The effect of isotype and the J kappa region on antigen binding and idiotype expression by antibodies binding alpha (1----6) dextran

Gene transfection and expression techniques have been used to produce three antibodies specific for alpha (1----6) linked dextran B512 with altered isotypes and J kappa regions. Expression of the L chain V region joined to J kappa 4 or J kappa 5 instead of to J kappa 2 reduced or abolished dextran binding. One antidextran with a reduced binding constant for dextran had the same combining site size as the parental mAb. Transfectoma Ig unreactive with dextran B512 did not bind to other class I or class II dextrans. Antibodies with J kappa 4-containing L chains expressed the 10.16.1 (anti-alpha(1----6) dextran) idiotype. In contrast variants expressing L chains with J kappa 5 lost idiotype expression, except when oligosaccharide is present on VH; all antibodies with J kappa 5 L chains continued to bind dextran but with reduced affinity. The presence of carbohydrate in VH may alter the conformation of both paratope and idiotope. Alteration of H chain isotype did not appear to significantly alter the ability of the antidextran to bind Ag; an exception may be that switching V regions to the IgM C region may decrease the apparent affinity for Ag.     

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.     

Sequences of variable regions of hybridoma antibodies to alpha (1----6) dextran in BALB/c and C57BL/6 mice

The variable region sequences of light and heavy chains of three hybridoma antibodies to alpha (1----6) dextran, two from BALB/c and one from C57BL/6 mice, were determined by cloning and sequencing their cDNA. The three kappa-light chains are identical in nucleotide and amino acid sequences, except for the use of different J by BALB/c and C57BL/6; all three had the germ-line sequence of antibodies to 2-phenyloxazolone (20). Nevertheless, 2-phenyloxazolone BSA did not cross-react in gel with antidextrans, nor did dextran react with anti-2-phenyloxazolone ascitic fluids. The heavy chains differed, the BALB/c hybridomas having only three amino acid differences in CDR2 and two in CDR3; the C57BL/6 hybridoma differed throughout the variable region. All three VH are members of the J558 family. The three identical V kappa sequences suggest a significant role in dextran binding, with the differences in CDR of VH and the various J mini-genes of VL and VH being responsible for only fine differences in specificity. Alternatively, the role of V kappa might be minor, with most of the complementarity ascribable to VH. Additional sequences are needed to evaluate whether these data are typical of the repertoire of anti-alpha (1----6) dextran-combining sites.     

GDP-fucose: beta-N-acetylglucosamine (Fuc to (Fuc alpha 1----6GlcNAc)-Asn-peptide)alpha 1----3-fucosyltransferase activity in honeybee (Apis mellifica) venom glands. The difucosylation of asparagine-bound N-acetylglucosamine

Incubation of honeybee (Apis mellifica) venom-gland extracts with GDP-[14C]fucose and GlcNAc beta 1----2Man alpha 1----6(GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----6)GlcNAc beta 1----N-Asn-peptide(NAc) gave a labeled product in 40% yield. Analysis by 500-MHz 1H-NMR spectroscopy indicated the transferred fucose-(Fuc) residue to be alpha 1----3-linked to the Asn-bound GlcNAc. Further proof was provided by one-dimensional and two-dimensional 1H-NMR analysis of the incubation mixture, after incubation with beta-N-acetylhexosaminidase. The established carbohydrate structure (formula; see text) proves the existence of a novel alpha 1----3-fucosyltransferase with the ability to effect difucosylation of the Asn-bound GlcNAc in N-glycans.     

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.     

p-Trifluoroacetamidophenyl O-alpha-D-mannopyranosyl-(1----3)-O-[alpha-D-mannopyranosyl- (1----6)]-beta-D-mannopyranoside

p-Nitrophenyl 2-O-benzyl-4,5-O-cyclohexylidene-beta-D-mannopyranoside (4) was condensed with tetra-O-benzoyl-alpha-D-mannopyranosyl bromide. The resulting, protected disaccharide was converted into p-nitrophenyl O-(2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl)-(1----3)-4-O-benzoyl-2-O- benzyl-beta-D-mannopyranoside (8), which was condensed with tetra-O-benzoyl-alpha-D-mannopyranosyl bromide to give p-nitrophenyl O-(2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl)-(1----3)-O -[2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1----6)]-4-O-benzoyl-2-O -benzyl-beta-D-mannopyranoside (9) in 75% yield. Conversion of the p-nitrophenyl group followed by deprotection then yielded the title compound, whose structure was confirmed by 1H- and 13C-n.m.r. spectroscopy.     

