Synthesis of N-glycoproteins with a native type of carbohydrate-peptide bond

Reaction of beta-oligoglycosylamines obtained from carbohydrate chains of N-glycoproteins (ovalbumin, ovomucoid, riboflavin-binding glycoprotein from hen egg white, and asialofetuin) with bovine serum albumin, lysozyme, and poly(L-Asp) in presence of water-soluble carbodiimide gave rise to a series of glycoconjugates, modelling natural N-glycoproteins. Carbohydrate-peptide bond was shown to be of N-glycosylamide type with participation of Asp and Glu residues. The method allows one to obtain synthetic N-glycoproteins from oligomannoside, complex and hybrid oligosaccharide chains, and may find application both in biochemistry and biotechnology for improvement of physico-chemical properties of unglycosylated proteins.     

Synthesis of a tetrasaccharide substrate of heparanase

A tetrasaccharide, corresponding to the heparan sulfate heparanase substrate, namely β-d-GlcA(2S)-(1→4)-α-d-GlcN(NS,6S)-(1→4)-β-d-GlcA-(1→4)-α-d-GlcN(NS,6S)-OMe, was synthesized in a convergent manner via coupling of a pair of the disaccharide building blocks as a key step.     

Synthesis of a tetrasaccharide fragment of mycobacterial arabinogalactan

The arabinogalactan of mycobacteria contains both monosaccharides in the furanose ring form, which are absent in mammals. We report here the first synthesis of the tetrasaccharide fragment alpha-D-Araf-(1-->5)-beta-D-Galf-(1-->5)-beta-D-Galf-(1-->6)-D-Galf, conveniently derivatized for further elongation. The strategy relied on the use of suitably substituted D-galactono-1,4-lactones as precursors for the galactofuranose units. Reduction of lactone tetrasaccharide 9 with disiamylborane afforded the tetrasaccharide synthon 1. The tetrasaccharide contains the linker unit of the arabinan to the galactan.     

Conformation analysis of modified tetrasaccharide sequences of the N-glycoprotein type--problem of the alpha-(1 to 6)-glycosidic bond

The conformational analysis of the recently synthesized tetrasaccharides alpha-D-Manp (1----3)-[alpha-D-Manp-(1----6)]-4-deoxy-beta-D-lyx-hexp+ ++-(1----4)-D-GlcNAc (2) and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)-D-GlcNAc (3) will be described. The preferred solution conformation of 2 and 3 is a gt-conformation, which is nearly identical with the preferred conformation of the naturally occurring tetrasaccharide alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-D-GlcNAc (1). The main structural feature is the backfolding of the alpha-(1----6)-linked D-Man to the reducing D-GlcNAc unit. Conformational analysis of the tetrasaccharides alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-1,6- anhydro-beta-D-GlcNAc (4), alpha-D-Manp-(1----3)-alpha-D-Manp-(1----6)]-4-deoxy-beta-D- lyx-hexp-(1----4)- 1,6-anhydro-beta-D-GlcNAc (5), and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)- 1,6-anhydro-beta-D-GlcNAc (6) gave additional proof for this backfolding. The substitution of the reducing unit leads to a smaller amount of gt- and a greater amount of gg-conformers. The method used for conformational analysis of 2-6 is a combination of n.m.r.-experiments and HSEA-calculations with the program GESA. Concerning the application of new 2D-techniques, the COLOC-experiment turned out to be extremely useful in sequencing oligosaccharides.     

Synthesis of a potential tetrasaccharide ligand for E-selectin

A potential tetrasaccharide ligand for E-selectin, (Na(+-)O(3)SO-3)Galbeta-(1-->4)[Fucalpha-(1-->3)]Glcbeta-(1-->6)Gal, an analogue of the ovarian cystadenoma glycoprotein tetrasaccharide fragment, was synthesized in a highly practical way.     

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.     

Synthesis of the starfish ganglioside LLG-3 tetrasaccharide

The first synthesis of the ganglioside LLG-3 tetrasaccharide, which has attractive biological activities as well as a unique structure, is described. A C8-methoxy decorated sialic acid building block was initially prepared and a glycolic acid moiety was then introduced by sialylation. Amide condensation between the sialyl glycolic acid and an amino group at C5 on the sialyllactoside unit afforded the fully protected LLG-3 tetrasaccharide. Finally, the desired tetrasaccharide part of LLG-3 was obtained after careful global deprotection.     

