Synthesis Of Neoglycoproteins Research Articles

The myrosinase-glucosinolate system to generate neoglycoproteins: A case study targeting mannose binding lectins

A convenient strategy for a ‘one-pot’ synthesis of neoglycoproteins (NGP) was developed using the myrosinase-glucosinolate couple, a natural enzyme-substrate system. This enzymatic reaction allowed us to generate an isothiocyanate in situ which then reacted with the lysine residues of bovine serum albumin protein (BSA) to produce multivalent neoglycoproteins. Using two models, glucomoringin which is a natural glucosinolate bearing a l-rhamnose unit, and an artificial glucosinolate specifically designed for mannose type lectins, an average of up to 17.8 and 28.7 carbohydrate residues could be respectively grafted onto the BSA protein. This process is comparable to commercial approaches using BSA-ManC without the disadvantage of handling harmful chemical reagents. Lectin binding screening (GLYcoPROFILE®) showed that among all NGPs synthesized, BSA-Man 16 gave similar and in some cases better affinities in comparison with commercial BSA-Manc towards various mannose-specific lectins.

ChemInform Abstract: Glycosidases in Carbohydrate Synthesis: When Organic Chemistry Falls Short

AbstractReview: 104 refs.

Synthesis of neoglycoproteins containing O-methylated trisaccharides related to excretory/secretory antigens of Toxocara larvae

The disaccharides allyl β- d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-β- and α- d-galactopyranoside 10a and 10b and the trisaccharides allyl 2- O-methyl-α- l-fucopyranosyl-(1→2)-β- d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-β- and α- d-galactopyranoside 18a and 18b have been prepared using stepwise assembly of the sugar units. The glycosidic linkages were formed employing the trichloroacetimidate procedure for the attachment of the galactopyranosyl residue and N-iodosuccinimide/triflic acid activation of an ethyl 1-thiofucopyranoside donor for fucosylation. Deprotection furnished the allyl glycosides which were converted into cysteamine-spacered ligands, activated with thiophosgene and subsequently linked to bovine serum albumin. The neoglycoproteins serve as immunoreagents to determine epitope specificities of monoclonal antibodies directed against highly immunogenic O-glycans located at the surface of Toxocara larvae.

Structure-activity profiles of complex biantennary glycans with core fucosylation and with/without additional alpha 2,3/alpha 2,6 sialylation: synthesis of neoglycoproteins and their properties in lectin assays, cell binding, and organ uptake.

The consideration of oligosaccharides and glycoconjugates as biopharmaceuticals is an emerging topic in drug design. Chemoenzymatic synthesis of N-glycans was performed to examine the influence of N-glycan core fucosylation on lectin-binding properties and biodistribution. As a first step in a systematic comparison of N-glycans, the core fucose moiety was chemically introduced into a complex-type biantennary heptasaccharide azide. After deprotection and attachment of a spacer, the terminal sections of the N-glycan were elongated enzymatically. Conversion of the amino group in the spacer to an isothiocyanate gave derivatives allowing convenient ligand attachment to bovine serum albumin (BSA). The resulting neoglycoproteins contained an average of 2.9-4.6 chains per carrier molecule. Relative to unsubstituted biantennary complex-type N-glycans, the core fucosylation appears to favor the extended orientation of the alpha 1,6-arm. This was deduced from an up to 5-fold alteration of affinity for lectins in solid-phase assays. Marked differences were also found for cell surface binding of cultured tumor cells, for staining of tumor cells in lung sections, and in organ distribution. In vivo, the alpha 2,6-sialylated neoglycoproteins showed a reduced serum half-life in mice relative to the alpha 2,3-sialylated isomer and the non-fucosylated congeners. These results support the notion that changing the shape of a glycan provides a promising strategy to optimize the affinity of protein-carbohydrate interactions. Overall, our study underscores the importance of chemoenzymatic synthesis to define the effect of chain orientation on the ligand properties of N-glycans.

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-β- d- talo-oct-2-ulopyranos}onate, prepared from the 4- O- pNBz-protected Ko derivative, was elaborated into the α-Ko allyl ketoside, the reducing disaccharide α-Kdo p-(2→4)-Ko and the disaccharide α-Kdo p-(2→4)-Ko p-(2→OAll). Conversely, methyl[4,5,7,8-tetra- O-acetyl-3- O-(4-nitrobenzoyl)-α- d- glycero- d- talo-2-octulopyranosyl bromide]onate [ Carbohydr. Res., 244 (1993) 69–84], was coupled with a Kdo acceptor to give the disaccharide α-Ko p-(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.

Synthesis of neoglycoproteins containing L-glycero-α-D- manno-heptopyranosyl-(1→4)- and -(1→5)-linked 3-deoxy-α-D-manno-oct-2- ulopyranosylonic acid (Kdo) phosphate determinants

The disaccharide allyl glycosides 4, 8, 13 and the trisaccharide 33 have been prepared using heptopyranosyl trichloroacetimidate 1 or the disaccharide bromide 27 as glycosyl donors followed by efficient O-phosphorylation via the amidite procedure. The allyl glycosides 4, 8 and 33 are converted into 3-(2-aminoethylthio)propyl glycosides and are coupled to bovine serum albumin. The resulting neoglycoconjugates 6 and 35 containing spacer-linked Hep-(1→4)-Kdo and Hep-(1→5)-Kdo 4-phosphate-(2→6)-β-GlcNAc residues correspond to part structures of the inner core region in bacterial lipopolysaccharide, whereas compound 10 contains the artificial analogue Hep-(1→4)-Kdo 5-phosphate. The compounds may be used in immunochemical characterisation of monoclonal antibodies.

