Characterization of an Indole-3-Acetamide Hydrolase from Alcaligenes faecalis subsp. parafaecalis and Its Application in Efficient Preparation of Both Enantiomers of Chiral Building Block 23-Dihydro-14-Benzodioxin-2-Carboxylic Acid

摘要

Both the enantiomers of 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid are valuable chiral synthons for enantiospecific synthesis of therapeutic agents such as (S)-doxazosin mesylate, WB 4101, MKC 242, 2,3-dihydro-2-hydroxymethyl-1,4-benzodioxin, and N-[2,4-oxo-1,3-thiazolidin-3-yl]-2,3-dihydro-1,4-benzodioxin-2-carboxamide. Pharmaceutical applications require these enantiomers in optically pure form. However, currently available methods suffer from one drawback or other, such as low efficiency, uncommon and not so easily accessible chiral resolving agent and less than optimal enantiomeric purity. Our interest in finding a biocatalyst for efficient production of enantiomerically pure 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid lead us to discover an amidase activity from Alcaligenes faecalis subsp. parafaecalis, which was able to kinetically resolve 2,3-dihydro-1,4-benzodioxin-2-carboxyamide with E value of >200. Thus, at about 50% conversion, (R)-2,3-dihydro-1,4-benzodioxin-2-carboxylic acid was produced in >99% e.e. The remaining amide had (S)-configuration and 99% e.e. The amide and acid were easily separated by aqueous (alkaline)-organic two phase extraction method. The same amidase was able to catalyse, albeit at much lower rate the hydrolysis of (S)-amide to (S)-acid without loss of e.e. The amidase activity was identified as indole-3-acetamide hydrolase (IaaH). IaaH is known to catalyse conversion of indole-3-acetamide (IAM) to indole-3-acetic acid (IAA), which is phytohormone of auxin class and is widespread among plants and bacteria that inhabit plant rhizosphere. IaaH exhibited high activity for 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which was about 65% compared to its natural substrate, indole-3-acetamide. The natural substrate for IaaH indole-3-acetamide shared, at least in part a similar bicyclic structure with 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which may account for high activity of enzyme towards this un-natural substrate. To the best of our knowledge this is the first application of IaaH in production of industrially important molecules.