Synthesis of oligosaccharides corresponding to Streptococcus pneumoniae type 9 capsular polysaccharide structures

Two trisaccharides, alpha-D-Galp-(1-->3)-beta-D-ManpNAc-(1-->4)-beta-D-Glcp and alpha-D-Glcp-(1-->3)-beta-D-ManpNAc-(1-->4)-beta-D-Glcp, corresponding to structures from Streptococcus pneumoniae capsular polysaccharides type 9A, L, V and type 9N, respectively, have been synthesised as 2-aminoethyl glycosides and as protected TMSE glycosides. Ethyl thioglycosides were used as glycosyl donors and NIS/TfOH (in CH(2)Cl(2) for beta-linkages) and DMTST (in Et(2)O for alpha-linkages) as promoters in the glycosylations. The beta-ManNAc motif was introduced at the disaccharide level by azide displacement of a 2-O-triflate with beta-D-gluco configuration. The protecting group patterns allow continued syntheses of larger structures.     

Synthesis of tetra- and pentasaccharides corresponding to the capsular polysaccharide of Streptococcus pneumoniae type 9A&L, 9N and 9A

Two tetrasaccharides, alpha-D-GlcAp-(1-->3)-alpha-D-Galp-(1-->3)-beta-D-ManpNAc-(1-->4)-beta-D-Glcp and alpha-D-GlcAp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-ManpNAc-(1-->4)-beta-D-Glcp (protected form), and a pentasaccharide, alpha-D-Glcp-(1-->4)-alpha-D-GlcAp-(1-->3)-alpha-D-Galp-(1-->3)-beta-D-ManpNAc-(1-->4)-beta-D-Glcp have been synthesised from 2-aminoethyl glycoside trisaccharide acceptors in a linear approach via consecutive alpha-glycosylations. Ethyl thioglycosides were used as glycosyl donors and DMTST in Et(2)O or NIS/TfOH in CH(2)Cl(2) were employed as promoters.     

Variant synthetic pathway to glucuronic acid-containing di- and trisaccharide thioglycoside building blocks for continued synthesis of Cryptococcus neoformans capsular polysaccharide structures

An alternative pathway to glucuronic acid-containing di- and trisaccharide thioglycoside building blocks, suitable for the synthesis of Cryptococcus neoformans capsular polysaccharide structures, has been developed. As opposed to our earlier synthesis, this approach features the introduction of the glucuronic acid motif at the di- and trisaccharide level through oxidation of a glucose residue. This approach circumvents problems encountered in glycosylations with glucuronic acid donors and benzylation of glucuronic acid-containing derivatives. Selective protection of primary alcohols was obtained at the di- and trisaccharide stage using TBDMS or trityl protecting groups, respectively. After benzylation of the secondary hydroxyl groups and subsequent removal of the TBDMS or trityl group, oxidation of the free primary alcohols to carboxylic acids was performed in high yield using the TEMPO-BAIB reagent mixture, which does not tend to oxidize thioglycosides. The new approach requires a number of extra steps, but has proven to be more reliable and easily reproducible.     

A new route for the synthesis of Streptococcus pneumoniae 19F and 19A capsular polysaccharide fragments avoiding the β-mannosamine glycosylation step

The recently described [Attolino, E.; Bonaccorsi, F.; Catelani, G.; D’Andrea, F. Carbohydr. Res. 2008, 343, 2545–2556.] β-d-MaNAcp-(1→4)-β-d-Glcp thiophenyl glycosyl donor 3 was used in α-glycosylation reactions of OH-2 and OH-3 of the suitably protected p-MeO-benzyl α-l-rhamnopyranoside acceptors 7 and 8. Glycosylation of the axial OH-2 of 7 took place in high yield (76%) and with acceptable stereoselectivity (α/β = 3.4) leading to the protected trisaccharide α-11, corresponding to the repeating unit of Streptococcus pneumoniae 19F. The same reaction on equatorial OH-3 of acceptor 8 gave the trisaccharide α-15, a constituent of the repeating unit of S. pneumoniae 19A, but in lower yield (41%) and without stereoselection (α/β = 1:1.3). Utilizing the introduced orthogonal protection of OH-1 and OH-4″, the trisaccharide α-11 was transformed into a trisaccharide building block suitable for the synthesis of its phosphorylated oligomers.     

