Synthesis of the tetrasaccharide repeating unit of the o-specific polysaccharide from salmonella muenster and salmonella minneapolis

The tetrasaccharide β-D-Man-(1→4)-α-L-Rha-(1→3)-D-Gal-(4←1)-α-D-Glc the repeating unit of the O-specific polysaccharide chain of the lipopolysaccharides from Salmonella muenster and S, minneapolis was obtained by glycosylation of benzyl 2,6-di-O-benzyl-4-0(2,3,4-tri-O-benzyl-6-O-benzoyl-α-D-glucopyranosyl)-β-D-galactopyranoside with 3-O-acetyl-4-0-(2,3,4,6-tetra-O-acetyl-β-D-mannopyranosyl)- β-L-rhamnopyranose-1,2-(methylorthoacetate), followed by removal of protecting groups. The structure of the synthetic tetrasaccharide was confirmed by methylation analysis, CrO₃-AcOH oxidation and ¹³C-NMR.     

Efficient synthesis of methyl lycotetraoside, the tetrasaccharide constituent of the tomato defence glycoalkaloid alpha-tomatine

Branched oligosaccharide lycotetraose, beta-D-glucopyranosyl-(1-->2)-[beta-D-xylopyranosyl-(1-->3)]-beta-D-glucopyranosyl-(1-->4)-beta-D-galactopyranose, is a key constituent of many steroidal saponins, including glycoalkaloid alpha-tomatine, which is involved in protection of plants from invading pathogens. A new synthesis of the methyl beta-lycotetraoside () is described. Key steps of the synthesis include two successive glycosylation reactions of disaccharide acceptor methyl (4,6-O-benzylidene-3-O-p-methoxybenzyl-beta-D-glucopyranosyl)-(1-->4)-2,3,6-tri-O-benzyl-beta-D-galactopyranoside with readily available benzoylated trichloroacetimidates of alpha-D-glucopyranose and alpha,beta-D-xylopyranose. This scheme allows sequential glycosylation in one-pot on account of the convenient in situ removal of a p-methoxybenzyl protecting group under the acid conditions of the first glycosylation step. Following deprotection, tetrasaccharide was obtained in 19% yield over eight steps.     

Total synthesis of the Bacteroides fragilis zwitterionic polysaccharide A1 repeating unit

Nearly all bacteria capsular polysaccharides are T-cell-independent antigens that do not promote immunoglobulin class switching from IgM to IgG nor memory responses. In contrast, zwitterionic polysaccharides activate T-cell-dependent immune responses by major histocompatability complex class II presentation, a mechanism previously believed to be reserved for peptidic antigens. The best studied zwitterionic polysaccharide, polysaccharide A1 (PS A1) is found on the capsule of the commensal bacteria Bacteroides fragilis . Its potent immunomodulatory properties have been linked to postoperative intra-abdominal abscess formation. Here, we report the synthesis of the PS A1 tetrasaccharide repeating unit (2) as a tool to investigate the biological role of this polysaccharide. A modular synthetic strategy originating from the reducing end of the PS A1 repeating unit was unsuccessful and illustrated the limitations of glycosylation reactions between highly armed glycosylating agents and poor nucleophiles. Thus, a [3 + 1] glycosylation relying on trisaccharide 5 and pyruvalated galactose 6 was used to complete the first total synthesis of the PS A1 repeating unit (2).     

