produced 15. Meanwhile, there was no reaction between 15 and the glycosyl bromide donor 19.     

Synthesis of N-glycan oxazolines: donors for endohexosaminidase catalysed glycosylation

Oxazoline mono-, di-, tri- and hexasaccharides, corresponding to the core components of N-linked glycoprotein high mannose glycans, are synthesised as potential glycosyl donors for endohexosaminidase catalysed glycosylation of glycopeptides and glycoprotein remodelling. The crucial beta-D-Manp-(1-->4)-D-GlcpNAc linkage is synthesised via epimerisation of gluco disaccharide substrates by sequential triflation and nucleophilic substitution. Oxazolines are formed directly from the anomeric OPMP protected N-acetyl glucosamine derivatives. Efficient endohexosaminidase catalysed glycosylation of a synthetic beta-D-GlcpNAcAsn glycosyl amino acid is demonstrated with the trisaccharide oxazoline donor.     

A facile and effective synthesis of alpha-(1-->6)-linked mannose di-, tri-, tetra-, hexa-, octa-, and dodecasaccharides, and beta-(1-->6)-linked glucose di-, tri-, tetra-, hexa-, and octasaccharides using sugar trichloroacetimidates as the donors and unprotected or partially protected glycosides as the acceptors

Reaction of 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl trichloroimidate with allyl alpha-D-mannopyranoside in the presence of TMSOTf selectively gave allyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-alpha-D-mannopyranoside through an orthoester intermediate. Benzoylation of 3, followed by deallylation, and then trichloroimidation afforded the disaccharide donor 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroimidate, while benzoylation of 3 followed by selective removal of acetyl groups yielded the disaccharide acceptor allyl alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranoside. Coupling of 5 with 6 gave the tetrasaccharide allyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranoside, which were converted into the tetrasaccharide donor 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroimdate and the tetrasaccharide acceptor allyl alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranoside, respectively, by the same strategies as used for conversion of 3 into 5 and 6. Condensation of 5 with 13 gave the hexasaccharide 14, while condensation of 12 with 13 gave the octasaccharide 17. Dodecasaccharide 21 was obtained by the coupling of 12 with the octasaccharide acceptor 20. Similar strategies were used for the syntheses of beta-(1-->6)-linked glucose di-, tri-, tetra-, hexa-, and octamers. Deprotection of the oligosaccharides in ammonia-saturated methanol yielded the free alpha-(1-->6)-linked mannosyl and beta-(1-->6)-linked glucosyl oligomers.     

Synthesis of a heparan sulfate mimetic disaccharide with a conformationally locked residue from a common intermediate

A simple mimetic of a heparan sulfate disaccharide sequence that binds to the growth factors FGF-1 and FGF-2 was synthesized by coupling a 2-azido-2-deoxy-d-glucopyranosyl trichloroacetimidate donor with a 1,6-anhydro-2-azido-2-deoxy-β-d-glucopyranose acceptor. Both the donor and acceptor were obtained from a common intermediate readily obtained from d-glucal. Molecular docking calculations showed that the predicted locations of the disaccharide sulfo groups in the binding site of FGF-1 and FGF-2 are similar to the positions observed for co-crystallized heparin-derived oligosaccharides obtained from published crystal structures.     

Studies on the origin of stereoselectivity in the synthesis of 1,2-trans glycofuranosyl azides

The stereoselectivity of the 1,2-trans directed, Lewis acid-catalysed azidation of peracylated furanoses was found to depend on the reactivity of the azide donor (azide nucleophilicity) and the configuration at the anomeric centre relative to the neighbouring 2-O-acyl group. Reactions of 1,2-trans glycosyl esters with highly nucleophilic azide donors, generated from SnCl4 and Me3SiN3, were stereospecific. The results are interpreted in terms of the rapid reaction of the azide species with bicyclic 1,2-acyloxonium (1,2-O-alkyliumdiyl-D-glycofuranose) ions, which were the primarily formed reactive intermediates. When using 1,2-cis glycosyl esters as starting materials the selectivity was reduced (90-94% de); the same is true with 1,2-trans counterparts if less nucleophilic Me3SiN3 in combination with Me3SiOTf catalyst was used. This occurred due to the appearance of the more reactive but less selective oxocarbenium (glycofuranoxonium) ions either as primarily formed reactive intermediates in the former case or after equilibration with acyloxonium ions in the latter case. Protected 1,2-trans beta-D-glycofuranosyl azides with ribo, xylo and 3-deoxy-erythro-pento configurations were best prepared from the corresponding glycosyl esters using 0.05 equivalents of SnCl4, i.e., under anomerization-free conditions. Azidation of methyl glycofuranosides proceeds with inferior (80-90% de) and less predictable selectivity irrespective of the starting anomeric configuration.     