Synthesis of 3,6-branched arabinogalactan-type tetra- and hexasaccharides for characterization of monoclonal antibodies

Synthesis of tetra- and hexasaccharides built up from a β-(1→6)-linked galactopyranosyl backbone with arabinofuranosyl side chains at position 3 and with a 3-aminopropyl spacer related to arabinogalactans is described. These oligosaccharides were prepared for investigation of monoclonal antibodies raised against arabinogalactan proteins (AGPs) from pressed juice of Echinacea purpurea.     

Structure and Function of the Oxidoreductase DsbA1 from Neisseria meningitidis

Neisseria meningitidis encodes three DsbA oxidoreductases (NmDsbA1-NmDsbA3) that are vital for the oxidative folding of many membrane and secreted proteins, and these three enzymes are considered to exhibit different substrate specificities. This has led to the suggestion that each N. meningitidis DsbA (NmDsbA) may play a specialized role in different stages of pathogenesis; however, the molecular and structural bases of the different roles of NmDsbAs are unclear. With the aim of determining the molecular basis for substrate specificity and how this correlates to pathogenesis, we undertook a biochemical and structural characterization of the three NmDsbAs. We report the 2.0-Å-resolution crystal structure of the oxidized form of NmDsbA1, which adopted a canonical DsbA fold similar to that observed in the structures of NmDsbA3 and Escherichia coli DsbA (EcDsbA). Structural comparisons revealed variations around the active site and candidate peptide-binding region. Additionally, we demonstrate that all three NmDsbAs are strong oxidases with similar redox potentials; however, they differ from EcDsbA in their ability to be reoxidized by E. coli DsbB. Collectively, our studies suggest that the small structural differences between the NmDsbA enzymes and EcDsbA are functionally significant and are the likely determinants of substrate specificity.     

Crystal Structures of Penicillin-Binding Proteins 4 and 5 from Haemophilus influenzae

We have determined high-resolution apo crystal structures of two low molecular weight penicillin-binding proteins (PBPs), PBP4 and PBP5, from Haemophilus influenzae, one of the most frequently found pathogens in the upper respiratory tract of children. Novel β-lactams with notable antimicrobial activity have been designed, and crystal structures of PBP4 complexed with ampicillin and two of the novel molecules have also been determined. Comparing the apo form with those of the complexes, we find that the drugs disturb the PBP4 structure and weaken X-ray diffraction, to very different extents. PBP4 has recently been shown to act as a sensor of the presence of penicillins in Pseudomonas aeruginosa, and our models offer a clue to the structural basis for this effect. Covalently attached penicillins press against a phenylalanine residue near the active site and disturb the deacylation step. The ready inhibition of PBP4 by β-lactams compared to PBP5 also appears to be related to the weaker interactions holding key residues in a catalytically competent position.     

Synthesis and inhibitory activity of a di- and a trisaccharide corresponding to an erythrocyte glycolipid responsible for the nor polyagglutination

The polyagglutinable erythrocytes NOR contain unusual neutral glycolipids reactive with anti-NOR antibodies. The disaccharide alpha-D-Galp-(1-->4)-D-GalpNAc and the trisaccharide alpha-D-Galp-(1-->4)-beta-D-GalpNAc-(1-->3)-D-Gal corresponding to the non-reducing end of a NOR glycolipid (NOR1) were chemically synthesized. The syntheses were based on a common (1-->4)-beta-D-GalNAc precursor, and utilized benzyl glycoside and benzyl ether functions for persistent blocking of hydroxyls. The alpha-D-Galp-(1-->4)-beta-D-GalpNAc structural element has been found only recently in Nature, and derivatives thereof have not been synthesized before. Both the synthesized oligosaccharides inhibited specifically human anti-NOR antibodies, the trisaccharide being 300 times more active than the disaccharide.     