The Synthesis of Four Dideoxygenated Analogues of β-Maltosyl-(1→4)-Trehalose

Abstract

Four derivatives of β-maltosyl-(1→4)-trehalose were prepared, each with two deoxy functions in one of the constitutive disaccharide building blocks. 2,3-Di-O-acetyl-4,6-dideoxy-4,6-diiodo-α-D-galactopyranosyl- (1→4) ?1,2,3,6-tetra-O-acetyl-D-glucopyranose (3) was employed as a precursor for the 4?,6?-dideoxygenated tetrasaccharide 9: coupling of 3 with 2,3,6-tri-O-benzyl-α-D-glucopyranosyl 2,3,6-tri-O-benzylidene-α-D-glucopyranoside (4) furnished the tetrasaccharide 5 which was deiodinated and deprotected to yield the target tetrasaccharide 9. Secondly, the dideoxygenated maltose derivative 3-deoxy-4,6-O-isopropylidene-2-O-pivaloyl-β-D-glucopyranosyl- (1→4) ?1,6-anhydro-3-deoxy-2-O-pivaloyl-β-D-glucopyranose (10) was ring-opened to the anomeric acetate 11. A [2+2] block synthesis with 4 in TMS triflate mediated glycosylation gave a tetrasaccharide which was deprotected to the 3″,3?-dideoxygenated analogue of β-maltosyl-(1→4)-trehalose. For the third tetrasaccharide, 2,3,2″,3′-tetra-O-benzyl-α,α-trehalose was iodinated at the primary positions and deiodinated in the presence of palladium-on-carbon, then this acceptor was selectively glycosylated with hepta-O-acetyl-maltosyl bromide (20). Removal of protective groups furnished the maltosyl trehalose tetrasaccharide deoxygenated at positions C-6 and C-6′. to prepare a 3,3′-dideoxygenated trehalose, the free hydroxyl groups of 2-O-benzyl-4,6-O-(R)-benzylidene-α-D-glucopyranosyl 2-O-benzyl-4,6-O-(R)-benzylidene-α-D-glucopyranoside (25) were reduced by Barton-McCombie deoxygenation. One of the benzylidene groups was opened reductively with sodium cyanoborohydride. The resulting free hydroxyl group at the 4′-position was glycosylated in a Koenigs-Knorr reaction with 20 to yield the 3,3′-dideoxygenated tetrasaccharide 32, the fourth target oligosaccharide, after deprotection.     

Towards a synthetic glycoconjugate vaccine against Neisseria meningitidis A

Albumin conjugates of synthetic fragments of the capsular polysaccharide of the Gram-negative bacterium Neisseria meningitidis serogroup A were prepared. The fragments include monosaccharides 1 [alpha-D-ManpNAc-(1-->O)-(CH(2))(2)NH(2)] and 2 [6-O-P(O)(O(-))(2)-alpha-D-ManpNAc-(1-->O)-(CH(2))(2)NH(2)], disaccharide 3 [alpha-D-ManpNAc-[1-->O-P(O)(O(-))-->6]-alpha-D-ManpNAc-(1-->O)-(CH(2))(2)NH(2)], and trisaccharide 4 [alpha-D-ManpNAc-[1-->O-P(O)(O(-))-->6]-alpha-D-ManpNAc-[1-->O-P(O)(O(-))-->6]-alpha-D-ManpNAc-(1-->O)-(CH(2))(2)NH(2)]. Two monosaccharide blocks were employed as key intermediates. The reducing-end mannose unit featured the NHAc group at C-2, and contained the aminoethyl spacer as the aglycon for the final bioconjugation. The interresidual phosphodiester linkages were fashioned from an anomerically positioned H-phosphonate group in a 2-azido-mannose building block. The spacer-linked saccharides 1-4 were N-acylated with hepta-4,6-dienoic acid and the resulting conjugated diene-equipped saccharides were subjected to Diels-Alder-type addition with maleimidobutyryl-group functionalized human serum albumin to form covalent conjugates containing up to 26 saccharide haptens per albumin molecule. Complete (1)H, (13)C, and (31)P NMR assignments for 1-4 are given. Antigenicity of the neoglycoconjugates containing 1-4 was demonstrated by a double immunodiffusion assay which indicated that a fragment as small as a monosaccharide is recognized by a polyclonal meningococcus group A antiserum and that the O-acetyl group(s) present in the natural capsular material is not essential for antigenicity.     

