MICROFLUIDIC REACTOR TECHNOLOGY IN OLIGOSACCHARIDE SYNTHESIS

Although carbohydrates offer new therapeutic opportunities in biomedical field, the industrial implementation of carbohydrate-based drugs is yet greatly thwarted by the difficulties and challenges inherent in oligosaccharide synthesis, especially for large scale preparation. Any tool or new technology enabling a cost-effective improvement of the lead generation process is therefore highly desirable in order to reduce the manufacturing costs of carbohydrate drugs. During last years, continuous-flow synthesis in microreactors has gained a great deal of attention featuring practical advantages such as high reproducibility, easy scalability and fast reaction optimization using small amounts of reagents or synthetic intermediates. This technique may therefore offer an effective support to make carbohydrates more attractive targets for drug discovery processes. In addition, also basic research in academia can benefit from microreactor technology as a tool to improve the organic synthesis of oligosaccharides. Here I report a systematic exploration of the glycosylation reaction, the most important and difficult transformation in oligosaccharide synthesis, carried out in microreactors under continuous-flow conditions. Various trichloroacetimidates and thioglycosides have been investigated as glycosyl donors in this study, using both primary and secondary glycosyl acceptors. Each microfluidic glycosylation has been compared with the same reaction performed under traditional conditions, in order to highlight advantages and drawbacks of microreactors technology. As a significant example of multistep continuous-flow synthesis, we also describe the preparation of a trisaccharide by means of two consecutive glycosylations performed in two interconnected microreactors. Furthermore I report preliminary study on the synthesis of glycosyl phosphodiester under microfluidic conditions for the preparation of short oligomers of Neisseria Meningitidis type X capsular polysaccharide fragments

Exploring glycosylation reactions under continuous-flow conditions

The industrial development of carbohydrate-based drugs is greatly thwarted by the typical challenges inherent in oligosaccharide synthesis. The practical advantages of continuous-flow synthesis in microreactors (high reproducibility, easy scalability, and fast reaction optimization) may offer an effective support to make carbohydrates more attractive targets for drug-discovery processes. Here we report a systematic exploration of the glycosylation reaction carried out under microfluidic conditions. Trichloroacetimidates and thioglycosides have been investigated as glycosyl donors, using both primary and secondary acceptors. Each microfluidic glycosylation has been compared with the corresponding batch reaction, in order to highlight advantages and drawbacks of microreactors technology. As a significant example of multistep continuous-flow synthesis, we also describe the preparation of a trisaccharide by means of two consecutive glycosylations performed in interconnected microreactors

Synthesis and immunological evaluation of protein conjugates of neisseria meningitidis X capsular polysaccharide fragments

A vaccine to prevent infections from the emerging Neisseria meningitidis X (MenX) is becoming an urgent issue. Recently MenX capsular polysaccharide (CPS) fragments conjugated to CRM197 as carrier protein have been confirmed at preclinical stage as promising candidates for vaccine development. However, more insights about the minimal epitope required for the immunological activity of MenX CPS are needed. We report herein the chemical conjugation of fully synthetic MenX CPS oligomers (monomer, dimer, and trimer) to CRM197. Moreover, improvements in some crucial steps leading to the synthesis of MenX CPS fragments are described. Following immunization with the obtained neoglycoconjugates, the conjugated trimer was demonstrated as the minimal fragment possessing immunogenic activity, even though significantly lower than a pentadecamer obtained from the native polymer and conjugated to the same protein. This finding suggests that oligomers longer than three repeating units are possibly needed to mimic the activity of the native polysaccharide

Exploring new methods to control the stereoselectivity of glycosylation reactions

