Synthesis of N-Glycolylneuraminic Acid (Neu5Gc) and Its Glycosides.

Sialic acids constitute a family of negatively charged structurally diverse monosaccharides that are commonly presented on the termini of glycans in higher animals and some microorganisms. In addition to N-acetylneuraminic acid (Neu5Ac), N-glycolyl neuraminic acid (Neu5Gc) is among the most common sialic acid forms in nature. Nevertheless, unlike most animals, human cells loss the ability to synthesize Neu5Gc although Neu5Gc-containing glycoconjugates have been found on human cancer cells and in various human tissues due to dietary incorporation of Neu5Gc. Some pathogenic bacteria also produce Neu5Ac and the corresponding glycoconjugates but Neu5Gc-producing bacteria have yet to be found. In addition to Neu5Gc, more than 20 Neu5Gc derivatives have been found in non-human vertebrates. To explore the biological roles of Neu5Gc and its naturally occurring derivatives as well as the corresponding glycans and glycoconjugates, various chemical and enzymatic synthetic methods have been developed to obtain a vast array of glycosides containing Neu5Gc and/or its derivatives. Here we provide an overview on various synthetic methods that have been developed. Among these, the application of highly efficient one-pot multienzyme (OPME) sialylation systems in synthesizing compounds containing Neu5Gc and derivatives has been proven as a powerful strategy

Chemoenzymatic synthesis of glycosides containing legionaminic acid or 7-N-acetyl-modified sialic acid

Sialic acids (Sias) are common terminal monosaccharides on the cell surface glycoconjugates of vertebrates and some higher invertebrates. Being the outermost sugar residues, Sias are involved in many molecular recognition events including viral and bacterial infections, cell-cell interaction, immune regulation, inflammation, cancer metastasis, and others.Among the various post-glycosylation modifications of Sias, O-acetylation is the most-common. O-Acetylation of Sia has striking effects on its recognition by viruses, lectins, sialidases and plays an important role in biological and pathological processes. Among all the O-acetyl modifications of Sias, 7-O-acetylation of sialoglycans is among the ones that are the most difficult to study. In addition to the sensitivity of the O-acetyl group towards both pH changes and esterase, it can migrate to hydroxyls at neighboring C-8 and C-9, resulting in a mixture of O-acetylated sialoglycans. 7-O-Acetylation of sialoglycans regulates many biological phenomena, specifically microbe-host interactions, and regulation of immune responses. We have designed and developed a chemical biology approach to partially overcome this challenge by replacing the oxygen atom linked to the C7 in the 7-O-acetylated sialic acid by a nitrogen to form an amide in the chemically and biologically stable 7-acetamido-7-deoxy-N-acetylenuraminic acid (Neu5Ac7NAc). The resulting Neu5Ac7NAc-containing sialoglycans as the stable mimics of Neu5,7Ac2-containing sialosides are synthesized using a highly efficient one-pot multienzyme (OPME) chemoenzymatic method. A novel chemoenzymatic synthon strategy is developed to construct a comprehensive library of a2–3- and a2–6-linked sialosides containing 7-N- or 7,9-di-N-acetyl sialic acid. Mannose derivatives containing multiple azido groups such as diazido-mannose (Man2,4diN3) and triazido-mannose (Man2,4,6triN3) are chemically synthesized from inexpensive galactose in multiple steps. These synthesized mannose derivatives were used as synthons for sialosides using bacterial sialoside biosynthetic enzymes which showed remarkable substrate promiscuity. They are successfully used in one-pot multienzyme (OPME) sialylation systems for highly efficient synthesis of sialosides containing azido moieties. Conversion of the azido groups in the sialic acid to N-acetyl groups generates the desired sialosides. Sialidase assays using these compounds showed that the N-acetyl group at C-5 is required for the catalytic activity of all the sialidases tested while sialosides with N-acetyl at C-7 in the sialic acid were poor or unsuitable substrates for these sialidases. Legionaminic acid, Leg5,7Ac2, is a bacterial nonulosonic acid reported as the component of lipopolysaccharides (LPS) and capsular polysaccharides (CPS) of pathogenic bacteria such as Campylobacter jejuni, Enterobacter cloacae, Acinetobacter baumannii and Cronobacter turicensis. Its presence has been linked to bacteria virulence in humans. One efficient strategy to investigate its role and to fight bacterial infections is to develop synthetic oligosaccharides which mimics the LPS or CPS. We have chemically synthesized the six carbon precursors of Leg5,7Ac2 and Leg5,7diN3 derivatives from commercially available D-fucose. 6-Deoxy Man2,4diN3 is used in an efficient one-pot multi-enzyme (OPME) chemoenzymatic methods for synthesizing a library of a2-3/6-linked glycans containing Leg5,7diN3. The Leg5,7diN3 in glycosides was chemically converted in one step to Leg5,7Ac2. An extensive library of glycosides containing Leg5,7Ac2 and underlying glycans may present novel targets for enabling study of its role in bacterial pathogenesis and physiology. In summary, during my doctoral research, I have worked on design and synthesis of stable mimics of unstable 7-O-acetyl sialic acids and 7-O-acetylated sialoglycans to explore their biology. I have also designed and synthesized glycans containing bacterial nonulosonic acids and derivatives to develop important probes and potential anti-bacterial therapeutics.