Total synthesis of sialylated and sulfated oligosaccharide chains from respiratory mucins

The total syntheses of several complex oligosaccharide moieties that occur in the core structure of sulfated mucins are reported. A trisaccharide acceptor was obtained through regio- and stereoselective sialylation of methyl (6-O-pivaloyl-beta-D-galactopyanosyl)(1-->3)-4,6-O-benzylidene-2-a cetamido-2-deoxy-alpha-D-galactopyranoside with a novel sialyl donor. A tetrasaccharide, pentasaccharide, and hexasaccharide were constructed in predictable and controlled manner with high regio- and stereoselectivity after the successful preparation and employment of a disaccharide donor, trisaccharide donor, disaccharide acceptor, and trisaccharide acceptor building blocks. Finally, a mild oxidative cleaving method was adopted for the selective removal of 2-naphthylmethyl (NAP) in the presence of benzyl groups.     

A new strategy in oligosaccharide synthesis using lipophilic protecting groups: synthesis of a tetracosasaccharide

The use of lipophilic, acyl-type protecting groups in the synthesis of higher-membered oligosaccharides is described by the example of oligosaccharides corresponding to the O-specific polysaccharide (O-SP) of Shigella dysenteriae type 1. Thus, O-stearoylated and O-lauroylated l-rhamnose and d-galactose precursors, respectively, were synthesized and were combined together with a 2-azido-2-deoxy-d-glucopyranosyl donor to form a fully protected lipidated repeating unit of the O-SP. This module was condensed with another tetrasaccharide containing conventional blocking groups. The resulting lipidated octasaccharide was isolated in a pure form by adsorption to a reverse phase adsorbent from which it could be selectively desorbed by alcoholic solvents. Subsequent chain elongation using the conventionally protected tetrasaccharide module as glycosyl donor afforded oligosaccharides up to and including a tetracosasaccharide. The proposed approach can substantially alleviate the difficulties associated with the conventional silica gel chromatographic purification of protected oligosaccharide intermediates and utilizes environmentally friendly solvents that are less expensive than the solvents used for silica gel chromatography. A new, highly efficient method is also proposed for the synthesis of carbohydrate acetals and cyclic orthoesters employing scandium trifluoromethanesulfonate as the catalyst.     

Synthesis of a tetrasaccharide corresponding to the teichoic acid from the cell wall of Streptomyces sp. VKM Ac-2275

A concise chemical synthesis of a tetrasaccharide found in the teichoic acid from the cell wall of Streptomyces sp. VKM Ac-2275 was achieved in high yield. A [2+2] block synthetic strategy has been adopted for the construction of the target tetrasaccharide by exploiting the orthogonal property of a thioglycoside. For the first time, the 2-(4-methoxyphenoxy) ethyl group has been used as the anomeric protecting group to make the glycone moiety with a readily available linker for its conjugation to a protein without destroying the cyclic structure at the reducing end. Yields were high in all of the intermediate steps.     

Linear synthesis of a protected H-type II pentasaccharide using glycosyl phosphate building blocks.

A linear synthesis of a fully protected H-type II blood group determinant pentasaccharide utilizing glycosyl phosphate and glycosyl trichloroacetimidate building blocks is reported. Envisioning an automated solid-phase synthesis of blood group determinants, the utility of glycosyl phosphates in the stepwise construction of complex oligosaccharides, such as the H-type II antigen, is demonstrated. Installation of the central glucosamine building block required the screening of a variety of nitrogen protecting groups to ensure good glucosamine donor reactivity and protecting group compatibility. The challenge to differentiate C2 of the terminal galactose in the presence of other hydroxyl and amine protecting groups prompted us to introduce the 2-(azidomethyl)benzoyl group as a novel mode of protection for carbohydrate synthesis. The compatibility of this group with traditionally employed protecting groups was examined, as well as its use as a C2 stereodirecting group in glycosylations. The application of the 2-(azidomethyl)benzoyl group along with a systematic evaluation of glycosyl donors allowed for the completion of the pentasaccharide and provides a synthetic strategy that is expected to be generally amenable to the solid support synthesis of blood group determinants.     