Construction of a novel shuttle vector for use in Haemophilus influenzae and H. parainfluenzae

Haemophilus influenzae is an important human pathogen. A number of complete genome sequences of various haemophili are available; however, functional studies have been limited by the lack of an effective shuttle vector which functions in all strains. Here, we have constructed a shuttle vector, pEJ6, which transfers genes between Escherichia coli and H. influenzae and H. parainfluenzae. The vector contains an origin of replication from pLS88 which is functional in E. coli and H. influenzae. In addition it contains an RP4 mobilisation region. The vector can be introduced by electroporation and conjugation into capsulate and non-typeable H. influenzae and is functional for allelic replacement and mutant complementation. The vector will be useful for investigating gene function in Haemophilus spp.     

Synthesis of mucin O-glycan core structures as their p-nitro- and p-aminophenyl glycosides

For the investigation of glycosidases, and for the construction of glycan arrays the p-nitrophenyl- and p-aminophenyl glycosides of mucin O-glycan core structures 1–7 and the 2,6-ST-antigen have been chemically synthesized using d-galactose as a precursor for GalNAc residues. GlcNAc residues have partly been introduced using a 4,6-di-O-benzoyl-2,3-N,O-oxazolidinone-protected donor, which allowed deprotection of the formed di- and tri-saccharides in one step using sodium methoxide.     

Biochemical and Structural Study of the Homologues of the Thiol-Disulfide Oxidoreductase DsbA in Neisseria meningitidis

Bacterial virulence depends on the correct folding of surface-exposed proteins, a process catalyzed by the thiol-disulfide oxidoreductase DsbA, which facilitates the synthesis of disulfide bonds in Gram-negative bacteria. The Neisseria meningitidis genome possesses three genes encoding active DsbAs: DsbA1, DsbA2 and DsbA3. DsbA1 and DsbA2 have been characterized as lipoproteins involved in natural competence and in host interactive biology, while the function of DsbA3 remains unknown.This work reports the biochemical characterization of the three neisserial enzymes and the crystal structures of DsbA1 and DsbA3. As predicted by sequence homology, both enzymes adopt the classic Escherichia coli DsbA fold. The most striking feature shared by all three proteins is their exceptional oxidizing power. With a redox potential of − 80 mV, the neisserial DsbAs are the most oxidizing thioredoxin-like enzymes known to date. Consistent with these findings, thermal studies indicate that their reduced form is also extremely stable. For each of these enzymes, this study shows that a threonine residue found within the active-site region plays a key role in dictating this extraordinary oxidizing power. This result highlights how residues located outside the CXXC motif may influence the redox potential of members of the thioredoxin family.     

Synthesis of 3,6-branched oligomannoside fragments of the mannan from Candida albicans cell wall corresponding to the antigenic factor 4

3-Aminopropyl glycosides of 3,6-branched penta- and hexamannoside fragments of the cell wall mannan from Candida albicans, corresponding to the antigenic factor 4, have been synthesized. Subsequent coupling of both oligosaccharides with BSA using the squarate procedure provided corresponding neoglycoconjugates.     

Defective B cell response to TLR9 ligand (CpG-ODN), Streptococcus pneumoniae and Haemophilus influenzae extracts in common variable immunodeficiency patients

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinaemia and antibody deficiency to both T dependent and independent antigens. Patients suffer from recurrent sinopulmonary infections mostly caused by Streptococcus pneumoniae and Haemophilus influenzae, but also gastrointestinal or autoimmune symptoms. Their response to vaccination is poor or absent. In this study we investigated B cell activation induced by the TLR9 specific ligand (CpG-ODN) and bacterial extracts from S. pneumoniae and H. influenzae known to stimulate several TLR. We found that B cells from CVID patients express lower levels of CD86 after stimulation with CpG-ODN, S. pneumoniae and H. influenzae extracts in combination with anti-IgM antibody and also display a lower proliferative index when stimulated with bacterial extracts. Our results point to a broad TLR signalling defect in B lymphocytes from CVID patients that may be related to the hypogammaglobulinaemia and poor response to vaccination characteristic of these patients.     

