73 research outputs found
Synthesis of C-Glycosyl Amino Acid Building Blocks Suitable for the Solid-Phase Synthesis of Multivalent Glycopeptide Mimics
No full textFive C-glycosyl functionalized lysine building blocks, featuring C-glycosidic derivatives of α-rhamnose, α-mannose, α-galactose, β-galactose, and β-N-acetyl glucosamine have been designed and synthesized. These derivatives, equipped with acid-labile protecting groups, are eminently suitable for solid-phase synthesis of multivalent glycopeptides. The lysine building blocks were prepared from C-allyl glycosides that underwent a Grubbs cross-metathesis with an acrylate, followed by a reduction of the C=C double bond in the resulting α,β-unsaturated esters, and liberation of the carboxylate to allow condensation with a lysine side chain. The thus obtained C-glycosides, five in total, were applied in the solid-phase peptide synthesis (SPPS) of three glycopeptides, showing the potential of the described building blocks in the assembly of well-defined mimics of homo- and heteromultivalent glycopeptides and glycoclusters
Synthetic Oligomers Mimicking Capsular Polysaccharide Diheteroglycan are Potential Vaccine Candidates against Encapsulated Enterococcal Infections
No full textInfections caused by Enterococcus spp. are a major concern in the clinical setting. In Enterococcus faecalis, the capsular polysaccharide diheteroglycan (DHG), composed of ß-d-galactofuranose-(1 → 3)-ß-d-glucopyranose repeats, has been described as an important virulence factor and as a potential vaccine candidate against encapsulated strains. Synthetic structures emulating immunogenic polysaccharides present many advantages over native polysaccharides for vaccine development. In this work, we described the synthesis of a library of DHG oligomers, differing in length and order of the monosaccharide constituents. Using suitably protected thioglycoside building blocks, oligosaccharides up to 8-mer in length built up from either Galf-Glcp or Glcp-Galf dimers were generated, and we evaluated their immunoreactivity with antibodies raised against DHG. After the screening, we selected two octasaccharides, having either a galactofuranose or glucopyranose terminus, which were conjugated to a carrier protein for the production of polyclonal antibodies. The resulting antibodies were specific toward the synthetic structures and mediated in vitro opsonophagocytic killing of different encapsulated E. feacalis strains. The evaluated oligosaccharides are the first synthetic structures described to elicit antibodies that target encapsulated E. faecalis strains and are, therefore, promising candidates for the development of a well-defined enterococcal glycoconjugate vaccine
Antibody Recognition of Different Staphylococcus aureus Wall Teichoic Acid Glycoforms
No full textWall teichoic acids (WTAs) are glycopolymers decorating the surface of Gram-positive bacteria and potential targets for antibody-mediated treatments against Staphylococcus aureus, including methicillin-resistant (MRSA) strains. Through a combination of glycan microarray, synthetic chemistry, crystallography, NMR, and computational studies, we unraveled the molecular and structural details of fully defined synthetic WTA fragments recognized by previously described monoclonal antibod-ies (mAbs 4461 and 4497). Our results unveiled the structural requirements for the discriminatory recognition of alpha- and beta-GlcNAc-modified WTA glycoforms by the complementarity-determining regions (CDRs) of the heavy and light chains of the mAbs. Both mAbs interacted not only with the sugar moiety but also with the phosphate groups as well as residues in the ribitol phosphate (RboP) units of the WTA backbone, highlighting their significant role in ligand specificity. Using elongated WTA fragments, containing two sugar modifications, we also demonstrated that the internal carbohydrate moiety of alpha-GlcNAc-modified WTA is preferentially accommodated in the binding pocket of mAb 4461 with respect to the terminal moiety. Our results also explained the recently documented cross-reactivity of mAb 4497 for beta-1,3/beta-1,4-GlcNAc-modified WTA, revealing that the flexibility of the RboP backbone is crucial to allow positioning of both glycans in the antibody binding pocket.Bio-organic Synthesi
Synthesis of Neisseria meningitidis serogroup A carba analogues as hydrolytically stable antigens for antimeningococcal glycoconjugate vaccines
No full textSynthesis of Neisseria meningitidis serogroup A carba analogues as hydrolytically stable antigens for antimeningococcal glycoconjugate vaccines
Ludovic Auberger, Jacopo Enotarpi, Jeroen Codeé, Roberto Adamo, Laura Polito, Luigi Lay
The Gram-negative encapsulated bacterium Neisseria meningitidis type A (MenA) is a major cause of meningitis in developing countries, especially in the sub-Saharan region of Africa [1]. The development and manufacture of an efficient glycoconjugate vaccine against MenA is largely hampered by the poor stability in water of the natural capsular polysaccharide (CPS)[2], composed of (1→6)-linked 2-acetamido-2-deoxy-α-D-mannopyranosyl phosphate repeating units, with acetyl substituents. As a consequence, most of MenA glycoconjugates currently available have been licensed as lyophilisates. The availability of MenA polysaccharide mimics resistant to hydrolysis, however, is highly attractive for the development of a more stable glycoconjugate vaccine in liquid formulation. To this end, we envisaged that the replacement of the endocyclic ring oxygen with a methylene group to get a carbocyclic analogue will lead to the loss of the acetalic character of the phosphodiester and consequently to the enhancement of its chemical stability [3], [4]. Furthermore, the 3-O-acetylation aims to strengthen the immunological profile of our neo-glycoconjugates, structurally designed even closer to the natural CPS MenA oligomer, partially acetylated at the position 3-OH with a rate of 75-95% [5].
