Anomeric O-Functionalization of Carbohydrates for Chemical Conjugation to Vaccine Constructs.

Carbohydrates mediate a wide range of biological interactions, and understanding these processes benefits the development of new therapeutics. Isolating sufficient quantities of glycoconjugates from biological samples remains a significant challenge. With advances in chemical and enzymatic carbohydrate synthesis, the availability of complex carbohydrates is increasing and developing methods for stereoselective conjugation these polar head groups to proteins and lipids is critically important for pharmaceutical applications. The aim of this review is to provide an overview of commonly employed strategies for installing a functionalized linker at the anomeric position as well as examples of further transformations that have successfully led to glycoconjugation to vaccine constructs for biological evaluation as carbohydrate-based therapeutics

Biocatalyzed synthesis of glycostructures with anti-infective activity

Molecules containing carbohydrate moieties play essential roles in fighting a variety of bacterial and viral infections. Consequently, the design of new carbohydrate-containing drugs or vaccines has attracted great attention in recent years as means to target several infectious diseases. Conventional methods to produce these compounds face numerous challenges because their current production technology is based on chemical synthesis, which often requires several steps and uses environmentally unfriendly reactants, contaminant solvents, and inefficient protocols. The search for sustainable processes such as the use of biocatalysts and eco-friendly solvents is of vital importance. Therefore, their use in a variety of reactions leading to the production of pharmaceuticals has increased exponentially in the last years, fueled by recent advances in protein engineering, enzyme directed evolution, combinatorial biosynthesis, immobilization techniques, and flow biocatalysis. In glycochemistry and glycobiology, enzymes belonging to the families of glycosidases, glycosyltransferases (Gtfs), lipases, and, in the case of nucleoside and nucleotide analogues, also nucleoside phosphorylases (NPs) are the preferred choices as catalysts. In this Account, on the basis of our expertise, we will discuss the recent biocatalytic and sustainable approaches that have been employed to synthesize carbohydrate-based drugs, ranging from antiviral nucleosides and nucleotides to antibiotics with antibacterial activity and glycoconjugates such as neoglycoproteins (glycovaccines, GCVs) and glycodendrimers that are considered as very promising tools against viral and bacterial infections. In the first section, we will report the use of NPs and N-deoxyribosyltransferases for the development of transglycosylation processes aimed at the synthesis of nucleoside analogues with antiviral activity. The use of deoxyribonucleoside kinases and hydrolases for the modification of the sugar moiety of nucleosides has been widely investigated. Next, we will describe the results obtained using enzymes for the chemoenzymatic synthesis of glycoconjugates such as GCVs and glycodendrimers with antibacterial and antiviral activity. In this context, the search for efficient enzymatic syntheses represents an excellent strategy to produce structure-defined antigenic or immunogenic oligosaccharide analogues with high purity. Lipases, glycosidases, and Gtfs have been used for their preparation. Interestingly, many authors have proposed the use Gtfs originating from the biosynthesis of natural glycosylated antibiotics such as glycopeptides, macrolides, and aminoglycosides. These have been used in the chemoenzymatic semisynthesis of novel antibiotic derivatives by modification of the sugar moiety linked to their complex scaffold. These contributions will be described in the last section of this review because of their relevance in the fight against the spreading phenomenon of antibiotic resistance. In this context, the pioneering in vivo synthesis of novel derivatives obtained by genetic manipulation of producer strains (combinatorial biosynthesis) will be shortly described as well. All of these strategies provide a useful and environmentally friendly synthetic toolbox. Likewise, the field represents an illustrative example of how biocatalysis can contribute to the sustainable development of complex glycan-based therapies and how problems derived from the integration of natural tools in synthetic pathways can be efficiently tackled to afford high yields and selectivity. The use of enzymatic synthesis is becoming a reality in the pharmaceutical industry and in drug discovery to rapidly afford collections of new antibacterial or antiviral molecules with improved specificity and better metabolic stability

Characterization of Cell Glycocalyx with Mass Spectrometry Methods.

The cell membrane plays an important role in protecting the cell from its extracellular environment. As such, extensive work has been devoted to studying its structure and function. Crucial intercellular processes, such as signal transduction and immune protection, are mediated by cell surface glycosylation, which is comprised of large biomolecules, including glycoproteins and glycosphingolipids. Because perturbations in glycosylation could result in dysfunction of cells and are related to diseases, the analysis of surface glycosylation is critical for understanding pathogenic mechanisms and can further lead to biomarker discovery. Different mass spectrometry-based techniques have been developed for glycan analysis, ranging from highly specific, targeted approaches to more comprehensive profiling studies. In this review, we summarized the work conducted for extensive analysis of cell membrane glycosylation, particularly those employing liquid chromatography with mass spectrometry (LC-MS) in combination with various sample preparation techniques

Sugars : burden or biomaterials of the future?

