In situ proton NMR analysis of alpha-alkynoate biotransformations. From 'invisible' substrates to detectable metabolites.

Only 2% of the known natural products with acetylenic bonds are alpha-alkynoates. Their polarized, conjugated triple bond is an optimal target for an enzymic hydration. Therefore they are good substrates for the enzymes involved in metabolism of acetylenic compounds, resulting in products that are suitable for bacterial growth. We isolated a Pseudomonas putida strain growing on 2-butynedioate as well as on propynoate, and determined the metabolic pathways of these two alpha-alkynoates. The triple bonds in both compounds were initially hydrated and 2-ketobutandioate as well as 3-ketopropanoate were formed. These two beta-keto acids were decarboxylated resulting in pyruvate and acetaldehyde, respectively. Pyruvate was further hydrolysed mainly to acetate and formate, whereas minor amounts were reduced to lactate. In the other biotransformation, acetaldehyde was oxidized to acetate accompanied by the reduction of 3-ketopropanoate to 3-hydroxypropanoate. Analyses of these metabolic processes were performed by in situ 1H-NMR spectroscopy in 1H2O, although the substrates, propynoate and 2-butynedioate, carried only one or even no detectable protons, respectively. However, while protons from the solvent are incorporated in the course of the pathway, the metabolites can be detected and identified. Therefore a detailed determination of the metabolic process is possible.