Synthesis of a branched pentasaccharide sequence of "bisected" structure of N-glycoproteins

For the synthesis of the threefold-branched pentasaccharide, O-alpha-D-mannopyranosyl-(1----3)-O-[(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-(1----4)]-O-[alpha-D-mannopyranosyl-(1----6)]-O-beta-D- mannopyranosyl-(1----4)-2-acetamido-2-deoxy-D-glucopyranose (20), which is a part of the structure of the N-glycoproteins, the disaccharide 4-O-(4-O-acetyl-3,6-di-O-allyl-2-O-benzyl-beta-D-mannopyranosyl) -1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-beta-D-glucopyranose was synthesized as a key compound by use of the silver silicate-catalyst procedure. After elimination of the 4-O-acetyl group, a 2-acetamido-2-deoxy-beta-D-glucopyranosyl group was attached according to the phthalimido method. Further elimination of the allyl groups allowed the linkage of two alpha-D-mannopyranosyl groups in the presence of mercury salt. A deblocking sequence consisting of four steps gave 20.     

Synthesis of a tetrasaccharide fragment of cobra venom factor oligosaccharide.

Synthesis of a tetrasaccharide fragment, alpha-L-Fuc-(1-->3)-beta-D-GlcNAc-(1-->2)-alpha-D-Man-(1-->6)-alpha-D-Man- OMe of the cobra venom factor (CVF) oligosaccharide is described.     

Synthesis of a tetrasaccharide representing a minimal epitope of an arabinogalactan

The hydrophobic alkyl chain-containing tetrasaccharide, dodecyl beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranosyl-(1-->6)-[alpha-L - arabinofuranosyl-(1-->2)]-beta-D-galactopyranoside, was synthesized efficiently using a convergent strategy. In coupling reactions, protected trichloroacetimidates proved to be better donors than their corresponding bromides in the preparation of the dodecyl disaccharide and trisaccharide. Zemplén deacylation provided the target tetramer in good overall yield.     

Synthesis of 3-O-beta-N-acetylglucosaminyl cyclic tetrasaccharide through a lysozyme-catalyzed transfer reaction

Egg white lysozyme was found to catalyze the transfer of N-acetylglucosamine to cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] (CTS). Structural analysis showed that the transfer product was 3-O-beta-N-acetylglucosaminyl CTS, cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[beta-GlcNAc-(1-->3)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. This branched saccharide is anticipated to be a model compound of the sugar chains of glycoproteins.     

Synthesis of the Glc3Man N-glycan tetrasaccharide by iterative allyl IAD

The synthesis of the tetrasaccharide alpha-D-Glcp-(1-->2)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->3)-alpha-D-Manp-OMe, corresponding to the terminal tetrasaccharide portion of the glucose terminated arm of the N-glycan tetradecasaccharide, was achieved with complete stereocontrol by the use of iterative allyl protecting group mediated intramolecular aglycon delivery (allyl IAD) demonstrating the utility of intramolecular glycosylation for the stereocontrolled construction of multiple glycosidic linkages during the synthesis of an oligosaccharide.     

Synthesis of the tetrasaccharide core region of antigenic lipo-oligosaccharides characteristic of Mycobacterium kansasii

The oligosaccharide core region, beta-D-Glcp-(1----3)-beta-D-Glcp-(1----4)-alpha-D-Glcp- 1----1)-alpha-D-Glcp (1), of the lipo-oligosaccharide-type antigens isolated from M. kansasii has been synthesised from 2,3,2',3',4',6'-hexa-O-benzyl-6-O-(1-phenylethyl)-alpha, alpha-trehalose (4). Compound 4 was obtained by LiAlH4-AlCl3-type hydrogenolysis of 2,3,2',3',4',6'-hexa-O-benzyl-4,6-O-(S)-(1-phenylethylidene)-alpha , alpha-trehalose. The beta-laminaribiosyl part of the molecule was built-up by sequential glycosylation steps using 2,4,6-tri-O-acetyl-3-O-allyl-alpha-D-glucopyranosyl bromide in the presence of HgBr2 and methyl 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranoside promoted by methyl triflate. The complete a priori 13C-n.m.r. spectrum assignment of 1 was achieved by applying 2D methods.     

