Novel Methodologies for the Synthesis of Multivalent Glycoconjugates

<p>Glycoconjugates, such as glycolipids and glycoproteins, play an important role in health and disease. The synthesis and biological evaluation of these glycoconjugates allows for the development of novel carbohydrate-based therapeutics and analytical tools. Traditionally, the conjugation of glycans to other substrates required the installment of an anomeric linker during the total synthesis of the glycan, however, this strategy does not allow for the conjugation of naturally isolated glycans. To address this concern, glycan conjugation methodologies without the need for protecting groups have been developed, including the use of oxyamine conjugation methodologies. In particular, the synthesis of a variety of novel bi-functional oxyamine linkers enabled the rapid assembly of various types of glycoconjugates, including fluorescent- and biotinylated-glycans, glycoproteins and multivalent glycodendrons. The multivalent presentation of glycans to cell surface lectins is often required to induce a measurable biological response. This multivalent binding can be achieved by the presentation of glycans on dendrons as these ‘glycodendrons’ have increased affinity for their corresponding lectin compared to monovalent glycans. Moreover, these glycodendrons have several advantages, including high synthetic control, low cytotoxicity and in addition can be derivatised with molecular probes of choice, which can aid in the biological evaluation of these glycoconjugates. Accordingly, novel biotinylated and fluorescent dendrons were synthesised from a highly convergent second generation dendron core scaffold. These functionalised dendrons then allow for the nonavalent conjugation of carbohydrates, such as Lewis antigens, for their biological evaluation in the selective targeting of lectins. Lewis antigens play an important role in host cell recognition, but these glycans are also involved in disease, such as in cancer metastasis and HIV-infection. The synthesis of Lewis antigens allows for the biological evaluation of these glycans, and moreover, could be employed in the development of novel glycan-based therapeutics and analytical tools. Accordingly, a novel high-yielding and efficient synthesis of a crystalline trisaccharide building block is presented, which can then be utilised in the synthesis of most Type-2 Lewis antigens. In particular, the global deprotection of the crystalline material gave the LewisX glycan antigen, a natural ligand for the C-Type lectin DC-SIGN on dendritic cells and macrophages. Finally, the rapid assembling of complex multivalent glycodendrons is discussed by conjugating the glycan antigens to the functionalised multivalent dendrons through the use of the bi-functional oxyamine linker methodology. In particular, the synthesis of a fluorescent LewisX glycodendron is presented, and to demonstrate a potential biological application of this methodology, the fluorescent LewisX glycodendron is evaluated as a flow cytometry marker for the C-type lectin DC-SIGN on human macrophages.</p>

Novel Methodologies for the Synthesis of Multivalent Glycoconjugates

<p>Glycoconjugates, such as glycolipids and glycoproteins, play an important role in health and disease. The synthesis and biological evaluation of these glycoconjugates allows for the development of novel carbohydrate-based therapeutics and analytical tools. Traditionally, the conjugation of glycans to other substrates required the installment of an anomeric linker during the total synthesis of the glycan, however, this strategy does not allow for the conjugation of naturally isolated glycans. To address this concern, glycan conjugation methodologies without the need for protecting groups have been developed, including the use of oxyamine conjugation methodologies. In particular, the synthesis of a variety of novel bi-functional oxyamine linkers enabled the rapid assembly of various types of glycoconjugates, including fluorescent- and biotinylated-glycans, glycoproteins and multivalent glycodendrons. The multivalent presentation of glycans to cell surface lectins is often required to induce a measurable biological response. This multivalent binding can be achieved by the presentation of glycans on dendrons as these ‘glycodendrons’ have increased affinity for their corresponding lectin compared to monovalent glycans. Moreover, these glycodendrons have several advantages, including high synthetic control, low cytotoxicity and in addition can be derivatised with molecular probes of choice, which can aid in the biological evaluation of these glycoconjugates. Accordingly, novel biotinylated and fluorescent dendrons were synthesised from a highly convergent second generation dendron core scaffold. These functionalised dendrons then allow for the nonavalent conjugation of carbohydrates, such as Lewis antigens, for their biological evaluation in the selective targeting of lectins. Lewis antigens play an important role in host cell recognition, but these glycans are also involved in disease, such as in cancer metastasis and HIV-infection. The synthesis of Lewis antigens allows for the biological evaluation of these glycans, and moreover, could be employed in the development of novel glycan-based therapeutics and analytical tools. Accordingly, a novel high-yielding and efficient synthesis of a crystalline trisaccharide building block is presented, which can then be utilised in the synthesis of most Type-2 Lewis antigens. In particular, the global deprotection of the crystalline material gave the LewisX glycan antigen, a natural ligand for the C-Type lectin DC-SIGN on dendritic cells and macrophages. Finally, the rapid assembling of complex multivalent glycodendrons is discussed by conjugating the glycan antigens to the functionalised multivalent dendrons through the use of the bi-functional oxyamine linker methodology. In particular, the synthesis of a fluorescent LewisX glycodendron is presented, and to demonstrate a potential biological application of this methodology, the fluorescent LewisX glycodendron is evaluated as a flow cytometry marker for the C-type lectin DC-SIGN on human macrophages.</p>