Widening the antimicrobial spectrum of esters of bicyclic amines: In vitro effect on gram-positive Streptococcus pneumoniae and gram-negative non-typeable Haemophilus influenzae biofilms

Antibiotic resistance is a global current threat of increasing importance. Moreover, biofilms represent a medical challenge since the inherent antibiotic resistance of their producers demands the use of high doses of antibiotics over prolonged periods. Frequently, these therapeutic measures fail, contributing to bacterial persistence, therefore demanding the development of novel antimicrobials. Esters of bicyclic amines (EBAs), which are strong inhibitors of Streptococcus pneumoniae growth, were initially designed as inhibitors of pneumococcal choline-binding proteins on the basis of their structural analogy to the choline residues in the cell wall. However, instead of mimicking the characteristic cell chaining phenotype caused by exogenously added choline on planktonic cultures of pneumococcal cells, EBAs showed an unexpected lytic activity. In this work we demonstrate that EBAs display a second, and even more important, function as cell membrane destabilizers. We then assayed the inhibitory and disintegrating activity of these molecules on pneumococcal biofilms. The selected compound (EBA 31) produced the highest effect on S. pneumoniae (encapsulated and non-encapsulated) biofilms at very low concentrations. EBA 31 was also effective on mixed biofilms of non-encapsulated S. pneumoniae plus non-typeable Haemophilus influenzae, two pathogens frequently forming a self-produced biofilm in the human nasopharynx. These results support the role of EBAs as a promising alternative for the development of novel, broad-range antimicrobial drugs encompassing both Gram-positive and Gram-negative pathogens.     

Concise synthesis of two trisaccharides related to the cytotoxic triterpenoid saponin isolated from Pithecellobium lucidum

Convergent synthesis of two trisaccharides related to the cytotoxic triterpenoid saponin isolated from Pithecellobium lucidum is reported. The trisaccharides are synthesized in the form of their propargyl glycosides to leave the scope for further glycoconjugate formation through various multi-component reactions. A simple protecting group manipulation is followed using commercially available monosaccharides, d-glucose, d-xylose, d-fucose and l-rhamnose. H₂SO₄ immobilized on silica is used as the Brönsted acid source for the N-iodosuccinimide-mediated thioglycoside activation for stereoselective glycosylations and proved to be a better choice over traditional Lewis acid catalysts such as TMSOTf and TfOH.     

A dual role for the lex2 locus: identification of galactosyltransferase activity in non-typeable Haemophilus influenzae strains 1124 and 2019

Lipopolysaccharide (LPS) of Haemophilus influenzae comprises a conserved tri-l-glycero-d-manno-heptosyl inner-core moiety (l-α-d-Hepp-(1→2)-[PEtn→6]-l-α-d-Hepp-(1→3)-[β-d-GlcIp-(1→4)]-l-α-d-Hepp-(1→5)-α-Kdop) to which addition of β-d-Glcp to O-4 of GlcI in serotype b strains is controlled by the gene lex2B. In non-typeable H. influenzae strains 1124 and 2019, however, a β-d-Galp is linked to O-4 of GlcI. In order to test the hypothesis that the lex2 locus is involved in the expression of β-d-Galp-(1→4-β-d-Glcp-(1→ from HepI, lex2B was inactivated in strains 1124 and 2019, and LPS glycoform populations from the resulting mutant strains were investigated. Detailed structural analyses using NMR techniques and electrospray-ionisation mass spectrometry (ESIMS) on O-deacylated LPS and core oligosaccharide material (OS), as well as ESIMSⁿ on permethylated dephosphorylated OS, indicated both lex2B mutant strains to express only β-d-Glcp extensions from HepI. This provides strong evidence that Lex2B functions as a galactosyltransferase adding a β-d-Galp to O-4 of GlcI in these strains, indicating that allelic polymorphisms in the lex2B sequence direct alternative functions of the gene product.     