Synthesis of neoglycoproteins containing 5-O-phosphorylated Kdomonosaccharide, 4-O- and 5-O-phosphorylated α-Kd↬(2 6)-2-acetamido-2-deoxy- -D-glucopyranosyl disaccharide residues

Radical addition of cysteamine to anomeric allyl glycosides of Kdo 5-phosphates 1 and 4 afforded good yields of the corresponding 3-(2-aminoethylthio)propyl glycosides. Similar reactions with the α-allyl glycoside of Kdo 4-phosphate 7 led to substantial formation of the dephosphorylated product 10 through intramolecular hydrolysis of the 4-O-phosphomonoester by the terminal amino function of the spacer group. Similar side reactions occurred upon deblocking of the 4- O-phosphorylated 6-aminohexyl-α-glycoside lactone derivative 18. Allyl glycosides of 4'- O- and 5'- O-phosphorylated Kd↬ (2 6)-glucosamine derivatives were prepared in good yields via phosphorylation using the amidite procedure. The 4'-phosphate moiety was introduced following intramolecular protection of the 5'-OH group as a lactone. Subsequent deprotection gave the allyl glycosides 25 and 33 which were transformed into the corresponding stable spacer derivatives 26 and 34 by coupling with cysteamine. The ligands were activated with thiophosgene and reacted with bovine serum albumin to give the neoglycoconjugates 3, 6, 27 and 35 to be used in immunochemical studies of monoclonal antibodies directed against Haemophilus influenzae Re-mutant and related lipopolysaccharides.

ChemInform Abstract: Synthesis of Neoglycoproteins as Artificial Antigens.

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Synthesis of Neoglycoproteins as Artificial Antigens

Abstract The synthesis of various allyl glycosides of L-rhamnose containing ether and/or ester substituents are reported. Ozonolysis of allyl glycosides followed by reductive amination with ∊-amino groups of lysine residues in bovine serum albumin using sodium cyanoborohydride at pH 7.8 provided different structural neoglycoproteins as artificial antigens.

An effective method for the synthesis of neoglycoproteins and neogangliosideproteins by use of reductively aminated sulfhydryl-containing carbohydrate conjugates.

A highly effective method for the synthesis of neoglycoconjugates has been developed. The technique is based on two steps. First, a sulfhydryl group is introduced into the carbohydrate by reductive amination with cysteamine, and secondly the sulfhydryl group carrying carbohydrate is coupled to a protein or carrier via a disulfide bridge. The method was used to couple sialic acid-containing carbohydrates such as N-acetylneuraminyllactose and the carbohydrate moieties of gangliosides (GM1, GM3 and GD1b) to bovine serum albumin. Biological functionality of the generated GM1- and GD1b-neoglycoprotein was demonstrated by a choleratoxin-binding assay and an immunoassay, respectively. The applicability of neoglycoconjugates for the quantification of gangliosides in solution by a competitive assay is shown.

Synthesis ofNeoglycoproteins containing the 3,6-di-O-methyl-β-d-glucopyranosyl epitope and their use in serodiagnosis of leprosy

A stratagem for the synthesis ofneoglycoproteins suitable for the selective serodiagnosis of leprosy is described in which synthetic 3,6-di-O-methyl-β-d-glucopyranose, the epitope of phenolic glycolipid I fromMycobacterium leprae, was used. Condensation of 8-methoxycarbonyloctanol with the acetobromo derivative of 3,6-di-O-methylglucose gave 8-methoxycarbonyloctyl 2,4-di-O-acetyl-3,6-di-O-methyl-β-d-glucopyranoside in 65% yield, and with absolute stereospecificity for the β anomer. The deacylated product was converted to the crystalline hydrazide and coupled to bovine gamma globulin, bovine serum albumin and poly-d-lysinevia intermediate acyl azide formation to produce the 8-carbonyloctyl 3,6-di-O-methyl-β-d-glucopyranosyl polypeptides. Theneoglycoproteins were highly sensitive in ELISA and emulated the specificity of the native glycolipid in analysis of sera from patients throughout the spectrum of leprosy and from different geographical regions. The 8-carbonyloctyl 3,6-di-O-methyl-α-d-glucopyranoside-bovine serum albumin was also synthesized and shown to have about one-half the activity of the β-linkedneoglycoprotein. A different synthetic approach produced the 8-carbonyloctyl 4-O-(3′,6′-di-O-methyl-β-d-glucopyranosyl)-α-l-rhamnopyranoside-bovine serum albumin which was also highly sensitive and specific for the serodiagnosis of leprosy. The presence of the second sugar unit, similar to that in the native glycolipid but for the absence ofO-methyl groups, seemed to provide a probe with greater felicity for the serological detection of tuberculoid leprosy.