Design of Orthogonally Protected Tetrasaccharides, Antigen Previously Representative of a Selection of Shigella Flexneri Serotypes

Les maladies diarrhéiques sont la deuxième cause de mortalité chez les enfants de moins de cinq ans. Les entérobactéries Shigella flexneri sont les principales responsables de la forme endémique de la shigellose, une maladie diarrhéique importante dans les pays en développement et pour laquelle de nombreuses stratégies vaccinales sont à l’étude. La partie polysaccharidique (antigène O, Ag-O) du lipopolysaccharide de surface est l’une des cibles majeures d'immunité protectrice contre la réinfection. Une grande variété d’Ag-Os, reflétant la diversité sérotypique, a été identifiée. De façon intéressante ces Ag-Os se différencient par la nature des substituants portés par le tétrasaccharide ABCD qui définit leur squelette commun. Afin de développer un vaccin issu de sucres de synthèse à large couverture sérotypique contre S. flexneri, des stratégies de synthèse hautement convergente d’analogues orthogonalement protégés du tétrasaccharide ABCD ont été explorées. Elles prennent en compte les sites de -D-glucosylation et de O-acétylation spécifiques de sérotypes. Les approches mises en place s’appuient sur la synthèse d’une diversité de précurseurs en série L-rhamnopyranose et 2-N-acétyl-2-désoxy-D-glucopyranosamine et leurs combinaisons optimisées. Quelques exemples de glucosylation 1,2-cis régiosélective, chimique et/ou enzymatique, valident le concept.Diarrhoeal diseases are the second cause of death among children under five. Shigella flexneri enterobacteria are the main causative agents of the endemic form of shigellosis, a diarrhoeal disease of high prevalence in developing countries and one for which numerous vaccine strategies are under studied. The polysaccharide part (O-antigen, O-Ag) of the bacterial lipopolysaccharide is a major target of protective immunity against reinfection. A large variety of O-Ags, expressing serotypic diversity, has been identified. Interestingly, these O-Ags differ by the nature of the substitutions occurring on the ABCD tetrasaccharide, which defines their common backbone. In order to develop a synthetic carbohydrate-based vaccine with broad serotype coverage against S. flexneri, highly convergent synthetic strategies towards orthogonally protected analogs of tetrasaccharide ABCD were investigated, while taking into account serotype-specific -D-glucosylation and O-acetylation sites. The selected approaches feature the synthesis of a variety of suitable L-rhamnopyranose and 2-N-acetyl-2-deoxy-D-glucopyranosamine precursors and their optimized combinations. The concept is supported by selected examples of 1,2-cis chemical and/or enzymatic glucosylation

A convergent chemoenzymatic strategy to deliver a diversity of Shigella flexneri serotype-specific O-antigen segments from a unique lightly protected tetrasaccharide core

International audienceProgress in glycoscience is strongly dependent on the availability of broadly diverse tailored-made, well-defined and often complex oligosaccharides. Herein, going beyond natural resources and aiming to circumvent chemical boundaries in glycochemistry, we tackle the development of an in vitro chemoenzymatic strategy holding great potential to answer the need for molecular diversity characterizing microbial cell-surface carbohydrates. The concept is exemplified in the context of Shigella flexneri, a major cause of diarrheal disease. Aiming at a broad serotype coverage S. flexneri glycoconjugate vaccine, a non-natural lightly protected tetrasaccharide was designed for compatibility with (i) serotype-specific glucosylations and O-acetylations defining S. flexneri O-antigens, (ii) recognition by suitable α-transglucosylases, and (iii) programmed oligomerization post enzymatic -D-glucosylation. The tetrasaccharide core was chemically synthesized from two crystalline monosaccharide precursors. Six α-transglucosylases found in the Glycoside Hydrolase family 70 were shown to transfer glucosyl residues on the non-natural acceptor. The successful proof-of-concept is achieved for a pentasaccharide featuring the glucosylation pattern from the S. flexneri type IV O-antigen. It demonstrates the potential of appropriately planned chemo-enzymatic pathways involving non-natural acceptors and low-cost donor/transglucosylase systems to achieve the demanding regioselective -D-glucosylation of large substrates, paving the way to microbial oligosaccharides of vaccinal interest

Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens

International audienceThe (chemo-)enzymatic synthesis of oligosaccharides has been hampered by the lack of appropriate enzymatic tools with requisite regio-and stereo-specificities. Engineering of carbohydrate-active enzymes, in particular targeting the enzyme active site, has notably led to catalysts with altered regioselectivity of the glycosylation reaction thereby enabling to extend the repertoire of enzymes for carbohydrate synthesis. Using a collection of 22 mutants of ΔN 123-GBD-CD2 branching sucrase, an enzyme from the Glycoside Hydrolase family 70, containing between one and three mutations in the active site, and a lightly protected chemically synthesized tetrasaccharide as an acceptor substrate, we showed that altered glycosylation product specificities could be achieved compared to the parental enzyme. Six mutants were selected for further characterization as they produce higher amounts of two favored pentasaccharides compared to the parental enzyme and/or new products. The produced pentasaccharides were shown to be of high interest as they are precursors of representative haptens of Shigella flexneri serotypes 3a, 4a and 4b. Furthermore, their synthesis was shown to be controlled by the mutations introduced in the active site, driving the glucosylation toward one extremity or the other of the tetrasaccharide acceptor. To identify the molecular determinants involved in the change of ΔN 123-GBD-CD2 regioselectivity, extensive molecular dynamics simulations were carried out in combination with in-depth analyses of amino acid residue networks. Our findings help to understand the interrelationships between the enzyme structure, conformational flexibility and activity. They also provide new insight to further engineer this class of enzymes for the synthesis of carbohydrate components of bacterial haptens. Carbohydrate-active enzymes catalyze a wide range of chemical reactions. They have emerged as a practical alternative to chemical catalysts, avoiding multiple steps of protection and deprotection often required in chemical synthesis to control the reactivity of the sugar hydroxyl groups and regio-and stereo-selectivity of the reaction. Some of them are rather versatile biocatalysts often displaying naturally a relaxed substrate specificity. This promiscuity can be further exacerbated by enzyme engineering to either broaden or narrow down the range of recognized substrates and/or control the reaction selectivity 1. In particular, mutagenesis targeting the enzym