Thus, we describe our synthetic approaches to 3-O-acetylated phosphodiester-linked carba oligomers of N-acetyl mannosamine (the repeating unit of MenA CPS, containing up to 8 repeating units. The increased stability of the synthetic carba oligomers was first confirmed by an accelerated stability study, then selected fragments were conjugated to CRM197 (a diphtheria toxin mutant) as a protein carrier. Finally, the immunological profile of the resulting neo-glycoconjugates will be carefully investigated, with the purpose to highlight the effect of the carbohydrate chain length and of the 3-O-acetylation on the immunoactivity [6].
[1] Tan L. K. K.; Carlone G. M.; Borrow R. Advances in the development of vaccines against Neisseria meningitidis. N. Engl. J. Med. 2010, 362, 1511-1520.
[2] Frasch, C. E. Production and control of Neisseria meningitidis vaccines. Adv. Biotechnol. Processes 1990, 13, 123-145.
[3] Gao, Q.; Zaccaria, C.; Tontini, M.; Poletti, L.; Costantino, P.; Lay, L. Synthesis and preliminary biological evaluation of carba analogues from Neisseria meningitidis A capsular polysaccharide. Org. Biomol. Chem. 2012, 10, 6673-6681.
[4] Gao Q.; Tontini M.; Brogioni G.; Nilo A.; Filippini S.; Harfouche C.; Polito L.; Romano M. R.; Costantino P.; Berti F.; Adamo R.; Lay L. ACS Chem. Biol. 2013, 8, 2561-2567.
[5] Berry DS.; Lynn F.; Lee C-H.; Frasch CE.; Bash MC. Effect of O-Acetylation of Neisseria Meningitis serogroup A capsular polysaccharide on development of functional immune responses, Infection and Immunity, 2002, 70(7), 3707-3713.
[6] This project has received funding from the H2020-MSCA-ITN-2015 “Glycovax” under grant agreement No 675671
NMR-solution structures of fluoro-substituted beta-peptides: A 3 14-helix and a hairpin turn. The first case of a 90degree(s) O=C-C-F dihedral angle in an a-fluoro-amide group
No full textTo further study the preference of the antiperiplanar (ap) conformation in -fluoro-amide groups, two -peptides, 1 and 2, containing a (2-F)-3hAla and a (2-F)-2hPhe residue, have been synthesized. Their NMR-solution structures in CD3OH were determined and compared with those of non-F-substituted analogs, 3 and 4a. While we have found in a previous investigation (Helv. Chim. Acta 2005, 88, 266) that a stereospecifically introduced F-substituent in the central position of a -heptapeptide is capable of breaking the 314-helical structure by enforcing the FCCO ap-conformation, we could now demonstrate that the same procedure leads to a structure with the unfavorable ca. 90° FCCO dihedral angle, enforced by the 314-helical folding in a -tridecapeptide (cf. 1; Fig. 4). This is interpreted as a consequence of cooperative folding in the longer -peptide. A F-substituent placed in the turn section of a -peptidic hairpin turn was shown to be in an ap-arrangement with respect to the neighboring CO bond (cf. 2; Fig. 7). Analysis of the non-F-substituted -tetrapeptides (with helix-preventing configurations of the two central 2/3-amino acid residues) provides unusually tight hairpin structural clusters (cf. 3 and 4a; Figs. 8 and 9). The skeleton of the -tetrapeptide H-(R)3hVal-(R)2hVal-(R)3hAla-(S)3hPhe-OH (4a) is proposed as a novel, very simple backbone structure for mimicking -peptidic hairpin turns.Raveendra I. Mathad, Bernhard Jaun, Oliver Flögel, James Gardiner, Markus Löweneck, Jeroen D. C. Codée, Peter H. Seeberger, Dieter Seebach, Michael K. Edmonds, Florian H. M. Graichen, and Andrew D. Abel
Static 3D Triangle Mesh Compression Overview
