This work has described synthetic strategies towards well-defined structures resembling capsular polysaccharide (CPS) fragments, CPS mimics, teichoic acid (TA) fragments as well as a third... Show moreThis work has described synthetic strategies towards well-defined structures resembling capsular polysaccharide (CPS) fragments, CPS mimics, teichoic acid (TA) fragments as well as a third-generation ring-closing tandem metathesis (RCM) linker to better exploit the potential of automated synthesis. The synthesis of diheteroglycan (DHG) fragments of the Gram-positive Enterococcus faecalis have been described and preliminary biological properties shown. CPS-mimics of Neisseria meningitidis A, a Gram-negative bacterium and one of the major causes of bacterial meningitis, could provide an alternative to the inherent instability of current glycoconjugate vaccines. TA fragments presented in this thesis, typical for Gram-positive bacteria, have been equipped with labile D-alanine appendages to further investigate their biological relevance in interactions with the host immune system. A tandem-RCM linker has been developed and tested on a carbohydrate automated synthesizer, providing a proof of concept of its use. Show less
Group B Streptococcus (GBS) is a Gram-positive bacterium and the most common cause of neonatal blood and brain infections. At least 10 different serotypes exist, that are characterized by their... Show moreGroup B Streptococcus (GBS) is a Gram-positive bacterium and the most common cause of neonatal blood and brain infections. At least 10 different serotypes exist, that are characterized by their different capsular polysaccharides. The Group B carbohydrate (GBC) is shared by all serotypes and therefore attractive be used in a glycoconjugate vaccine. The GBC is a highly complex multiantennary structure, composed of rhamnose rich oligosaccharides interspaced with glucitol phosphates. We here report the development of a convergent approach to assemble a pentamer, octamer, and tridecamer fragment of the termini of the antennae. Phosphoramidite chemistry was used to fuse the pentamer and octamer fragments to deliver the 13-mer GBC oligosaccharide. Nuclear magnetic resonance spectroscopy of the generated fragments confirmed the structures of the naturally occurring polysaccharide. The fragments were used to generate model glycoconjugate vaccine by coupling with CRM197. Immunization of mice delivered sera that was shown to be capable of recognizing different GBS strains. The antibodies raised using the 13-mer conjugate were shown to recognize the bacteria best and the serum raised against this GBC fragment-mediated opsonophagocytic killing best, but in a capsule dependent manner. Overall, the GBC 13-mer was identified to be a highly promising antigen for incorporation into future (multicomponent) anti-GBS vaccines. Show less
Group B Streptococcus (GBS) is a Gram-positive bacterium and the most common cause of neonatal blood and brain infections. At least 10 different serotypes exist, that are characterized by their... Show moreGroup B Streptococcus (GBS) is a Gram-positive bacterium and the most common cause of neonatal blood and brain infections. At least 10 different serotypes exist, that are characterized by their different capsular polysaccharides. The Group B carbohydrate (GBC) is shared by all serotypes and therefore attractive be used in a glycoconjugate vaccine. The GBC is a highly complex multiantennary structure, composed of rhamnose rich oligosaccharides interspaced with glucitol phosphates. We here report the development of a convergent approach to assemble a pentamer, octamer, and tridecamer fragment of the termini of the antennae. Phosphoramidite chemistry was used to fuse the pentamer and octamer fragments to deliver the 13-mer GBC oligosaccharide. Nuclear magnetic resonance spectroscopy of the generated fragments confirmed the structures of the naturally occurring polysaccharide. The fragments were used to generate model glycoconjugate vaccine by coupling with CRM197. Immunization of mice delivered sera that was shown to be capable of recognizing different GBS strains. The antibodies raised using the 13-mer conjugate were shown to recognize the bacteria best and the serum raised against this GBC fragment-mediated opsonophagocytic killing best, but in a capsule dependent manner. Overall, the GBC 13-mer was identified to be a highly promising antigen for incorporation into future (multicomponent) anti-GBS vaccines. Show less