The synthesis of complex carbohydrate remains a challenge for synthetic chemists, especially in terms of stereocontrol (anomeric ratio) of glycosylation reactions. The stereoselective formation of glycosidic bonds depends indeed on numerous factors such as the reaction temperature, the solvent, the reagents concentration, the promoter and the coupling partners. In addition, the monosaccharide residues required for the assembly of larger saccharide structures are precious synthetic intermediates themselves as they require multistep synthesis. Large amount of starting material are usually consumed for the identification of the best reaction conditions using traditional batch procedures, and their optimization and subsequent scale-up of the optimized reaction conditions pose an additional hurdle. Continous-flow microfluidic devices offer a well-engineered approach to meet some of these challenges, especially in terms of better control of reaction parameters. Quite surprisingly, glycosylation reactions have been still little explored under continous-flow conditions, and only few example of this chemistry are reported in the literature.1 The aim of the present research project is to investigate the use of microfluidic reactor in carbohydrate chemistry area, with particular emphasis on the glycosylation reactions (especially in terms of stereoselection control). Initially, a panel of different glycosyl acceptors (primary and secondary) and different glycosyl donors (with or without neighbouring participating group at C-2 and with different leaving group at the anomeric carbon) were synthesized and employed in glycosylation reactions under batch conditions. Then, some glycosylation reactions were carried out under microfluidic conditions in order to compare their relative efficiencies. Stereochemical control of glycosylation reaction can be also achieved by using chiral promoter. Inspired by Fairbanks and co-workers who recently described glycosylation reactions promoted by a chiral Br\uf8nsted acid catalyst,2 we decided to explore the use of a chiral Lewis acid catalyst to induce stereoselective glycosylations. In particular, the stereochemical course of a model glycosylation reaction has been investigated using the trimethylsilyl esters derived from (R/S)-BINOL phosphoric acid

Major advances in the development of synthetic oligosaccharide-based vaccines

Because of their involvement in a variety of different biological processes and their occurrence onto pathogens and malignant cell surface, carbohydrates have been identified as ideal candidates for vaccine formulation. However, as free oligosaccharides are poorly immunogenic and do not induce immunological memory in the most at risk population (infants and young children, elderly and immunocompromised patients), glycoconjugate vaccines containing the same carbohydrate antigen covalently linked to an immunogenic carrier protein have gained a prominent role. Accordingly, a number of glycoconjugate vaccines mostly directed against infections caused by bacterial pathogens have been licensed and are currently available on the market. However, also glycoconjugate vaccines suffer from significant drawbacks. The challenging procedures required for the isolation and purification of the carbohydrate antigen from its natural source often lead to poor homogeneity and presence of biological contaminants, resulting in batch-to-batch variability. Moreover, in some cases, the overwhelming immunogenicity of the carrier protein may induce the carbohydrate epitope suppression, causing hyporesponsiveness. The development of synthetic oligosaccharide-based vaccine candidates, characterized by the presence of pure and well-defined synthetic oligosaccharide structures, is expected to meet the requirement of homogeneous and highly reproducible preparations. In the present chapter, we report on the major advances in the development of synthetic carbohydrate-based vaccines. First of all, we describe different strategies developed during the last years to circumvent the inherent difficulties of classical oligosaccharide synthesis, such as the one-pot glycosylation and the solid-phase synthesis, and their application to the preparation of carbohydrate antigens apt to conjugation with protein carriers. Next, we discuss the most representative methodologies employed for the chemical ligation of oligosaccharide structures to proteins. Finally, in the last section, we report significant examples of fully synthetic vaccines exploiting the multivalency effect. These constructs are based on the concept that the conjugation of multiple copies of synthetic oligosaccharide antigens to multivalent scaffolds, such as dendrimers, (cyclo)peptides, gold nanoparticles, and calixarenes, raises cooperative interactions between carbohydrates and immune receptors, leading to strong enhancement of the saccharide antigen immunogenicity

Synthesis of fragments of salmonella Typhi capsular polysaccharide and their zwitterionic analogues