Synthesis of the tetrasaccharide repeating-unit of the O-specific polysaccharide from Salmonella senftenberg

The oligosaccharide β-d-Man-(1 → 4)-α-l-Rha (1 → 3)-d-Gal-(6 ← 1)-α-d-Glc, which is the repeating unit of the O-specific polysaccharide chain of the lipopolysaccharide from Salmonella senftenberg, was obtained by glycosylation of benzyl 2,4-di-O-benzyl-6-O-(2,3,4-tri-O-benzyl-6-O-p-nitrobenzoyl-α-d-glucopyranosyl)-β-d-galactopyranoside or benzyl 2-O-acetyl-6-O-(2,3,4-tri-O-benzyl-6-O-p-nitrobenzoyl-α-d-glucopyranosyl)-β-d-galactopyranoside with 3-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-d-mannopyranosyl)-β-l-rhamnopyranose 1,2-(methyl orthoacetate) followed by removal of protecting groups.     

Synthesis of the 6-deoxytalose-containing tetrasaccharide of the glycopeptidolipid from Mycobacterium intracellare serotype 7

An efficient synthesis of 4-methoxyphenyl α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→2)-6-deoxy-α-l-Talp, the tetrasaccharide related to the GPLs of Mycobacterium intracellare serotype 7, was achieved with 4-methoxyphenyl 3,4-di-O-benzoyl-6-deoxy-α-l-talopyranoside (6c) as the key intermediate which was obtained through selective 3-O-benzoylation of 4-O-benzoyl-6-deoxy-α-l-taloside. Coupling of 6c with 3-O-allyloxycarbonyl-2,4-di-O-benzoyl-α-l-rhamnopyranosyl trichloroacetimidate followed by removal of the allyloxycarbonyl protecting group afforded the disaccharide acceptor 11. Condensation of 11 with 2,3,4-tri-O-benzoyl-α-l-rhamnopyranosyl-(1→3)-2,4-di-O-benzoyl-α-l-rhamnopyranosyl trichloroacetimidate and subsequent deprotection gave the target tetrasaccharide.     

Synthesis of a tetrasaccharide unit of the capsular polysaccharide of Streptococcus pneumoniae type 9V

In the synthesis of 8-methoxycarbonyloctyl O-(alpha-D-galactopyranosyl)-(1----3)-O-(2-acetamido-2-deoxy-beta-D- mannopyranosyl)-(1----4)-O-(beta-D-glucopyranosyl)-(1----4)-alpha-D- glucopyranoside, which represents a component of the capsular polysaccharide of Streptococcus pneumoniae type 9V, the key step was the coupling of alpha-D-Galp-(1----3)-beta-D-ManpNAc-(1----4)-D-Glc as glycosyl donor with 8-ethoxy-carbonyloctyl 6-O-acetyl-2,3-di-O-benzyl-alpha-D-glucopyranoside as glycosyl acceptor by use of the imidate method. Only the beta-imidate of the trisaccharide could be employed in this glycosidation reaction to give stereoselectively the tetrasaccharide in high yield. The alpha-imidate of the trisaccharide led to hydrolysis of the imidate group.     

Synthesis of the tetrasaccharide repeating-unit of the lipopolysaccharide isolated from pseudomonas maltophilia

The title tetrasacharide having the structure 3-O-Me-β- -Xylp-(1→4)-- -Rhap-(1→4)-- -Rhap-(1→2)- -Rhap was obtained by reaction of the -acetobromo derivative of 4-O-(3-O-methyl-β- -xylopyranosyl)- -rhamnopyranose and benzyl 3,4-di-O-benzyl-2-O-(2,3-O-isopropylidene-- -rhamnopyranosyl)-- -rhamnopyranoside, followed by removal of the protecting groups. The synthesised compounds were characterised on the basis of n.m.r. data.     

Melanoma antigens as modified normal gene sequences

Melanomas are highly aggressive tumors with a well-documented antigenic nature. Several melanoma antigens have been reported, four of which, p97, Ia-like antigen, B700, and A have been implicated as having regions in common with normally occurring proteins. P97 has partial sequence homology with transferrin and lactotransferrin, Ia-like antigen is immunologically cross-reactive with alpha and beta chains of Ia-like proteins, A is a variant of alpha actin, and B700 resembles a normal melanosomal membrane protein. In addition, B700 has partial sequence homology to serum albumins. These observations suggest that melanoma tumors can produce antigenic proteins by modification of normally occurring proteins. The possible mechanisms are discussed.