Helicobacter pylori BabA–SabA Key Roles in the Adherence Phase: The Synergic Mechanism for Successful Colonization and Disease Development

Helicobacter pylori is a pathogenic microorganism that successfully inhabits the human stomach, colonizing it by producing several virulence factors responsible for preventing host self-defense mechanisms. The adherence mechanism to gastric mucosal tissue is one of the most important processes for effective colonization in the stomach. The blood group antigen-binding adhesion (BabA) and sialic acid-binding adherence (SabA) are two H. pylori outer membrane proteins able to interact with antigens in the gastroduodenal tract. H. pylori possesses several mechanisms to control the regulation of both BabA and SabA in either the transcriptional or translational level. BabA is believed to be the most important protein in the early infection phase due to its ability to interact with various Lewis antigens, whereas SabA interaction with sialylated Lewis antigens may prove important for the adherence process in the inflamed gastric mucosal tissue in the ongoing-infection phase. The adherence mechanisms of BabA and SabA allow H. pylori to anchor in the gastric mucosa and begin the colonization process.

Expression of the Carbohydrate Lewis Antigen, Sialyl Lewis A, Sialyl Lewis X, Lewis X, and Lewis Y in the Placental Villi of Patients With Unexplained Miscarriages

BackgroundLewis antigens such as Sialyl Lewis A (sLeA), Sialyl Lewis X (sLeX), Lewis X (LeX), and Lewis Y (LeY) are a class of carbohydrate molecules that are known to mediate adhesion between tumor cells and endothelium by interacting with its selectin ligands. However, their potential role in miscarriage remains enigmatic. This study aims to analyze the expression pattern of sLeA, sLeX, LeX, and LeY in the placental villi tissue of patients with a medical history of unexplained miscarriages.MethodsParaffin-embedded slides originating from placental tissue were collected from patients experiencing a miscarriage early in their pregnancy (6–13 weeks). Tissues collected from spontaneous (n = 20) and recurrent (n = 15) miscarriages were analyzed using immunohistochemical and immunofluorescent staining. Specimens obtained from legally terminated normal pregnancies were considered as control group (n = 18). Assessment of villous vessel density was performed in another cohort (n = 10 each group) of gestation ages-paired placenta tissue. Protein expression was evaluated with Immunoreactive Score (IRS). Statistical analysis was performed by using Graphpad Prism 8.ResultsExpression of sLeA, sLeX, LeX, and LeY in the syncytiotrophoblast was significantly upregulated in the control group compared with spontaneous and recurrent miscarriage groups. However, no prominent differences between spontaneous and recurrent miscarriage groups were identified. Potential key modulators ST3GAL6 and NEU1 were found to be significantly downregulated in the recurrent miscarriage group and upregulated in the spontaneous group, respectively. Interestingly, LeX and LeY expression was also detected in the endothelial cells of villous vessels in the control group but no significant expression in miscarriage groups. Furthermore, assessment of villous vessel density using CD31 found significantly diminished vessels in all size groups of villi (small villi &lt;200 µm, P = 0.0371; middle villi between 200 and 400 µm, P = 0.0010 and large villi &gt;400 µm, P = 0.0003). Immunofluorescent double staining also indicated the co-localization of LeX/Y and CD31.ConclusionsThe expression of four mentioned carbohydrate Lewis antigens and their potential modulators, ST3GAL6 and NEU1, in the placenta of patients with miscarriages was significantly different from the normal pregnancy. For the first time, their expression pattern in the placenta was illustrated, which might shed light on a novel understanding of Lewis antigens’ role in the pathogenesis of miscarriages.