Conformational Analysis of Neomycin B and Its Derivative

Neomycin B is an aminoglycoside antibiotic, topically used to prevent and treat infections. When ingested, Neomycin cannot be absorbed by the intestine and therefore can be prescribed during liver failure or before GI surgery to keep ammonia levels and bacterial levels low and prevent hepatic encephalopathy, which is loss of brain function due to a buildup of toxins. Neomycin B binds to a specific protein in the bacterial ribosome and interferes with bacterial protein synthesis, which leads to destruction of the bacterium. In addition, neomycin B is known to bind to RNA as well as triple-strand-DNA. The exact location of binding in the latter case is not known and is subject to ongoing research in multiple groups. The effectiveness of binding (matched molecular “handshake” or “key-lock” match) is dependent on neomycin’s solution conformation. Even if new chemical structure is added to neomycin (“key chain”), neomycin’s structure (“key”) must remain unaltered to fit the binding site (“lock”). The objective of this project was to characterize the solution geometry of neomycin B and an EDTA-derivative thereof. To our knowledge, neomycin has not been fully characterized in recent years. Quantitative information about its conformation in solution and the effects of attached substituents on the structure guides scientists in deciphering neomycin’s mode of action. In the present work, complete conformational analysis was accomplished by nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations. We assigned all 1H-, 13C, and 15N-resonances unambiguously, confirmed by short- and long-distance correlations. Quantitative coupling constants and qualitative 1H-1H-distances were extracted from J-resolved HMBC and NOE experiments. The compounds 2-(hydroxymethyl)-tetrahydrofuran and D-ribose were used as model compounds before analyzing neomycin and its EDTA-derivative. MD simulations were carried out in AMBER 14 with the GLYCAM06 force field. Preliminary data indicated that experimental and computational data agree well

Conformational Analysis of Neomycin B and Its Derivative

Neomycin B is an aminoglycoside antibiotic, topically used to prevent and treat infections. When ingested, Neomycin cannot be absorbed by the intestine and therefore can be prescribed during liver failure or before GI surgery to keep ammonia levels and bacterial levels low and prevent hepatic encephalopathy, which is loss of brain function due to a buildup of toxins. Neomycin B binds to a specific protein in the bacterial ribosome and interferes with bacterial protein synthesis, which leads to destruction of the bacterium. In addition, neomycin B is known to bind to RNA as well as triple-strand-DNA. The exact location of binding in the latter case is not known and is subject to ongoing research in multiple groups. The effectiveness of binding (matched molecular “handshake” or “key-lock” match) is dependent on neomycin’s solution conformation. Even if new chemical structure is added to neomycin (“key chain”), neomycin’s structure (“key”) must remain unaltered to fit the binding site (“lock”). The objective of this project was to characterize the solution geometry of neomycin B and an EDTA-derivative thereof. To our knowledge, neomycin has not been fully characterized in recent years. Quantitative information about its conformation in solution and the effects of attached substituents on the structure guides scientists in deciphering neomycin’s mode of action. In the present work, complete conformational analysis was accomplished by nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations. We assigned all 1H-, 13C, and 15N-resonances unambiguously, confirmed by short- and long-distance correlations. Quantitative coupling constants and qualitative 1H-1H-distances were extracted from J-resolved HMBC and NOE experiments. The compounds 2-(hydroxymethyl)-tetrahydrofuran and D-ribose were used as model compounds before analyzing neomycin and its EDTA-derivative. MD simulations were carried out in AMBER 14 with the GLYCAM06 force field. Preliminary data indicated that experimental and computational data agree well