Synthesis of a 2,3,4-triglycosylated rhamnoside fragment of rhamnogalacturonan-II side chain A using a late stage oxidation approach

Pectic polysaccharide RG-II, a key component of plant primary cell walls, is known to exist as a dimer formed by means of borate diester cross-links between apiosyl residues of one of its constituent side-chain oligosaccharides. Described herein is the strategy for the synthesis of the branched tetrasaccharide alpha-d-GalA-(1-->2)-[beta-D-GalA-(1-->3)]-[alpha-L-Fuc-(1-->4)]-alpha-L-Rha-OMe, an RG-II fragment that is linked to the apiosyl residue that is thought to be responsible for the borate complexation in RG-II dimer. Iterative glycosylation of the rhamnoside acceptors derived from the key 2,3-orthoacetate of methyl 4-O-methoxybenzyl-alpha-d-rhamnopyranoside afforded the protected tetrasaccharide. The target dicarboxylic acid saccharide was subsequently prepared by removal of protecting groups followed by TEMPO-mediated oxidation of galactopyranosyl residues to galactopyranosyluronic acids.     

Synthesis of the core tetrasaccharide of Trypanosoma cruzi glycoinositolphospholipids: Manp(alpha1-->6)-Manp(alpha1-->4)-6-(2-aminoethylphosphonic acid)-GlcNp(alpha1-->6)-myo-Ins-1-PO4

[structure: see text] Synthesis of the core tetrasaccharide Manp(alpha1-->6)-Manp(alpha1-->4)-6-(2-aminoethylphosphonic acid)-GlcNp(alpha1-->6)-myo-Ins-1-PO4, found in glycoinositolphospholipids of Trypanosoma cruzi parasites, is described. The key building block, 6-O-(2-azido-3-O-benzyl-6-O-((2-benzyloxycarbonylaminoethyl)phosphonic acid benzyl ester)-2-deoxy-alpha-D-glucopyranosyl)-1-di-O-benzylphosphoryl-4,5-O-isopropylidene-2,3-O-(D-1,7,7-trimethyl[2,2,1]bicyclohept-6-ylidene)-D-myo-inositol, was synthesized using a partially protected glucosyl D-camphorinositolphosphate and a (2-benzyloxycarbonylaminoethyl)phosphonic acid derivative in a regioselective phosphonate esterfication. Elongation with ethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzyl-1-alpha-D-thiomannopyranoside using dimethyl(methylthio)sulfonium trifluoromethanesulfonate gave a fully protected tetrasaccharide which was successfully deprotected subsequently with sodium methoxide, sodium in liquid ammonia, and aq hydrochloric acid to give title compound.     

Convergent synthesis of a pentasaccharide repeating unit corresponding to the cell wall O-antigen of Salmonella enterica O44

A convergent synthetic strategy has been developed for the synthesis of a pentasaccharide fragment corresponding to the O-antigen of Salmonella enterica O44 strain. An intermediate tetrasaccharide derivative was prepared by a [2+2] block glycosylation of two disaccharide derivatives. The p-methoxybenzyl (PMB) group has been used as the in situ temporary protecting group minimizing the number of functional group manipulation steps. The application of the armed–disarmed glycosylation concept reduced the number of steps in the synthetic strategy. The glycosylation steps were highly stereoselective and high yielding.     

Synthesis of a Tetrasaccharide Related to the Repeating Unit of the Antigen from Shigella dysenteriae Type 9 in the Form of Its 2‐(Trimethylsilyl)ethyl Glycoside

Abstract

Starting from D‐mannose, D‐galactose and D‐glucosamine hydrochloride, two disaccharide blocks were synthesized. Schmidt's inverse addition technique for trichloroacetimidate was utilized for the construction of a disaccharide with a β‐mannosidic linkage in good yield. The two disaccharides in the appropriate form were then allowed to react in the presence of N‐iodosuccinimide (NIS) and trifluoromethanesulfonic acid (TfOH) to give the tetrasaccharide derivative from which removal of protecting groups gave the desired tetrasaccharide in the form of its 2‐(trimethylsilyl)ethyl glycoside.     