Exploration of the use of an acylsulfonamide safety-catch linker for the polymer-supported synthesis of hyaluronic acid oligosaccharides

The synthesis of hyaluronic acid oligosaccharides on polyethylene glycol (PEG) using an acylsulfonamide linker has been explored. Hyaluronic acid is a challenging synthetic target that usually involves the condensation of highly disarmed glucuronic acid building blocks. Amine-ended PEG monomethyl ether was efficiently functionalized with a hydroxyl-terminated acylsulfonamide linker. Suitably protected d-glucosamine (GlcN) and d-glucuronic acid (GlcA) monosaccharide building blocks were coupled to the polymer acceptor using the trichloroacetimidate glycosylation method. The sulfonamide safety-catch linker enables simultaneous cleavage of the monosaccharide from the polymer and orthogonal functionalization for further (bio)-conjugation of the sugar sample. Subsequent glycosylation of PEG-bound glycosyl acceptor to generate hyaluronic acid oligosaccharide chain failed. Model glycosylation experiments in solution and on soluble support using the same unreactive acceptors and donors allows for the synthesis of an orthogonally protected hyaluronic acid disaccharide and suggest that the encountered difficulties could be attributed to the presence of the N-acylsulfonamide.     

Regioselective glycosylation of 4,6-O-benzylidenated glucopyranosides

Regioselective glycosylation with allyl 4,6-O-benzylidene-alpha,beta-D-glucopyranoside or methyl 4,6-O-benzylidene-alpha,beta-D-glucopyranoside as the acceptor was investigated. It was found that the regioselectivity depends upon donor size and anomeric configuration of the acceptor, i.e., with a monosaccharide donor and an alpha-form acceptor, the (1-->3)-linked product was obtained predominantly or exclusively, while with disaccharide or trisaccharide donors and either an alpha or beta form acceptor, the (1-->2)-linked oligosaccharides were the only products.     

Synthesis of glycosyl diglycerides

Synthesis of mono- and diglycosyl digly cerides with natural structure from 1,2-di-O-acyl-sn-glycerols, 1,2-O-isopropylidene-sn-glycerol, 2,5-methylene-D-mannitol by the orthoester method of glycosylation is reported.     

Synthesis of an L-rhamnose tetrasaccharide, the common and major structure of the repeating unit of the O-antigenic polysaccharide of a strain of Klebsiella pneumoniae and Pseudomonas holci.

A tetrasaccharide, alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-L-Rhap, the common and major structure of the repeating unit of the O-antigenic polysaccharide of a strain of Klebsiella pneumoniae and Pseudomonas holci was synthesized as its methyl and octyl glycosides. Selective 3-O-glycosylation of allyl alpha-L-rhamnopyranoside with 2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl trichloroacetimidate gave allyl 2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl-(1-->3)-alpha-L-rhamnopyranoside (3). Benzoylation, deallylation, and trichloroacetimidation afforded 2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl-(1-->3)-2,4-di-O-benzoyl-alpha-L-rhamnopyranosyl trichloroacetimidate (6). Self condensation of 3,4-di-O-benzoyl-beta-L-rhamnopyranose 1,2-methyl orthoester or 1,2-octyl orthoester gave methyl or octyl 2-O-acetyl-3,4-di-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->2)-3,4-di-O-benzoyl-alpha-L-rhamnopyranoside (16 or 17), and subsequent selective deacetylation gave the disaccharide acceptor (18 or 19). Coupling of 6 with 18 (or 19), followed by deacylation in ammonia-saturated methanol, produced the target tetrasacharide.     

Synthesis of two heptasaccharide analogues of the lentinan repeating unit

beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->6)]-beta-D-Glcp (18) and the allyl glycoside of beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)[-beta-D-Glcp-(1-->6)]-alpha-D-Glcp (29) were synthesized as the analogues of the lentinan repeating heptaose by building the pentasaccharide backbones first, followed by attaching the side chains. 4,6-O-benzylidenated mono-13 or disaccharide 8 were used as the acceptor to ensure the beta linkage in the synthesis of 18, while 4,6-O-benzylidenated disaccharides 21 and 23 were used as the donor and acceptor, respectively, to ensure the beta linkage in the synthesis of 29.     

Synthesis of heparin-like oligosaccharides on polymer supports

The biological functions of a variety of proteins are regulated by heparan sulfate glycosaminoglycans. In order to facilitate the elucidation of the molecular basis of glycosaminoglycan-protein interactions we have developed syntheses of heparin-like oligosaccharides on polymer supports. A completely stereoselective strategy previously developed by us for the synthesis of these oligosaccharides in solution has been extended to the solid phase using an acceptor-bound approach. Both a soluble polymer support and a polyethylene glycol-grafted polystyrene resin have been used and different strategies for the attachment of the acceptor to the support have been explored. The attachment of fully protected disaccharide building blocks to a soluble support through the carboxylic group of the uronic acid unit by a succinic ester linkage, the use of trichloroacetimidates as glycosylating agents and of a functionalized Merryfield type resin for the capping process allowed for the construction of hexasaccharide and octasaccharide fragments containing the structural motif of the regular region of heparin. This strategy may facilitate the synthesis of glycosaminoglycan oligosaccharides by using the required building blocks in the glycosylation sequence.     