Enzymatic modular assembly of hybrid Lewis antigens

Facile enzymatic modular assembly of 3 complex hybrid Lewis antigens.

Complementary Use of Carbohydrate Antigens Lewis a, Lewis b, and Sialyl-Lewis a (CA19.9 Epitope) in Gastrointestinal Cancers: Biological Rationale towards a Personalized Clinical Application

Carbohydrate antigen 19.9 (CA19.9) is used as a tumor marker for clinical and research purposes assuming that it is abundantly produced by gastrointestinal cancer cells due to a cancer-associated aberrant glycosylation favoring its synthesis. Recent data has instead suggested a different picture, where immunodetection on tissue sections matches biochemical and molecular data. In addition to CA19.9, structurally related carbohydrate antigens Lewis a and Lewis b are, in fact, undetectable in colon cancer, due to the down-regulation of a galactosyltransferase necessary for their synthesis. In the pancreas, no differential expression of CA19.9 or cognate glycosyltransferases occurs in cancer. Ductal cells only express such Lewis antigens in a pattern affected by the relative levels of each glycosyltransferase, which are genetically and epigenetically determined. The elevation of circulating antigens seems to depend on the obstruction of neoplastic ducts and loss of polarity occurring in malignant ductal cells. Circulating Lewis a and Lewis b are indeed promising candidates for monitoring pancreatic cancer patients that are negative for CA19.9, but not for improving the low diagnostic performance of such an antigen. Insufficient biological data are available for gastric and bile duct cancer. Studying each patient in a personalized manner determining all Lewis antigens in the surgical specimens and in the blood, together with the status of the tissue-specific glycosylation machinery, promises fruitful advances in translational research and clinical practice.

Glycan-dependent two-step cell adhesion mechanism of Tc toxins

Toxin complex (Tc) toxins are virulence factors widespread in insect and human bacterial pathogens. Tcs are composed of three subunits: TcA, TcB and TcC. TcA facilitates receptor-toxin interaction and membrane permeation, TcB and TcC form a toxin-encapsulating cocoon. While the mechanisms of holotoxin assembly and prepore-to-pore transition have been well-described, little is known about receptor binding and cellular uptake of Tcs. Here, we identify two classes of glycans, heparins/heparan sulfates and Lewis antigens, that act as receptors for different TcAs from insect- and human pathogenic bacteria. Glycan array screening and electron cryo microscopy (cryo-EM) structures reveal that all tested TcAs bind unexpectedly with their α-helical part of the shell domain to negatively charged heparins. In addition, TcdA1 from the insect-pathogen Photorhabdus luminescens binds to Lewis antigens with micromolar affinity. A cryo-EM structure of the TcdA1-Lewis X complex reveals that the glycan interacts with the receptor-binding domain D of the toxin. Our results suggest a two-step association mechanism of Tc toxins involving glycans on the surface of host cells.

Automated glycan assembly of Lewis type I and II oligosaccharide antigens

Lewis antigens are fucosylated oligosaccharides that play crucial roles in various biological processes. Here, we illustrate how automated glycan assembly (AGA) provides quick access to a series of more than ten defined Lewis type-I and type-II antigens.

Lectin Activity of the TcdA and TcdB Toxins ofClostridium difficile

ABSTRACTClostridium difficileis a major cause of hospital-acquired antibiotic-associated diarrhea.C. difficileproduces two cytotoxins, TcdA and TcdB; both toxins are multidomain proteins that lead to cytotoxicity through the modification and inactivation of small GTPases of the Rho/Rac family. Previous studies have indicated that host glycans are targets for TcdA and TcdB, with interactions thought to be with both α- and β-linked galactose. In the current study, screening of glycan arrays with different domains of TcdA and TcdB revealed that the binding regions of both toxins interact with a wider range of host glycoconjugates than just terminal α- and β-linked galactose, including blood groups, Lewis antigens,N-acetylglucosamine, mannose, and glycosaminoglycans. The interactions of TcdA and TcdB with ABO blood group and Lewis antigens were assessed by surface plasmon resonance (SPR). The blood group A antigen was the highest-affinity ligand for both toxins. Free glycans alone or in combination were unable to abolish Vero cell cytotoxicity by TcdB. SPR competition assays indicate that there is more than one glycan binding site on TcdB. Host glycoconjugates are common targets of bacterial toxins, but typically this binding is to a specific structure or related structures. The binding of TcdA and TcdB is to a wide range of host glycans providing a wide range of target cells and tissuesin vivo.