Development and validation of high-performance size exclusion chromatography methods to determine molecular size parameters of Haemophilus influenzae type b polysaccharides and conjugates

Current vaccines against Haemophilus influenzae type b (Hib) consist of the polyribosyl ribitol phosphate (PRP) capsular polysaccharide chemically conjugated to a carrier protein. Among the various biological and physical analyses to be performed on these vaccines, the determination of the molecular size of the polysaccharide preparations throughout the conjugation process is particularly relevant. Comparison of results from high-performance size exclusion chromatography (HPSEC) with those routinely obtained using conventional gel permeation chromatography (CGPC) methods highlights the correlation between the two methods for determining the values of the chromatographic distribution coefficient (K_D) of native and activated polysaccharides. The resulting data showed that the K_D value is sufficient to characterize these polysaccharides using an HPSEC method. However, additional molecular size parameters (i.e., molar mass and hydrodynamic radius) are necessary for a reliable characterization of the tetanus conjugate (PRP-T), certainly due to the lattice-like structure of the conjugate. In practice, an absolute detection system in HPSEC composed of a low-angle light scattering detector, a viscometer, and a refractive index (RI) detector was used. As demonstrated, these HPSEC methods are rapid, accurate, and reproducible for the polysaccharides and their glycoconjugates and provide a relevant and more informative alternative to the current CGPC methods.     

An integrated approach to the ligand binding specificity of Neisseria meningitidis M1 alanine aminopeptidase by fluorogenic substrate profiling,inhibitory studies and molecular modeling

Neisseria meningitides is a gram-negative diplococcus bacterium and is the main causative agent of meningitis and other meningococcal diseases. Alanine aminopeptidase from N. meningitides (NmAPN) belongs to the family of metallo-exopeptidase enzymes, which catalyze the removal of amino acids from the N-terminus of peptides and proteins, and are found among all the kingdoms of life. NmAPN is suggested to be mostly responsible for proteolysis and nutrition delivery, similar to the orthologs from other bacteria.     

Synthesis of a tetrasaccharide related to the O-antigen from Azospirillum lipoferum SR65

Concise synthesis of a tetrasaccharide repeating unit of the LPS isolated from Azospirillum lipoferum SR65 has been accomplished through suitable protecting group manipulations and stereoselective glycosylation starting from commercially available l-rhamnose and d-glucose. The target oligosaccharide in the form of its p-methoxyphenyl glycoside is suitable for further glycoconjugate formation via selective cleavage of the OMP glycoside. Plant growth-promoting bacteria (PGPB) of genus Azospirillum plays important roles in the growth and development of plants. The interaction between the roots of the plants and the microbes is governed by the cell surface carbohydrate polymers (CPS, LPS, etc.). The present synthetic-based study elucidates aspects of plant-microbe interaction and future biofertiliser design.     

Synthesis of a tetrasaccharide donor corresponding to the O-specific polysaccharide of Shigella dysenteriae type 1

O-(2,4-Di-O-chloroacetyl-α-l-rhamnopyranosyl)-(1 → 2)-O-(3,4,6-tri-O-benzoyl-α-d-galactopyranosyl)-(1 → 3)-O-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glycopyranosyl)-(1 → 3)-2,4-di-O-benzoyl-α-l-rhamnopyranosyl trichloroacetimidate (1) was synthesized in a stepwise manner, using the following monosaccharide units: 2-(trimethylsilyl)ethyl 2,4-di-O-benzoyl-α-l-rhamnopyranoside, 2-azido-4,6-O-benzylidene-3-O-chloroacetyl-2-deoxy-β-d-glycopyranosyl chloride, methyl 3,4,6-tri-O-benzoyl-2-O-(4-methoxybenzyl)-1-thio-β-d-galactopyranoside, and 2,4-di-O-benzoyl-3-O-chloroacetyl-α-l-rhamnopyranosyl chloride. Compound 1 corresponds to a complete tetrasaccharide repeating unit of the O-specific polysaccharide of the lipopolysaccharide of Shigella dysenteriae type 1.