Chemical Synthesis Elucidates the Immunological Importance of a Pyruvate Modification in the Capsular Polysaccharide of Streptococcus pneumoniae Serotype 4

Carbohydrate modifications are believed to strongly affect the immunogenicity of glycans. Capsular polysaccharides (CPS) from bacterial pathogens are frequently equipped with a pyruvate that can be placed across the 4,6‐, 3,4‐, or 2,3‐positions. A trans‐2,3‐linked pyruvate is present on the CPS of the Gram‐positive bacterium Streptococcus pneumoniae serotype 4 (ST4), a pathogen responsible for pneumococcal infections. To assess the immunological importance of this modification within the CPS repeating unit, the first total synthesis of the glycan was carried out. Glycan microarrays containing a series of synthetic antigens demonstrated how antibodies raised against natural ST4 CPS specifically recognize the pyruvate within the context of the tetrasaccharide repeating unit. The pyruvate modification is a key motif for designing minimal synthetic carbohydrate vaccines for ST4.     

Chemical Synthesis Elucidates the Key Antigenic Epitope of the Autism-Related Bacterium Clostridium bolteae Capsular Octadecasaccharide

The gut pathogen Clostridium bolteae has been associated with the onset of autism spectrum disorder (ASD). To create vaccines against C. bolteae, it is important to identify exact protective epitopes of the immunologically active capsular polysaccharide (CPS). Here, a series of C. bolteae CPS glycans, up to an octadecasaccharide, was prepared. Key to achieving the total syntheses is a [2+2] coupling strategy based on a β-d -Rhap-(1→3)-α-d -Manp repeating unit that in turn was accessed by a stereoselective β-d -rhamnosylation. The 4,6-O-benzylidene-induced conformational locking is a powerful strategy for forming a β-d -mannose-type glycoside. An indirect strategy based on C2 epimerization of β-d -quinovoside was efficiently achieved by Swern oxidation and borohydride reduction. Sequential glycosylation, and regioselective and global deprotection produced the disaccharide and tetrasaccharide, up to the octadecasaccharide. Glycan microarray analysis of sera from rabbits immunized with inactivated C. bolteae bacteria revealed a humoral immune response to the di- and tetrasaccharide, but none of the longer sequences. The tetrasaccharide may be a key motif for designing glycoconjugate vaccines against C. bolteae.     

The 2-naphthylmethyl (NAP) group in carbohydrate synthesis: first total synthesis of the GlyCAM-1 oligosaccharide structures

Total syntheses of the GlyCAM-1 (glycosylation-dependent cell adhesion molecule-1) oligosaccharide structures: [alpha-NeuAc-(2 --> 3)-beta-Gal-(1 --> 4)-[alpha-Fuc-(1 --> 3)]-beta-(6-O-SO3Na)-GlcNAc-(1 --> 6)]-[alpha-NeuAc-(2 --> 3)-beta-Gal-(1 --> 3)]-alpha-GalNAc-OMe (1) and [alpha-NeuAc-(2 --> 3)-beta-Gal-(1 --> 4)-[alpha-Fuc-(1 --> 3)]-beta-GlcNAc-(1 --> 6)]-[alpha-NeuAc-(2 3)-beta-Gal-(1 --> 3)]-alpha-GalNAc-OMe (2) through a novel sialyl LewisX tetrasaccharide donor are described. Employing sequential glycosylation strategy, the starting trisaccharide was regio- and stereoselectively constructed through coupling of a disaccharide imidate with the monosaccharide acceptor phenyl-6-O-naphthylmethyl-2-deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside with TMSOTf as a catalyst without affecting the SPh group. The novel sialyl Lewisx tetrasaccharide donor 3 was then obtained by alpha-L-fucosylation of trisaccharide acceptor with the 2,3,4-tri-O-benzyl-1-thio-beta-L-fucoside donor. The structure of the novel sialyl Lewisx tetrasaccharide was established by a combination of 2D DQF-COSY and 2D ROESY experiments. Target oligosaccharides 1 and 2 were eventually constructed through heptasaccharide which was obtained by regioselective assembly of advanced sialyl Lewisx tetrasaccharide donor 3 and a sialylated trisaccharide acceptor in a predictable and controlled manner. Finally, target heptasaccharides 1 and 2 were fully characterized by 2D DQF-COSY, 2D ROESY, HSQC, HMBC experiments and FAB mass spectroscopy.     