There is a vast genomic resource for enzymes active on carbohydrates. Lagging far behind, however, are functional chemical tools for the rapid characterization of carbohydrate-active enzymes.... Show moreThere is a vast genomic resource for enzymes active on carbohydrates. Lagging far behind, however, are functional chemical tools for the rapid characterization of carbohydrate-active enzymes. Activity-based probes (ABPs) offer one chemical solution to these issues with ABPs based upon cyclophellitol epoxide and aziridine covalent and irreversible inhibitors representing a potent and widespread approach. Such inhibitors for enzymes active on polysaccharides are potentially limited by the requirement for several glycosidic bonds, themselves substrates for the enzyme targets. Here, it is shown that non-hydrolysable trisaccharide can be synthesized and applied even to enzymes with challenging subsite requirements. It was found that incorporation of carbasugar moieties, which was accomplished by cuprate-assisted regioselective trans-diaxial epoxide opening of carba-mannal synthesised for this purpose, yields inactivators that act as powerful activity-based inhibitors for alpha-1,6 endo-mannanases. 3-D structures at 1.35-1.47 angstrom resolutions confirm the design rationale and binding to the enzymatic nucleophile. Carbasugar oligosaccharide cyclophellitols offer a powerful new approach for the design of robust endoglycosidase inhibitors, while the synthesis procedures presented here should allow adaptation towards activity-based endoglycosidase probes as well as configurational isosteres targeting other endoglycosidase families. Show less
Berni, F.; Enotarpi, J.; Voskuilen, T.; Li, S.; Marel, G.A. van der; Codée, J.D.C. 2020
Glycopolymers are found surrounding the outer layer of many bacterial species. The first uses as immunogenic component in vaccines are reported since the beginning of the XX century, but it is only... Show moreGlycopolymers are found surrounding the outer layer of many bacterial species. The first uses as immunogenic component in vaccines are reported since the beginning of the XX century, but it is only in the last decades that glycoconjugate based vaccines have been effectively applied for controlling and preventing several infectious diseases, such as H. influenzae type b (Hib), N. meningitidis, S. pneumoniae or group B Streptococcus. Methicillin resistant S. aureus (MRSA) strains has been appointed by the WHO as one of those pathogens, for which new treatments are urgently needed. Herein we present an overview of the carbohydrate-based cell wall polymers associated with different S. aureus strains and the related affords to deliver well-defined fragments through synthetic chemistry. Show less
Enotarpi, J.; Tontini, M.; Balocchi, C.; Es, D. van der; Auberger, L.C.; Balducci, E.; ... ; Adamo, R. 2020
Correction to: Nature Communicationshttps://doi.org/10.1038/s41467-020-18279-x, published online 7 September 2020.The original version of this Article contained errors in Fig. 1. The label ‘a’ was... Show moreCorrection to: Nature Communicationshttps://doi.org/10.1038/s41467-020-18279-x, published online 7 September 2020.The original version of this Article contained errors in Fig. 1. The label ‘a’ was omitted for the left panel and the label ‘b’ was omitted for the right panel. In Fig. 1b, the right parenthesis was incorrectly positioned indicating a 4-carbon linker, rather than the correct 6-carbon linker. These errors have been corrected in both the PDF and HTML versions of the Article. Show less
Infections 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... Show moreInfections 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 the 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. Resulting antibodies were specific towards the synthetic structures and mediated in-vitro opsonophagocytic killing of different encapsulated E. feacalis strains. The evaluated structures 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. Show less
Wang, L.; Berni, F.; Enotarpi, J.; Overkleeft, H.S.; Marel, G.A. van der; Codée, J.D.C. 2020
The stereoselective construction of 1,2-cis-glycosidic linkages is key in the assembly of biologically relevant glycans, but remains a synthetic challenge. Reagent-controlled glycosylation... Show moreThe stereoselective construction of 1,2-cis-glycosidic linkages is key in the assembly of biologically relevant glycans, but remains a synthetic challenge. Reagent-controlled glycosylation methodologies, in which external nucleophiles are employed to modulate the reactivity of the glycosylation system, have become powerful means for the construction of 1,2-cis-glycosidic linkages. Here we establish that nucleophilic additives can support the construction of α-1,2-glucans, and apply our findings in the construction of a d-alanine kojibiose functionalized glycerol phosphate teichoic acid fragment. This latter molecule can be found in the cell wall of the opportunistic Gram-positive bacterium, Enterococcus faecalis and represents a structural element that can possibly be used in the development of therapeutic vaccines and diagnostic tools. Show less