Capsular polysaccharides (CPS) are T-independent antigens, and therefore they are not able to induce the formation of memory B cells. Their immunogenicity can be enhanced by conjugation with a immunogenic protein, forming a T-dependent glycoconjugate. Recently it has been discovered a new group of CPS that can directly activate T cells through the traditional MHC-II-dependent mechanism. Although these molecules show a wide diversity of chemical structure, they share the common characteristic of presenting a zwitterionic charge motif distributed along the chain, i.e. they contain both positive (e.g. NH3+) and negative (e.g. phosphate or carboxylate) charge centers within a repeating unit structure (zwitterionic polysaccharides, ZPS). This zwitterionic charge motif is believed to be responsible of their peculiar immunological activity, which is unique among bacterial polysaccharides. ZPS might offer previously unrecognized opportunities for the design of new classes of vaccines, based on the artificial introduction of a zwitterionic charge motif by chemical modification of surface glycans of pathogens. The design of new T cell dependent, ZPS-based antigens for vaccine formulation, however, requires a better understanding of how ZPS antigens stimulate the host immune system and a correlation of the ZPS structural and conformational properties with their biological activity. We report the preparation of fully synthetic oligomers of Salmonella enterica serovar Typhi (often called S. Typhi) CPS, and their zwitterionic analogues. S. Typhi is a motile Gram-negative bacterium, whose CPS (often referred to as Vi antigen) is an anionic polymer composed by \u3b1-(1-4)-linked N-acetyl galactosaminuronic acid repeating units predominantly O-acetylated at position 3. The synthesis of Vi oligomers required a strategy based on two versatile intermediates suitably protected for the systematic introduction of charge centres on each repeating unit, and containing non participating protecting groups onto 2-amino functions to allow the formation of 1,2-cis glycosidic linkages. Both are easily attainable from commercially available D-galactosamine hydrochloride. We employed the non participating azide group, which can be converted into the amino group (ZPS) or into the acetamido function (natural Vi) in the late stages of the synthesis. After extensive exploration, the glycosylation reactions were carried out using N-phenyl-trifluoroacetimidates as glycosyl donors, yielding stereoselectively the desired \u3b1 product. The immunological properties of the synthetic oligomers will be investigated in order to correlate the structural features with their biological behaviour

Synthesis of fragments of Salmonella thyphi capsular polysaccharide and their zwitterionic analogues

Capsular polysaccharides (CPS) are T-independent antigens, and therefore they are not able to induce the formation of memory B cells. Their immunogenicity can be enhanced by conjugation with a immunogenic protein, forming a T-dependent glycoconjugate. Recently it has been discovered a new group of CPS that can directly activate T cells through the traditional MHC-II-dependent mechanism. Although these molecules show a wide diversity of chemical structure, they share the common characteristic of presenting a zwitterionic charge motif distributed along the chain, i.e. they contain both positive (e.g. NH3+) and negative (e.g. phosphate or carboxylate) charge centers within a repeating unit structure (zwitterionic polysaccharides, ZPS). This zwitterionic charge motif is believed to be responsible of their peculiar immunological activity, which is unique among bacterial polysaccharides. ZPS might offer previously unrecognized opportunities for the design of new classes of vaccines, based on the artificial introduction of a zwitterionic charge motif by chemical modification of surface glycans of pathogens. The design of new T cell dependent, ZPS-based antigens for vaccine formulation, however, requires a better understanding of how ZPS antigens stimulate the host immune system and a correlation of the ZPS structural and conformational properties with their biological activity. We report the preparation of fully synthetic oligomers of Salmonella enterica serovar Typhi (often called S. typhi) CPS, and their zwitterionic analogues. S. Typhi is a motile Gram-negative bacterium, whose CPS (often referred to as Vi antigen) is an anionic polymer composed by \u3b1-(1-4)-linked N-acetyl galactosaminuronic acid repeating units predominantly O-acetylated at position 3. The synthesis of Vi oligomers required a strategy based on two versatile intermediates suitably protected for the systematic introduction of charge centres on each repeating unit, and containing non participating protecting groups onto 2-amino functions to allow the formation of 1,2-cis glycosidic linkages. Both are easily attainable from commercially available D-galactosamine hydrochloride. We employed the non participating azide group, which can be converted into the amino group (ZPS) or into the acetamido function (natural Vi) in the late stages of the synthesis. After extensive exploration, the glycosylation reactions were carried out using N-phenyl-trifluoroacetimidates as glycosyl donors, yielding stereoselectively the desired \u3b1 product. The immunological properties of the synthetic oligomers will be investigated in order to correlate the structural features with their biological behaviour

