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