No full text3D triangle meshes are extremely used to model discrete surfaces, and almost always represented with two tables: one for geometry and another for connectivity. While the raw size of a triangle mesh is of around 200 bits per vertex, by coding cleverly (and separately) those two distinct kinds of information it is possible to achieve compression ratios of 15:1 or more. Different techniques must be used depending on whether single-rate vs. progressive bitstreams are sought; and, in the latter case, on whether or not hierarchically nested meshes are desirable during reconstructio
P. aeruginosa SGNH hydrolase-like proteins AlgJ and AlgX have similar topology but separate and distinct roles in alginate acetylation
No full textThe O-acetylation of polysaccharides is a common modification used by pathogenic organisms to protect against external forces. Pseudomonas aeruginosa secretes the anionic, O-acetylated exopolysaccharide alginate during chronic infection in the lungs of cystic fibrosis patients to form the major constituent of a protective biofilm matrix. Four proteins have been implicated in the O-acetylation of alginate, AlgIJF and AlgX. To probe the biological function of AlgJ, we determined its structure to 1.83 Å resolution. AlgJ is a SGNH hydrolase-like protein, which while structurally similar to the N-terminal domain of AlgX exhibits a distinctly different electrostatic surface potential. Consistent with other SGNH hydrolases, we identified a conserved catalytic triad composed of D190, H192 and S288 and demonstrated that AlgJ exhibits acetylesterase activity in vitro. Residues in the AlgJ signature motifs were found to form an extensive network of interactions that are critical for O-acetylation of alginate in vivo. Using two different electrospray ionization mass spectrometry (ESI-MS) assays we compared the abilities of AlgJ and AlgX to bind and acetylate alginate. Binding studies using defined length polymannuronic acid revealed that AlgJ exhibits either weak or no detectable polymer binding while AlgX binds polymannuronic acid specifically in a length-dependent manner. Additionally, AlgX was capable of utilizing the surrogate acetyl-donor 4-nitrophenyl acetate to catalyze the O-acetylation of polymannuronic acid. Our results, combined with previously published in vivo data, suggest that the annotated O-acetyltransferases AlgJ and AlgX have separate and distinct roles in O-acetylation. Our refined model for alginate acetylation places AlgX as the terminal acetlytransferase and provides a rationale for the variability in the number of proteins required for polysaccharide O-acetylation
Elucidating the role of N-acetylglucosamine in Group A Carbohydrate for the development of an effective glycoconjugate vaccine against Group A Streptococcus
No full textGroup A Carbohydrate (GAC), conjugated to an appropriate carrier protein, has been proposed as an attractive
vaccine candidate against Group A Streptococcus infections. Native GAC consists of a polyrhamnose (polyRha)
backbone with N-acetylglucosamine (GlcNAc) at every second rhamnose residue. Both native GAC and the
polyRha backbone have been proposed as vaccine components. Here, chemical synthesis and glycoengineering
were used to generate a panel of different length GAC and polyrhamnose fragments. Biochemical analyses were
performed confirming that the epitope motif of GAC is composed of GlcNAc in the context of the polyrhamnose
backbone. Conjugates from GAC isolated and purified from a bacterial strain and polyRha genetically expressed
in E. coli and with similar molecular size to GAC were compared in different animal models. The GAC conjugate
elicited higher anti-GAC IgG levels with stronger binding capacity to Group A Streptococcus strains than the
polyRha one, both in mice and in rabbits.
This work contributes to the development of a vaccine against Group A Streptococcus suggesting GAC as
preferable saccharide antigen to include in the vaccine
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