Synthesis of Polyacrylamide Copolymers Containing (1→6)-Branched (1→3)-β-D-Linked Tri- and Tetra-Saccharides Related to Schizophyllan

Abstract

The allyl β-glycosides of a trisaccharide O-β-D-Glcp-(1→3)-O-[β-D-Glcp-(1→6)]-β-D-Glcp and of a tetrasaccharide O-β-D-Glqp-(1→3)-O-[β-D-Glqp-(1→6)]-O-β-D-Glcp-(1→3)-β-D-Glcp, corresponding to the branching point or the repeating unit of antitumor (1→6)-branched-(1→3)-β-D-glucans, have been synthesized starting from ethyl 2-O-benzoyl-4,6-O-benzylidene-l-thio-α-D-glucopyranoside and copolymerized in a radical reaction with acrylamide to obtain polyacrylamide copolymers containing the tri-and tetra-saccharides for immunochemical studies of schizophyllan.     

Large oligosaccharide-based glycodendrimers

Carbohydrate-based dendritic structures composed of 21 and 27 monosaccharide residues have been synthesized in a convergent manner from trisaccharide building blocks. The oligosaccharide AB2 monomers are based on a maltosyl beta(1-->6)galactose structure, which has been modified to include two methylamino groups at the primary positions of the glucosyl residues. Reductive alkylation of the secondary amino groups, with the innate formyl function of a second oligosaccharide monomer, allows for the chemoselective construction of dendritic wedges, while employing a minimal number of protecting groups. The first-generation dendron can be coupled either to another AB2 monomer, to give a second-generation dendron, or to a tris[2-(methylamino)ethyl]amine-based core moiety, to provide a carbohydrate-based dendrimer. Alternating alpha- and beta-glucosyl residues in the monomers and dendrons, simplifies 1H NMR spectra as a consequence of spreading out the anomeric proton signals. Monomers and dendrons were characterized by extensive one- and two-dimensional NMR spectroscopy in addition to FAB, electrospray, and MALDI-TOF mass spectrometry. Molecular dynamics simulations revealed similar conformations in the dendrons as in the isolated trisaccharide repeating units.     

Synthesis of a chito-tetrasaccharide β-1,4-GlcNAc-β-1,4-GlcN repeating unit

Abstract  

tert-Butyldimethylsilyl (4-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-β-d-glucopyranosyl)-(1 → 4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d-glucopyranoside (Kawada and Yoneda [MOCHEM-D-09-00120], 2009), designed as a repeating disaccharide unit in a β-glucan having two different faces, was converted into a glycosyl donor and an acceptor. The glycosyl acceptor was glycosylated with the donor to afford a chito-tetrasaccharide derivative in good yield. Phthalimido and azido groups in the tetrasaccharide were successively converted into acetamido and free amino groups, and all other protecting groups were cleaved to obtain the chito-tetrasaccharide (2-amino-2-deoxy-β-d-glucopyranosyl)-(1 → 4)-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1 → 4)-(2-amino-2-deoxy-β-d-glucopyranosyl)-(1 → 4)-2-acetamido-2-deoxy-d-glucopyranose.