P450 BM3-Catalyzed Regio- and Stereoselective Hydroxylation Aiming at the Synthesis of Phthalides and Isocoumarins.

Cytochrome P450 BM3 monooxygenases are able to catalyze the regio- and stereoselective oxygenation of a broad range of substrates, with promising potential for synthetic applications. To study the suitability of P450 BM3 variants for stereoselective benzylic hydroxylation of 2-alkylated benzoic acid esters, the biotransformation of methyl 2-ethylbenzoate, resulting in both enantiomeric forms of 3-methylphthalide, was investigated. In the case of methyl 2-propylbenzoate as a substrate the regioselectivity of the reaction was shifted towards ß-hydroxylation, resulting in the synthesis of enantioenriched R- and S-configured 3-methylisochroman-1-one. The potential of P450 BM3 variants for regio- and stereoselective synthesis of phthalides and isocoumarins offers a new route to a class of compounds that are valuable synthons for a variety of natural compounds.

Chemoenzymatic Total Synthesis of the Proposed Structures of Putaminoxins B and D.

Different enzymatic and nonenzymatic approaches were tested and compared to afford enantiopure homoallylic and allylic alcohols as building blocks in a total synthesis showcase. Thereby, highly enantioselective alcohol dehydrogenases and the P450 BM3 monooxygenase variant A74G L188Q were compared to classical asymmetric reagent-controlled allyl additions. Thus, the first total syntheses of the proposed structures for putaminoxins B/D and their respective enantiomers were accomplished. Detailed spectroscopic analysis of the newly synthesized compounds unraveled a discrepancy with respect to the reported structures of putaminoxins B/D. Furthermore, it was demonstrated that total synthesis is generally required for unequivocal assignment of configuration, because purely comparative NMR studies and judgment by analogy can lead to false predictions.

The Interplay between a Multifunctional Dehydratase Domain and a C-Methyltransferase Effects Olefin Shift in Ambruticin Biosynthesis.

The olefin shift is an important modification during polyketide biosynthesis. Particularly for type I cis-AT PKS, little information has been gained on the enzymatic mechanisms involved. We present our in vitro investigations on the olefin shift occurring during ambruticin biosynthesis. The unique, multifunctional domain AmbDH4 catalyzes consecutive dehydration, epimerization, and enoyl isomerization. The resulting 3-enethioate is removed from the equilibrium by α-methylation catalyzed by the highly specific C-methyltransferase AmbM. This thermodynamically unfavorable overall process is enabled by the high, concerted substrate specificity of the involved enzymes. AmbDH4 shows close relationship to DH domains and initial mechanistic studies suggest that the olefin shift occurs via a similar proton-shuttling mechanism as previously described for EI domains from trans-AT-PKS.