Immunochemical similarity of synthetic alpha-(1----3)-branched D-glucans to dextran B1375

Anti-dextran B1375 antibodies were raised in rabbits by injecting formalin-killed Leuconostoc mesenteroides strain NRRL B1375, and the anti-dextran serum was used to examine native dextran B1375, and synthetic linear and four alpha-(1----3)-branched alpha-(1----6)-D-glucopyranans for similarities. The antiserum reacted with the homologous dextran B1375 and also with all the synthetic linear and branched glucans. Precipitation and precipitation-inhibition studies indicated that the antiserum contained at least three groups of antibodies with different specificities, the first specific for linear alpha-(1----6)-D-glucopyranan structure, the second specific for alpha-D-glycopyranosyl-(1----3)-branching and the last specific for another, unknown structure present in the dextran B1375 molecule. Two samples of the synthetic branched glucans were shown to be immunochemically the most similar to natural dextran B1375 by inhibition experiments.     

Total synthesis of the mollu-series glycosyl ceramides alpha-D-Manp-(1----3)-beta-D-Manp-(1----4)-beta-D-Glcp-(1----1)-Cer and alpha-D-Manp-(1----3)-[beta-D-Xylp-(1----2)]-beta-D-Manp-(1----4)-beta- D-Glcp-(1----1)-Cer

The mollu-series glycosphingolipids, O-alpha-D-mannopyranosyl-(1----3)-O-beta-D-mannopyranosyl-(1----4)-O-bet a-D-glucopyranosyl-(1----1)-2-N-tetracosanoyl-(4E)-sphingeni ne and O-alpha-D-mannopyranosyl-(1----3)-O-[beta-D-xylopyranosyl-(1----2])-O- beta-D-mannopyranosyl-(1----4)-O-beta-D-glucopyranosyl-(1----1)-2-N- tetracosanoyl-(4E)-sphingenine, were synthesized for the first time by using 2,3,4-tri-O-acetyl-D-xylopyranosyl trichloroacetimidate, methyl 2,3,4,6-tetra-O-acetyl-1-thio-alpha-D-mannopyranoside, benzyl O-(4,6-di-O-benzyl-beta-D-mannopyranosyl)-(1----4)-2,3,6-tri-O-benzyl-be ta-D- glucopyranoside 9, and (2S,3R,4E)-2-azido-3-O-(tert-butyldiphenylsilyl)-4-octade cene-1,3-diol 6 as the key intermediates. The hexa-O-benzyl disaccharide 9 was prepared by coupling two monosaccharide synthons, namely, 2,3-di-O-allyl-4,6-di-O-benzyl-alpha-D-mannopyranosyl bromide and benzyl 2,3,6-tri-O-benzyl-beta-D-glucopyranoside. It was demonstrated that azide 6 was highly efficient as a synthon for the ceramide part in the coupling with both glycotriaosyl and glycotetraosyl donors, particularly in the presence of trimethylsilyl triflate.     

An immunochemical study of the combining site specificities of C57BL/6J monoclonal antibodies to alpha (1----6)-linked dextran B512

This is the first report of an immunochemical study of the combining site specificities of a set of monoclonal antibodies to dextran B512 from C57BL/6J mice. The results confirm previous observations on antidextran combining sites and reveal specificities not seen earlier extending the observed repertoire of antibody combining sites to the single alpha (1----6)-linked glucosyl antigenic determinant. Eight C57BL/6J anti-dextran B512 hybridomas, four IgM,kappa and four IgA,kappa, were produced by PEG fusion of immune spleen cells with the nonproducer myeloma cell line P3X63Ag8 6.5.3. Antibody combining site specificities were determined by quantitative precipitin assays with 14 dextrans. Native dextrans with high percentages of linear alpha (1----6)-linked glucoses, similar to the immunogen B512, were the best precipitinogens; dextrans with alternating alpha (1----3), alpha (1----6) linkages, and highly branched dextrans were less effective. All antibodies precipitated with a synthetic, unbranched alpha (1----6)-linked dextran, suggesting their combining sites were "groove-like" and directed toward internal sequences of alpha (1----6)-linked residues, rather than "cavity-like" and directed toward a nonreducing terminal glucose. Two of the IgA hybridomas gave biphasic precipitin curves with dextran B512; this was shown to be due to differences in the precipitability of IgA monomers and polymers. Differences were observed in the reactivities of several dextrans considered previously to be structurally similar, and a newly proposed structural model of dextran B1299S was assessed. Quantitative precipitin inhibition studies with alpha (1----6)-linked isomaltosyl (IM) oligosaccharides, IM2 to IM9, showed that maximum inhibition was reached with IM6 or IM7, consistent with earlier estimates of the upper limit for the sizes of anti-B512 combining sites. Two IgM hybridomas showed a unique pattern, with inhibition being obtained only with IM5 or larger IM oligosaccharides. Association constants of the antidextrans for dextran B512 and for IM7, determined by affinity gel electrophoresis, ranged from 10(2) to 10(4) ml/g, comparable to earlier findings with antidextrans and other anticarbohydrate antibodies.     

