Synthesis of carbohydrate based tools to explore the biosynthesis of and develop detection methods for prymnesin toxins

Hems, Edward (2017) Synthesis of carbohydrate based tools to explore the biosynthesis of and develop detection methods for prymnesin toxins. Doctoral thesis, University of East Anglia.

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    Abstract

    Prymnesium parvum is a harmful microalga which produces glycosylated ichthyotoxic metabolites called prymnesins. This thesis describes the synthesis of chemical tools to explore the biosynthesis and detection of prymnesins. Sugar-nucleotides proposed to be involved in the glycosylation of prymnesins were synthesised. UDP-α-ᴅ-galactofuranose was biosynthesised from chemosynthetic Galf-1-P
    using galactose-1-phosphate uridylyltransferase. GDP-α-ᴅ-arabinopyranose and GDP-β-ʟ- xylopyranose were proposed to be involved in the biogenesis of ʟ-xylofuranose, and were
    stereoselectively synthesised by direct displacement of an acylated glycosyl bromide with GDP. Multiple reaction monitoring transitions for the NDP-sugars were recorded using porous graphitic carbon column based LC-MS, as standards for profiling algal cell extracts.
    Prymnesins share a conserved terminal bis-alkyne, which may prove a useful biomarker. Bisalkyne standards were synthesised by Cadiot-Chodkiewicz coupling, and used to show that there was no appreciable difference in reactivity between terminal alkynes and bis-alkynes under CuAAC conditions. CuAAC based toxin detection shows potential; coupling of authentic prymnesins with 3-azido-7-hydroxycoumarin gave fluorescent species which were separable by TLC and visible under UV-light. Raman detection was also explored, but was dismissed due to fluorescent quenching by algal pigments.
    Sugar-glycerol compounds inspired by prymnesin’s glycosylated backbone were chemically synthesised. Neighbouring group participation was utilised to synthesise 1,2-trans glycosides. SnCl2 promoted glycosylation with furanosyl fluorides gave 1,2-cis furanosides with moderate stereocontrol, whilst TMSOTf promoted glycosylation with pyranosyl imidates gave 1,2-cis pyranosides with excellent stereocontrol. 1,2-trans Sugar-glycerol fragments gave NMR signals closer to prymnesin literature values than 1,2-cis fragments.
    Two fragments of prymnesin-1 glycosylated with α-ʟ-arabinopyranose and α-ᴅ-ribofuranose were chemically synthesised. Possible CuAAC and carbodiimide conjugation of the fragments to a carrier protein for anti-prymnesin-1 antibody production was considered. Because the stereochemistry of the prymnesin backbones at this region is undefined, both the 2R- and 2S- glycerol isomers were synthesised. The separated isomers were distinguished by comparing NOESY NMR with computational models.

    Item Type: Thesis (Doctoral)
    Faculty \ School: Faculty of Science > School of Chemistry
    Depositing User: Brian Watkins
    Date Deposited: 29 Nov 2017 15:01
    Last Modified: 29 Nov 2017 15:01
    URI: https://ueaeprints.uea.ac.uk/id/eprint/65624
    DOI:

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