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.     

Mucin biosynthesis. Characterization of UDP-galactose: alpha-N-acetylgalactosaminide beta 3 galactosyltransferase from human tracheal epithelium

We have characterized the UDP-galactose: alpha-N-acetylgalactosaminide beta 3 galactosyltransferase in human tracheal epithelium using asialo ovine submaxillary mucin as the acceptor. Maximal enzyme activity was obtained at pH 6.0-7.5 and at 20-25 mM MnCl2 and at 2% Triton X-100. Cd2+ could substitute for Mn2+ as the divalent ion cofactor. Spermine, spermidine, putrecine, cadaverine, and poly-L-lysine stimulated the enzyme activity at low (2.5 mM) MnCl2 concentration. The apparent Michaelis constants for N-acetylgalactosamine, asialo ovine submaxillary mucin, and UDP-galactose were 15.5, 1.14, and 1.36 mM, respectively. The enzyme activity was not affected by alpha-lactalbumin. The alpha-N-acetygalactosaminide beta 3 galactosyltransferase was shown to be different from the N-acetylglucosamine galactosyltransferase by acceptor competition studies. The product of galactosyltransferase was identified as Gal beta 1 leads to 3GalNAc alpha Ser (Thr) by (a) isolation of [14C]Gal-GalNAc-H2 after alkaline borohydride treatment of the 14C-labeled product, (b) establishment of the beta-configuration of the newly synthesized glycosidic bond by its complete cleavage by bovine testicular beta-galactosidase, and (c) assignment of the 1 leads to 3 linkage by identification of threosaminitol obtained from the oxidation of the disaccharide with periodic acid followed by reduction with sodium borohydride, hydrolysis in 4 N HCl, and analysis on an amino acid analyzer. The 1 leads to 3 linkage was confirmed by its resistance to jack bean beta-galactosidase and by the presence of a m/e 307 ion fragment and the absence of a m/e 276 ion by gas-liquid chromatography-mass spectrometry analysis. When acid and beta-galactosidase-treated human tracheobronchial mucin was used as the acceptor, 3.3% of the product was found as [14C]Gal-GalNAc-H2. The remainder of the [14C]Gal was found in longer oligosaccharides formed by a different beta-galactosyltransferase. This galactosyltransferase is slightly inhibited by alpha-lactalbumin and stimulated by spermine.     

Syntheses of beta-(1-->6)-branched beta-(1-->3)-linked d-galactans that exist in the rhizomes of Atractylodes lancea DC

Effective syntheses of galactose hepta-, octa-, nona-, and decasaccharides that exist in the rhizomes of Atractylodes lancea DC were achieved with 2,3,4,6-tetra-O-benzoyl-alpha-d-galactopyranosyl trichloroacetimidate (1), 4-methoxyphenyl 2,3,4-tri-O-benzoyl-beta-d-galactopyranoside (2), 6-O-acetyl-2,3,4-tri-O-benzoyl-alpha-d-galactopyranosyl trichloroacetimidate (5), 4-methoxyphenyl 6-O-acetyl-2,4-di-O-benzoyl-beta-d-galactopyranoside (22), and 4-methoxyphenyl 2,4,6-tri-O-benzoyl-beta-d-galactopyranoside (26) as the key synthons. Coupling of 2 with 1, followed by oxidative cleavage of 1-OMP and subsequent trichloroacetimidate formation gave the beta-(1-->6)-linked disaccharide donor 4. Condensation of 2 with 5 and subsequent selective deacetylation by methanolysis produced the beta-(1-->6)-linked disaccharide acceptor 7. Reaction of 7 with 4, oxidative cleavage of 1-OMP, and trichloroacetimidate formation produced the tetrasaccharide donor 9. The penta- (15), the hexa- (17), and the heptasaccharide donor 19 were synthesized similarly. Meanwhile, treatment of 1 with 22 yielded beta-(1-->3)-linked disaccharide 23 and alpha-(1-->3)-linked disaccharide 25. Oxidative cleavage of 1-OMp of 23 followed by trichloroacetimidate formation produced the disaccharide donor 24. Coupling of 26 with 24, again, gave beta-linked 27 and alpha-linked 29. Selective 6-O-deacetylation of 27 afforded the trisaccharide acceptor 28. TMSOTf-promoted condensation 28 of with the tetra- (9), penta- (15), hexa-(17), and heptasaccharide donor 19, followed by deprotection, gave the target compounds.