The synthesis of chiral malonic acids and their application for the investigation of the steric course of enzymic reactions
The synthesis of chiral malonic acids and their application for the investigation of the steric course of enzymic reactions
The pro-pro-chiral centre (Caabb) of malonic acid was made chiral by labelling with stable isotopes of hydrogen and carbon. Using a combination of chemical and enzymic steps (/?)-{l-13C;2-2H]malonic acid and (S)-[l-lsC;2-2H]malonic acid were synthesised in approximately 35% yield. The absolute stereochemistry and enantiomeric purity of the chiral malonic acids were determined by first converting them into their malonyl-CoA derivatives using succinyl-CoA transferase followed by transformation into palmitic acid with homogeneous fatty acid synthase isolated from yeast that is known to remove H^, from malonyl-CoA. A novel analysis method was developed by which the labelled palmitic acid which arises from the (R)-[l-lsC;2-2HJmalonic acid could be distinguished from that derived from the (S)-[l- C;2-2H]malonic acid by mass spectrometry. This method was extended to investigate the steric course of homogeneous fatty acid synthases isolated from rat liver and Penicillium patulum. From these investigations it was concluded that all three fatty acid synthases investigated catalyse the reaction by the same steric course. In this the hydrogen atom originating from Hg,- in malonyl-CoA is retained during the dehydration of the /9-hydroxyacyl-CoA intermediate during each of the enzyme catalytic cycles. The extent of the exchange of the sensitive methylene hydrogen atoms of malonyl-CoA during the reaction were rigorously established.
The related polyketide synthase, 6-methylsalicylic acid synthase, was isolated in homogeneous form from Penicillium patulum and its properties were investigated. The stereochemical mechanism of 6-methylsalicylic acid synthase was investigated using the chiral malonic acids after their activation to malonyl-CoA derivatives and the 6-methylsalicylic acid produced was analysed by mass spectrometry. The results indicated that during the formation of the aromatic ring, hydrogen atoms with opposite configurations are retained at the 3- and 5-positions. Further investigations using non-labelled acetoacetyl-CoA, together with chiral malonic acids, proved that the label incorporated into the 3-position of the 6-methylsalicylic acid arises from the HRf of malonyl-CoA. Thus the hydrogen at the 5-position of 6-methylsalicyclic acid must arise from HSl- of malonyl-CoA. Mechanistic possibilities for the biosynthesis of 6-methylsalicylic acid are discussed.
University of Southampton
1990
Spencer, Jonathan Brian
(1990)
The synthesis of chiral malonic acids and their application for the investigation of the steric course of enzymic reactions.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The pro-pro-chiral centre (Caabb) of malonic acid was made chiral by labelling with stable isotopes of hydrogen and carbon. Using a combination of chemical and enzymic steps (/?)-{l-13C;2-2H]malonic acid and (S)-[l-lsC;2-2H]malonic acid were synthesised in approximately 35% yield. The absolute stereochemistry and enantiomeric purity of the chiral malonic acids were determined by first converting them into their malonyl-CoA derivatives using succinyl-CoA transferase followed by transformation into palmitic acid with homogeneous fatty acid synthase isolated from yeast that is known to remove H^, from malonyl-CoA. A novel analysis method was developed by which the labelled palmitic acid which arises from the (R)-[l-lsC;2-2HJmalonic acid could be distinguished from that derived from the (S)-[l- C;2-2H]malonic acid by mass spectrometry. This method was extended to investigate the steric course of homogeneous fatty acid synthases isolated from rat liver and Penicillium patulum. From these investigations it was concluded that all three fatty acid synthases investigated catalyse the reaction by the same steric course. In this the hydrogen atom originating from Hg,- in malonyl-CoA is retained during the dehydration of the /9-hydroxyacyl-CoA intermediate during each of the enzyme catalytic cycles. The extent of the exchange of the sensitive methylene hydrogen atoms of malonyl-CoA during the reaction were rigorously established.
The related polyketide synthase, 6-methylsalicylic acid synthase, was isolated in homogeneous form from Penicillium patulum and its properties were investigated. The stereochemical mechanism of 6-methylsalicylic acid synthase was investigated using the chiral malonic acids after their activation to malonyl-CoA derivatives and the 6-methylsalicylic acid produced was analysed by mass spectrometry. The results indicated that during the formation of the aromatic ring, hydrogen atoms with opposite configurations are retained at the 3- and 5-positions. Further investigations using non-labelled acetoacetyl-CoA, together with chiral malonic acids, proved that the label incorporated into the 3-position of the 6-methylsalicylic acid arises from the HRf of malonyl-CoA. Thus the hydrogen at the 5-position of 6-methylsalicyclic acid must arise from HSl- of malonyl-CoA. Mechanistic possibilities for the biosynthesis of 6-methylsalicylic acid are discussed.
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Published date: 1990
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Local EPrints ID: 460427
URI: http://eprints.soton.ac.uk/id/eprint/460427
PURE UUID: 0e3c982c-dbca-43d9-870d-e11661985df5
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Date deposited: 04 Jul 2022 18:22
Last modified: 04 Jul 2022 18:22
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Author:
Jonathan Brian Spencer
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