Chemoenzymatic Synthesis and NMR Conformational Studies of Sialosides and Sialidase Inhibitors

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  • Chemoenzymatic Synthesis and NMR Conformational Studies of Sialosides and Sialidase Inhibitors Book Detail

  • Author : Wanqing Li
  • Release Date : 2019
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  • ISBN 13 : 9781658414036
  • File Size : 34,34 MB
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Chemoenzymatic Synthesis and NMR Conformational Studies of Sialosides and Sialidase Inhibitors by Wanqing Li PDF Summary

Book Description: Sialic acids (Sia) are monosaccharides commonly presented at the termini of the carbohydrate moieties of cell surface glycoconjugates. Sialic acids with or without diverse post-glycosylational modifications are directly involved in many molecular recognition events including immune regulation, cell-cell interaction, inflammation, cancer metastasis, bacterial and viral infection. Sialidases are the key enzymes involved in the catabolism of sialic acid-containing oligosaccharides or glycoconjugates by catalyzing the cleavage of terminal sialic acids, and thus play a role in regulating the level of sialic acids on cell surface. During my doctoral research, I aimed at developing stable mimics of unstable sialic acid forms for understanding their biological roles and developing sialic acid-derived sialidase inhibitors using one-pot multienzyme (OPME) synthetic methods and Nuclear Magnetic Resonance (NMR)-based analysis. Among numerous post-glycosylational modifications, O-acetylation is a common biologically important modification of the glycans on glycoconjugates. O-Acetylation of sialic acid (Sia) modulates its recognition by sialic acid-binding proteins and plays an important role in biological and pathological processes. 9-O-Acetylation is the most common modification of sialic acid in human and has striking effects on many biological phenomena, including microbe-host interactions, complement action, regulation of immune responses, sialidase action, cellular apoptosis, and tumor immunology. However, study of O-acetylated sialoglycans has been hampered due to the instability of O-acetyl group including sensitivity towards both pH changes and esterases. We have demonstrated a chemical biology strategy to partially solve this problem by replacing the oxygen atom in the C9 ester group of sialic acid by a nitrogen to form an amide. Chemically and biologically stable 9-acetamido-9-deoxy-N-acetylenuraminic acid (Neu5Ac9NAc)-containing sialoglycans have been proposed as stable mimics of Neu5,9Ac2-containing sialosides, and synthesized using efficient one-pot multienzyme (OPME) methods. Similar biological actions of Neu5Ac9NAc and Neu5,9Ac2 have been confirmed by glycan microarray studies, mammalian cell incorporation and surface expression studies, as well as sialidase cleavage activities. To study the structural resemblance, a systematic Nuclear Magnetic Resonance (NMR) spectroscopic and molecular dynamics (MD) simulation study was undertaken for Neu5,9Ac2-containing GM3 ganglioside glycan (GM3-glycan) and its Neu5Ac9NAc analog. GM3-glycan with Neu5Ac as the non-O-acetyl form of Neu5,9Ac2 was used as a control. Complete 1H and 13C NMR chemical shift assignments, three-bond 1H-13C trans-glycosidic coupling constants (3J[subscript CH]), accurate 1H-1H coupling constants (3J[subscript HH]), nuclear Overhauser effects, and hydrogen bonding detection were carried out. Results show that structural modification (O- or N-acetylation) on the C-9 of Neu5Ac in GM3 glycan does not cause significant conformational changes on either its glycosidic dihedral angles or its secondary structure. All structural differences are confined to the Neu5Ac glycerol chain and minor temperature-dependent changes are seen in the aglycone portion. Furthermore, OH chemical shifts were assigned at -10 °C and no evidence of intramolecular hydrogen bond was observed. The results provide additional evidence regarding structural similarities between sialosides containing 9-N-acetylated and 9-O-acetylated Neu5Ac and support the opportunity of using 9-N-acetylated Neu5Ac as a stable mimic to study the biochemical role of 9-O-acetylated Neu5Ac. In addition to studying the O-acetylated sialoglycans, sialic acid derivatives as potential bacterial sialidase inhibitors were explored. Structure-based drug design has led to potent inhibitors against neuraminidases influenza A viruses that have been used successfully as approved therapeutics. However, selective and effective inhibitors against bacterial and human sialidases are still being actively pursued. Guided by crystal structural analysis, several derivatives of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en or DANA) were designed and synthesized as triazole-linked transition state analogs. Inhibition studies revealed that glycopeptide analog E-(TriazoleNeu5Ac2en)-AKE and compound (TriazoleNeu5Ac2en)-A were selective inhibitors against Vibrio cholerae sialidase, while glycopeptide analog (TriazoleNeu5Ac2en)-AdE selectively inhibited V. cholerae and Arthrobacter ureafaciens sialidases. Besides sialidase inhibitors designed based on transition-state analog, a library of 2(equatorial),3(axial or equatorial)-difluorosialic acids with their C-5 and/or C-9 derivatives were chemoenzymatically synthesized as mechanism-based sialidase inhibitors. Inhibition studies against several bacterial sialidases and a recombinant human cytosolic sialidase hNEU2 indicated that sialidase inhibition was affected by the C-3 fluorine stereochemistry, as well as functional group derivatization at C-5 and/or C-9. Compounds with an axial fluorine at C-3 were better inhibitors (up to 100-fold) against bacterial sialidases compared to their 3F (equatorial) counterparts. While C-5-modified compounds were less efficient inhibitors against bacterial sialidases, 9-N3-modified 2,3-difluoro-Neu5Ac showed increased inhibitory activity against bacterial sialidases. Among inhibitors tested, 9-azido-3-deoxy-2-equatorial-3-axial-difluro-N-acetylneuraminic acid (2e3aDFNeu5Ac9N3) was a low nanomolar inhibitor selectively against bacterial sialidases from Streptococcus pneumoniae (SpNanA), Clostridium perfringens (CpNanI), and A. ureafaciens. In addition, I have developed an efficient 2-step synthetic route for gram-scale production of sialic acid derivative 2,7-anhydro-N-acetylneuraminic acid (2,7-anhydro-Neu5Ac) for exploring its biological significance. Efficient gram-scale enzymatic synthesis of 2,7-anhydro-Neu5Ac from N-acetylneuraminic acid (Neu5Ac) has been achieved in a 2-step reaction cycle, through one-pot 2-enzyme-catalyzed [alpha]2-3-sialoside formation reaction followed by S. pneumoniae sialidase SpNanB-catalyzed reaction. The 2-step enzymatic strategy used only water, ethanol and acetonitrile in the reaction and purification processes, and produced gram-scale 2,7-anhydro-Neu5Ac in over 90% yield with recycled use of the acceptor.

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