During the past few years, the field of tissue engineering (TE) has been shifting from replacement to regenerative strategies. Following this tendency, the requirements for biomaterials to be used in TE have been also changing. While a few decades ago bioinert materials that do not provoke undesired body responses were in the focus of material sciences, nowadays third generation biomaterials mimicking the nanoscale mechanisms of the interactions between cells and their in vivo environment are the target of material design. Although these mechanisms involve different bioactive molecules, until now mainly strategies involving small peptide epitopes that copycat specific sequences of complex proteins have been exploited. The breakthroughs that such approaches brought to biomaterials and TE fields are undeniable. Nevertheless, the important role that carbohydrates play in tissue structuring and function is still poorly explored and exploited in this context and we believe that this is one of the missing pieces in the TE puzzle. Carbohydrates are an integral part of our life. We are literally covered by them: from bacteria to mammalian cells, the molecular landscape of the cell surface is coated with sugars forming the so-called glycocalyx. This strategic placement of the sugars makes them crucial for the development, growth, function and/or survival of an organism. It is believed that the structural diversity of carbohydrates is the key for understanding and controlling those processes because of the huge number of ligand structures, which sugars can display in molecular recognition systems. However, their main advantages: the intricacy and the large natural diversity have turned against the scientists and have hampered their study. As a result, the field of glycomics is much less developed compared to its counterparts genomics and proteomics within TE. Recent advances in carbohydrate synthesis, sensing technologies and processing methodologies are inducing rapid changes in this field and will be discussed in this paThe authors acknowledge the funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. NMP4-SL-2009-229292

Glycation of Animal Proteins Via Maillard Reaction and Their Bioactivity

Nowadays there has been an increase in the need to incorporate foods in our diets that have optimal and palatable organoleptic characteristics as well as complex interaction in human biological processes that provide beneficial properties to human health. Animal foods and their by-products are an important source of macro- and micronutrients and also a great protein source; nevertheless the consumption of these products has been decreasing since they have been associated with the generation of chronic degenerative diseases; therefore the food industry has sought to innovate toward the generation of healthier foods. This chapter presents an overview of the glycation of proteins of animal origin via the Maillard reaction emphasizing on their posttranslational modifications and their possible uses in food, based on their bioactivity

Combined chemical synthesis and tailored enzymatic elongation provide fully synthetic and conjugation-ready Neisseria meningitidis serogroup X vaccine antigens

Studies on the polymerization mode of Neisseria meningitidis serogroup X capsular polymerase CsxA recently identified a truncated construct that can be immobilized and used for length controlled on-column production of oligosaccharides. Here, we combined the use of a synthetic acceptor bearing an appendix for carrier protein conjugation and the on-column process to a novel chemo-enzymatic strategy. After protein coupling of the size optimized oligosaccharide produced by the one-pot elongation procedure, we obtained a more homogeneous glycoconjugate compared to the one previously described starting from the natural polysaccharide. Mice immunized with the conjugated fully synthetic oligomer elicited functional antibodies comparable to controls immunized with the current benchmark MenX glycoconjugates prepared from the natural capsule polymer or from fragments of it enzymatically elongated. This pathogen-free technology allows the fast total in vitro construction of predefined bacterial polysaccharide fragments. Compared to conventional synthetic protocols, the procedure is more expeditious and drastically reduces the number of purification steps to achieve the oligomers. Furthermore, the presence of a linker for conjugation in the synthetic acceptor minimizes manipulations on the enzymatically produced glycan prior to protein conjugation. This approach enriches the methods for fast construction of complex bacterial carbohydrates

Impact and Control of Sugar Size in Glycoconjugate Vaccines

Glycoconjugate vaccines have contributed enormously to reducing and controlling encapsulated bacterial infections for over thirty years. Glycoconjugate vaccines are based on a carbohydrate antigen that is covalently linked to a carrier protein; this is necessary to cause T cell responses for optimal immunogenicity, and to protect young children. Many interdependent parameters affect the immunogenicity of glycoconjugate vaccines, including the size of the saccharide antigen. Here, we examine and discuss the impact of glycan chain length on the efficacy of glycoconjugate vaccines and report the methods employed to size polysaccharide antigens, while highlighting the underlying reaction mechanisms. A better understanding of the impact of key parameters on the immunogenicity of glycoconjugates is critical to developing a new generation of highly effective vaccines

From structural to functional glycomics: core substitutions as molecular switches for shape and lectin affinity of N-glycans

Glycan epitopes of cellular glycoconjugates act as versatile biochemical signals (sugar coding). Here, we test the hypothesis that the common N-glycan modifications by core fucosylation and introduction of the bisecting N-acetylglucosamine moiety have long-range effects with functional consequences. Molecular dynamics simulations indicate a shift in conformational equilibria between linear extension or backfolding of the glycan antennae upon substitution. We also present a new fingerprint-like mode of presentation for this multi-parameter system. In order to delineate definite structure-function relationships, we strategically combined chemoenzymatic synthesis with bioassaying cell binding and the distribution of radioiodinated neoglycoproteins in vivo. Of clinical relevance, tailoring the core region affects serum clearance markedly, e. g., prolonging circulation time for the neoglycoprotein presenting the N-glycan with both substitutions. alpha 2,3-Sialylation is another means toward this end, similarly seen for type II branching in triantennary N-glycans. This discovery signifies that rational glycoengineering along the given lines is an attractive perspective to optimize pharmacokinetic behavior of glycosylated pharmaproteins. Of general importance for the concept of the sugar code, the presented results teach the fundamental lesson that N-glycan core substitutions convey distinct characteristics to the concerned oligosaccharide relevant for cis and trans biorecognition processes. These modifications are thus molecular switches