Chemoenzymatic Synthesis of Sialosides Containing 7‑N- or 7,9-Di‑N‑acetyl Sialic Acid as Stable O‑Acetyl Analogues for Probing Sialic Acid-Binding Proteins

A novel chemoenzymatic synthon strategy has been developed to construct a comprehensive library of α2-3- and α2-6-linked sialosides containing 7-N- or 7,9-di-N-acetyl sialic acid, the stable analogue of naturally occurring 7-O-acetyl- or 7,9-di-O-acetyl-sialic acid. Diazido and triazido-mannose derivatives that were readily synthesized chemically from inexpensive galactose were shown to be effective chemoenzymatic synthons. Together with bacterial sialoside biosynthetic enzymes with remarkable substrate promiscuity, they were successfully used in one-pot multienzyme (OPME) sialylation systems for highly efficient synthesis of sialosides containing multiple azido groups. Conversion of the azido groups to N-acetyl groups generated the desired sialosides. The hydrophobic and UV-detectable benzyloxycarbonyl (Cbz) group introduced in the synthetic acceptors of sialyltransferases was used as a removable protecting group for the propylamine aglycon of the target sialosides. The resulting N-acetyl sialosides were novel stable probes for sialic acid-binding proteins such as plant lectin MAL II, which bond strongly to sialyl T antigens with or without an N-acetyl at C7 or at both C7 and C9 in the sialic acid

Sialosides Containing 7‑N‑Acetyl Sialic Acid Are Selective Substrates for Neuraminidases from Influenza A Viruses

Sialidases or neuraminidases are sialic-acid-cleaving enzymes that are expressed by a broad spectrum of organisms, including pathogens. In nature, sialic acids are monosaccharides with diverse structural variations, but the lack of novel probes has made it difficult to determine how sialic acid modifications impact the recognition by sialidases. Here, we used a chemoenzymatic synthon strategy to generate a set of α2-3- and α2-6-linked sialoside probes that contain 7-N-acetyl or 7,9-di-N-acetyl sialic acid as structure mimics for those containing the less stable naturally occurring 7-O-acetyl- or 7,9-di-O-acetyl modifications. These probes were used to compare the substrate specificity of several sialidases from different origins. Our results show that 7-N-acetyl sialic acid was readily cleaved by neuraminidases from H1N1 and H3N2 influenza A viruses, but not by sialidases of human or bacterial origin, thereby indicating that the influenza enzymes possess a distinctive and more promiscuous substrate binding pocket

One‐pot Multienzyme (OPME) Chemoenzymatic Synthesis of Brain Ganglioside Glycans with Human ST3GAL II Expressed in E. coli

A human sialyltransferase ST3GAL II (hST3GAL II) was successfully expressed in Escherichia coli as an active soluble fusion protein with an N-terminal maltose-binding protein (MBP) and a C-terminal hexa-histidine tag. It was used as an efficient catalyst in a one-pot multienzyme (OPME) sialylation system for high-yield production of the glycans of ganglioside GM1b and highly sialylated brain gangliosides GD1a and GT1b. Further sialylation of GM1b and GD1a glycans using a bacterial α2-8-sialyltransferase in another OPME sialylation reaction led to the formation of the glycans of GD1c and brain ganglioside GT1a, respectively. The lower reverse glycosylation activity of the recombinant hST3GAL II compared to its bacterial sialyltransferase counterpart simplifies the handling of enzymatic synthetic reactions and has an advantage for future use in automated chemoenzymatic synthetic processes