Biosynthesis of fluoroacetate and 4-fluorothreonine in Streptomyces cattleya

Abstract

This thesis describes results on the biosynthesis of fluoroacetate and 4-fluorothreonine by the bacterium Streptomyces cattleya. Exploratory (^14)C-radiolabelling experiments, designed to determine the relative incorporation of a range of metabolites into fluoroacetate, demonstrated that substrates such as glucose, glycine and serine were efficient precursors. [2-(^13)C]-Glycolate, [l,2-(^13)C(_2)]-glycolate, [2,2-(^2)H(_2)]-glycolate, DL-[3,4,4,4-(^2)H(_4)]-threonine, DL-[4,4,4-(^2)H(_3)]-threonine, [3,3,3-(^2)H(_3)]-alanine, [3-(^2)H]-fluoropyruvate and [3-(^2)H]-3-fluorolactate were synthesised and characterised. Incorporations of synthesised and commercially available putative precursors, [l-(^13)C]-glycine, [2-(^13)C]-glycine, [1,2-(^13)C(_2) glycine, [3-(^13)C]-serine, [l-(^13)C]-pyruvate, [2-(^13)C]-pyruvate, [3-(^13)C]-pyruvate, [2-(^13)C]-acetate, [3-(^13)C]-alanine, [2,3,3-(^2)H(_3)]- aspartate, [2,2,3,3-(^2)H(_4)]-succinate and [2-(^13)C]-glycerol into fluoroacetate and 4-fluorothreonine were assessed. [2-(^13)C]-Glycerol appeared to be the highest incorporated precursor of those tested. The (^13)C label from [2-(^13)C]-glycerol predominated at G-1 (-56%) of fluoroacetate and C-3 (57%) of 4-fluorothreonine. (^19)F-NMR emerged as a powerful analytical tool by which to determine the regiospecific incorporation of '^G and into the fluorometabolites. A conclusion to emerge from this study is that a glycolytic intermediate in S. cattleya appears to be a subsfrate for the bio-fluorination process. Incubation studies with [3-(^2)H]-fluoropyruvate and [3-(^2)H]-3-fluorolactate, tested as possible initial fluorination products, showed low incorporation of deuterium atoms into the fluorometabolites which did not reinforce a role for 2-phosphoglycerate,3- phosphoglycerate and glycerol-3-phosphate as substrates for the fluorinating enzyme. The incorporations which are almost identical into the two fluorometabolites strongly indicate that the assembly of the carbon skeleton of fluoroacetate and C-3 and C-4 of4- fluorothreonine involves a common biosynthetic intermediate. Thus the close metabolic relationship between the two fluorometabolites indicates the presence of a single fluorinating enzyme in S. cattleya. A three step route to 4-(25;55)-fluorothreonine was developed by a modification of Seebach's imidazolidinone methodology for the preparation of L-threonine and is the subject of Chapter 4. An X-ray crystal structure of the synthetic 4-(2S,3S)- fluorothreonine assisted in assignment and confirmation of the absolute stereochemistry of the natural product. This modified methodology is amenable to isotope labelling by employing NaB(^2)H(_4) to generate [3-(^2)H]-4-fluorothreonine. Moreover it offers an alternative route to threo-amino acids and has the advantage of using acid chlorides in place of aldehydes when the required aldehyde is unavailable.