Cloning and characterization of DNA complementary to human UDP-GalNAc: Fuc alpha 1----2Gal alpha 1----3GalNAc transferase (histo-blood group A transferase) mRNA

Based on the partial amino acid sequence, the cDNA encoding UDP-GalNAc:Fuc alpha 1----2Gal alpha 1----3GalNAc transferase, the specific primary gene product of histo-blood group A gene (A transferase), was cloned and sequenced. Poly(A)+ RNA from human stomach cancer cell line MKN45, expressing high levels of A antigen, was used for construction of a lambda gt10 cDNA library. Degenerate synthetic oligodeoxynucleotides were used for polymerase chain reactions to detect the presence of the sequence of interest in cDNA (presence test) and to identify the correct clones (identification test) after screening the library with a radiolabeled polymerase chain reaction amplified fragment. Nucleotide sequence analysis revealed a coding region of 1062 base pairs encoding a protein of 41 kDa. Hydrophobicity plot analysis shows the existence of three domains: N-terminal short stretch, transmembranous hydrophobic region, and a long C-terminal domain (a feature common to all glycosyltransferases cloned so far). Southern hybridization analysis has shown that this DNA does not represent a multigene family. No restriction fragment length polymorphism was found to correlate with ABO blood group type. Bands were detected in Northern hybridization of mRNAs from cell lines expressing A, B, AB, or H antigens. These results suggest that sequences of ABO genes are essentially very similar (with minimal differences), and the inability of the O gene to encode A or B transferases is probably due to structural differences rather than A or B transferase expression failure.     

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.     

Regulation of the anti-alpha(1----3) dextran IgG antibody response of BALB/c mice by idiotype-specific T suppressor lymphocytes.

The immune response of BALB/c mice against the so-called thymus-independent bacterial Ag alpha(1----3) dextran (Dex) is restricted to the expression of few major idiotypes (Id). It is furthermore under the control of T lymphocytes which regulate the isotype expression in such a way that they prevent anti-Dex IgG antibody production upon immunization. At the same time these T cells are part of a regulatory system for Dex-specific B cell memory formation. The underlying Ts cell activity has previously been analyzed by using euthymic and athymic congenic animals. Now we have isolated CD4-positive Id-specific T cell lines and clones which by several criteria are representatives of the above Ts cells. They inhibit in vitro proliferation and antibody secretion of Dex-specific hybridoma B cells. They prevent Id-restricted in vivo IgG anti-Dex antibody formation in T cell-reconstituted BALB/c nu/nu mice. At the same time they enforce, again Id-specific, accumulation of Dex-specific B memory cells. As has been shown previously under the influence of splenic Ts cells, these B memory cells are arrested in the original host but can be expanded and activated for anti-Dex IgG antibody formation upon adoptive transfer into X-irradiated allotype congenic nonresponder BALB.Ighb mice. The data show that the regulatory influence of T cells on the anti-Dex response is Id specific. It can now be studied by means of cloned Ts cells.     

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.     

Cross-reaction of synthetic alpha-(1----3)-branched glucans with rabbit anti-N4 dextran

Cross-reactions of four synthetic branched glucans (3-O-alpha-D-glucopyranosyl-(1----6)-alpha-D-glucopyranans: V39, V17, V37, and V32, each containing one unit glucose branches amounting to 11-12%, 33-43%, 50-54%, and 71-100%, respectively) with rabbit anti-N4 dextran were examined. All four samples precipitated antibodies raised in rabbits by injecting N4 dextran-concanavalin A conjugate. The ability of glucans to precipitate antibody depended on the quantity of branches, samples with more branches precipitating less antibody nitrogen under the same conditions. This may indicate an inhibitory effect of the branches on precipitation. Oligosaccharide inhibition assay showed that the precipitation reactions were specific for (1----6)-alpha-D-glucopyranosyl linkages, and the maximum size of the alpha-(1----6)-specific antibody combining site corresponded to isomaltopentaose. Determination of antibody nitrogen and glucan in the precipitates indicated that the ratios of one combining site of antibody to numbers of glucose residues were 1:9 (V39), 1:11 (V17), and 1:16 (V37) in the extreme antibody excess region. A synthetic sample of manno-glucan ((1----6)-alpha-D-glucopyranan containing about 27% of randomly linked 3-O-alpha-D-mannopyranosyl side chains) also reacted with the same antibody.     