Synthesis of fragments of Salmonella Typhi capsular polysaccharide and their zwitterionic analogues

Capsular polysaccharides (CPS) are T-independent antigens, and therefore they are not able to induce the formation of memory B cells. Their immunogenicity can be enhanced by conjugation with a immunogenic protein, forming a T-dependent glycoconjugate. Recently it has been discovered a new group of CPS that can directly activate T cells through the traditional MHC-II-dependent mechanism. Although these molecules show a wide diversity of chemical structure, they share the common characteristic of presenting a zwitterionic charge motif distributed along the chain, i.e. they contain both positive (e.g. NH3+) and negative (e.g. phosphate or carboxylate) charge centers within a repeating unit structure (zwitterionic polysaccharides, ZPS). This zwitterionic charge motif is believed to be responsible of their peculiar immunological activity, which is unique among bacterial polysaccharides. ZPS might offer previously unrecognized opportunities for the design of new classes of vaccines, based on the artificial introduction of a zwitterionic charge motif by chemical modification of surface glycans of pathogens. The design of new T cell dependent, ZPS-based antigens for vaccine formulation, however, requires a better understanding of how ZPS antigens stimulate the host immune system and a correlation of the ZPS structural and conformational properties with their biological activity. We report the preparation of fully synthetic oligomers of Salmonella enterica serovar Typhi (often called S. Typhi) CPS, and their zwitterionic analogues. S. Typhi is a motile Gram-negative bacterium, whose CPS (often referred to as Vi antigen) is an anionic polymer composed by \u3b1-(1-4)-linked N-acetyl galactosaminuronic acid repeating units predominantly O-acetylated at position 3. The synthesis of Vi oligomers required a strategy based on two versatile intermediates suitably protected for the systematic introduction of charge centres on each repeating unit, and containing non participating protecting groups onto 2-amino functions to allow the formation of 1,2-cis glycosidic linkages. Both are easily attainable from commercially available D-galactosamine hydrochloride. We employed the non participating azide group, which can be converted into the amino group (ZPS) or into the acetamido function (natural Vi) in the late stages of the synthesis. After extensive exploration, the glycosylation reactions were carried out using N-phenyl-trifluoroacetimidates as glycosyl donors, yielding stereoselectively the desired \u3b1 product. The immunological properties of the synthetic oligomers will be investigated in order to correlate the structural features with their biological behaviour

Synthesis of fragments of Salmonella Typhi capsular polysaccharide and their zwitterionic analogues

Salmonella enterica serovar Typhi (often called S. Typhi) is a motile Gram-negative bacterium, whose capsular polysaccharide (often referred to as Vi antigen) is an anionic polymer composed of \u3b1-(1-4)-linked N-acetyl-galactosaminuronic acid repeating units predominantly O-acetylated at position 3. The degree of acetylation was found to be crucial for the immunogenicity. We investigated two different synthetic routes for the oligomers assembly: a late stage post-glycosylation oxidation approach and a pre-glycosylation oxidation strategy, the latter employing suitably protected uronates donor and acceptor. Glycosylation reactions were carried out using N-phenyltrifluoroacetimidates as glycosyl donors, and experimental conditions were carefully screened in order to ensure the stereoselective formation of the desired \u3b1 product. The azido group at C-2 can be converted into either an acetamido function (natural Vi) or a free amino group (zwitterionic derivatives). Finally, a pentenyl linker was installed at C-1 of the reducing end in order to facilitate the subsequent conjugation to protein carrier and/or multivalent scaffolds