Speare_2004_Org.Biomol.Chem_2_2942

A chemical synthesis of the 2-hydroxy-6-ketohexa-2,4-dienoic acid intermediates on bacterial meta-cleavage pathways has been established, using a Heck coupling strategy. Coupling of ethyl 3-bromo-2-acetoxyacrylate with 1-aryl vinyl ketals or 1-aryl allylic alcohols proceeded in 70-90% yield. Heck coupling with an alkyl vinyl ketal was also successful, allowing the synthesis of an alkyl-substituted ring fission intermediate. The synthetic ring fission intermediates were used to investigate the enzymatic reaction catalysed by C-C hydrolase BphD from Pseudomonas LB400. A reduced substrate analogue 2,6-dihydroxy-6-phenylhexa-2,4-dienoic acid was processed enzymatically to benzaldehyde by C-C hydrolase BphD, consistent with a catalytic mechanism involving general base-catalysed attack of water to give a gem-diol intermediate, and not consistent with a nucleophilic mechanism. A series of para-substituted 2-hydroxy-6-keto-6-phenylhexa-2,4-dienoic acid substrates were assayed against BphD, and the derived Hammett plot (rho=-0.71) is consistent with a departing carbanion in the transition state for C-C cleavage.