Murine monoclonal antibodies directed to the human histo-blood group A transferase (UDP-GalNAc:Fuc alpha 1----2Gal alpha 1----3-N-acetylgalactosaminyltransferase) and the presence therein of N-linked histo-blood group A determinant

Mouse MAbs (WKH-1 through -3) to the human histo-blood group A glycosyltransferase (Fuc alpha 1----2Gal alpha 1----3 galactosaminyltransferase) were established by immunization with the purified native A transferase protein. Hybridomas were selected on the basis of solid-phase reactivity with the purified native A transferase, cell immunofluorescence and immunoprecipitation of transferase activity, and absence of reactivity with blood group ABH carbohydrate determinants. Three MAbs, thus selected, were found most likely to react with the protein epitopes unrelated to carbohydrate epitopes of purified A transferase. The MAbs reacted with cells having high A transferase activity and immunoprecipitated the A transferase activity as well as the 40,000 MW iodinated transferase protein. The antibodies were shown, however, to immunoprecipitate and partially inhibit not only A1 and A2 but also B transferase activity from plasma and A transferase from human lung, and to react with B cells expressing B transferase, thus indicating a cross-reactivity with B transferase. In contrast, they showed no reactivity with various cells having the O phenotype and did not immunoprecipitate the A transferase from porcine submaxillary glands or the alpha 1----2fucosyltransferase from Colo205 cells. The purified A glycosyltransferase was found to carry blood group A carbohydrate determinants by immunochemical detection with a panel of anti-carbohydrate MAbs. These determinants are believed to be N-linked, since treatment of the purified A transferase with N-glycanase removed activity. Immunohistological studies of three epithelial tissues showed that the antibodies stained the Golgi area of cells in epithelia from A and B, but not O, individuals.     

Variable region cDNA sequences and antigen binding specificity of mouse monoclonal antibodies to isomaltosyl oligosaccharides coupled to proteins. T-dependent analogues of alpha(1----6)dextran

Four mouse hybridomas specific for alpha(1----6)dextran, 16.4.12E (IgA kappa, C57BL/6), 28.4.10A (IgM kappa, BALB/c), 35.8.2H (IgG1 kappa, BALB/c), and 36.1.2D (IgM kappa, BALB/c) were obtained by immunization with the T-dependent Ag isomaltohexaose or isomaltotriose coupled to keyhole limpet hemocyanin or to BSA. Immunochemical characterization of the hybridoma antibodies showed that 16.4.12E and 36.1.2D had cavity-type combining sites, recognizing the terminal non-reducing end of alpha(1----6)dextran, whereas 28.4.10A and 35.8.2H had groove-type sites, recognizing internal linear segments of the dextran. The V region cDNA of the H and L chains of the antibodies were cloned and sequenced. VH of 16.4.12E and VH of 36.1.2D belonged to the X24 and Q52 germ-line gene families, respectively. The VH and V kappa sequences of 16.4.12E and V kappa sequence of 36.1.2D were highly homologous to those of W3129, the only anti-alpha(1----6)dextran mAb with a cavity-type site thus far sequenced; 16.4.12E differed from W3129 in the D, JH, and J kappa. VH genes of 28.4.10A and 35.8.2H were homologous to those of several anti-alpha(1----6)dextrans with groove-type sites, but belonged to the J558 germ-line gene family, differed from the other J558 anti-alpha(1----6)dextrans, probably representing a different germ-line subfamily. The L chain sequence of 28.4.10A encoded by V kappa-Ars and J kappa 2 was almost identical to other groove-type anti-alpha(1----6)dextrans obtained by immunizing with the T-independent glycolipid Ag, stearyl-isomaltotetraose. Use of T-dependent Ag such as isomaltosyl oligosaccharide-protein conjugates provides an additional parameter for probing the fine structure of antibody combining sites and evaluating the V-gene repertoire of anti-alpha(1----6)dextrans.     

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.     

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.     

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.