Biomimetic Macromolecules for Macrophage Targeting and Modulation

Carbohydrate recognition has come to the forefront of biological aiming to uncover the mechanisms of physiological and pathological processes. Cell surface glycans are involved in processes including cellular adhesion, cell signaling, and immune response. A new approach for profiling cell surface glycans has great potential for a wide range of biomedical applications. Lectins have been conventionally used to determine the structure and function of glycoproteins, however, their numbers are still restricted compared to the number of glycan structures. Boronic acid has proven a remarkable small molecule capable of binding diols in aqueous solution. This interaction indicates boronic acid derived molecules may serve as lectin mimetics for profiling and targeting cell surface glycans. In the first part of this dissertation study the specific binding site of boronic acid to individual pyranosides was confirmed followed by the synthesis and evaluation of protein-boronic acid conjugates as lectin mimetics. 3-aminophenylboronic acid was conjugated to gluco-, manno- and galactopyranosides, followed by methylation, both under basic conditions. Based on a specific permethylation product for the carbohydrate, boronic acid specificity towards 1,2 and 1,3 diol configurations was confirmed by 1H, 13C NMR, and mass spectrometry. As a result, unique binding profiles were observed for each pyranoside. Next, bovine serum albumin (BSA)-PBA conjugates were synthesized in a density controlled affording multivalent lectin mimetics. The resultant BSA-PBA conjugates were characterized by SDS-PAGE and MALDI-TOF MS. Cell surface glycan binding capacity was confirmed by a competitive lectin assay examined by flow cytometry. Macrophages express lectins as receptors for specific immune responses. Synthetic glycans are candidates for targeting cell surface lectins and for immunomodulation applications. In the second part of this dissertation, novel N-glycan polymers were synthesized and their immunomodulation effects were examined. N-linked glycopolymers were synthesized via cyanoxyl-mediated free radical polymerization (CMFRP). Then, their cytotoxicity and cell activation abilities against RAW 264.7 cells were examined. As a result, N-glycan polymers showed no cytotoxicity at a concentration of 1,250 mg/mL except the N-alpha-2,6-sialolactosyl polymer, which proved cytotoxic at 1250 µg/mL. N-alpha-2,3-sialolactosyl polymer showed the strongest activity for inducing cell surface marker expression compared to controls, indicating high macrophage modulation activity

Biomimetic Macromolecules for Macrophage Targeting and Modulation

Carbohydrate recognition has come to the forefront of biological aiming to uncover the mechanisms of physiological and pathological processes. Cell surface glycans are involved in processes including cellular adhesion, cell signaling, and immune response. A new approach for profiling cell surface glycans has great potential for a wide range of biomedical applications. Lectins have been conventionally used to determine the structure and function of glycoproteins, however, their numbers are still restricted compared to the number of glycan structures. Boronic acid has proven a remarkable small molecule capable of binding diols in aqueous solution. This interaction indicates boronic acid derived molecules may serve as lectin mimetics for profiling and targeting cell surface glycans. In the first part of this dissertation study the specific binding site of boronic acid to individual pyranosides was confirmed followed by the synthesis and evaluation of protein-boronic acid conjugates as lectin mimetics. 3-aminophenylboronic acid was conjugated to gluco-, manno- and galactopyranosides, followed by methylation, both under basic conditions. Based on a specific permethylation product for the carbohydrate, boronic acid specificity towards 1,2 and 1,3 diol configurations was confirmed by 1H, 13C NMR, and mass spectrometry. As a result, unique binding profiles were observed for each pyranoside. Next, bovine serum albumin (BSA)-PBA conjugates were synthesized in a density controlled affording multivalent lectin mimetics. The resultant BSA-PBA conjugates were characterized by SDS-PAGE and MALDI-TOF MS. Cell surface glycan binding capacity was confirmed by a competitive lectin assay examined by flow cytometry. Macrophages express lectins as receptors for specific immune responses. Synthetic glycans are candidates for targeting cell surface lectins and for immunomodulation applications. In the second part of this dissertation, novel N-glycan polymers were synthesized and their immunomodulation effects were examined. N-linked glycopolymers were synthesized via cyanoxyl-mediated free radical polymerization (CMFRP). Then, their cytotoxicity and cell activation abilities against RAW 264.7 cells were examined. As a result, N-glycan polymers showed no cytotoxicity at a concentration of 1,250 mg/mL except the N-alpha-2,6-sialolactosyl polymer, which proved cytotoxic at 1250 µg/mL. N-alpha-2,3-sialolactosyl polymer showed the strongest activity for inducing cell surface marker expression compared to controls, indicating high macrophage modulation activity