Enzyme-Catalyzed Asymmetric Hydration of C=C Bonds

The enzyme-catalyzed hydration of C=C bonds in a stereoselective manner represents a highly value- adding transformation. The addition of the rather simple molecule `water` goes along with the introduction of a new chiral centre in 100% atom efficiency thus converting prochiral alkenes to the corresponding nonracemic alcohols. No generally applicable protocol has been established in traditional chemistry (acid-catalyzed 1,2-Markovnikov-type addition of water across nonconjugated C=C bonds and the complementary base-catalyzed 1,4-Michael-type addition to activated C=C bonds) with few exceptions based on metal-catalyzed stereoselective hydration reactions. Aside from this, a handful of indirect methods have been shown which require complex protocols. In contrast, hydration reactions play an essential role in nature and are usually catalyzed by hydratases (also termed hydro- lyases). However, these enzymes are in general highly substrate specific and in some cases oxygen- sensitive, which strongly limits their potential for biocatalytic applications. Recently, we were able to show the proof-of-concept for the asymmetric hydration of styrene-type substrates catalyzed by phenolic acid decarboxylases due to their catalytic promiscuity. Based on preliminary results (see Angew. Chem. Int. Ed. 2013, 52, 2293) I plan to investigate this novel enzymatic reaction in detail in order to fully understand and exploit this biotransformation focussing on: i) extension of the enzymatic toolbox (sequence-, mechanism- and structure-based search for new enzyme candidates), ii)evaluation of the substrate scope (structurally and electronically diverse classes of compounds), iii) enlargement of the substrate tolerance (mutant design), iv)reaction engineering to optimize the biocatalytic performance (increase of conversion and optical purity of hydration products), v) use of non-natural nucleophiles to generate chiral N-, S- and O-synthons, vi)elucidation of the catalytic mechanism (active site variants), vii) use of various bicarbonate-surrogates. During the course of this project I will work in close cooperation with Prof. K. Gruber and his coworkers (University of Graz) taking advantage of their expertise in structural biology and molecular modeling. Furthermore, I plan to extend my cooperation with Prof. A. Liese (Technical University Hamburg-Harburg) to benefit from his expertise in bioprocess technology. Overall, this multidisciplinary approach (biocatalysis, structural biology, enzyme engineering, process engineering and organic synthesis) provides optimal conditions in order to assure a most efficient outcome for the applied project.

The development of novel, sustainable biocatalytic concepts for the synthesis of value-added compounds constituted the major focus of this project. The regioselective para-carboxylation of electron-rich aromatic compounds using CO2 as alternative carbon source as well as the stereoselective addition of water and non-natural nucleophiles to C=C bonds, both reactions catalyzed by decarboxylases have been investigated in great detail. Great efforts are currently being made in academia and the chemical industry in order to develop environmentally friendly and energy-efficient concepts for the production of chemical compounds. The utilization of renewable resources, mild reaction/process conditions and the reduction of problematic (toxic) waste display key challenges for the design of novel processes. In recent years, biocatalysis has been explored as very promising and sustainable alternative to chemical methods.The key feature in this innovative technology displays the application of enzymes (biocatalysts) which are able to operate under ambient reaction conditions (aqueous medium, room temperature, atmospheric pressure etc.) in a highly regio- and/or stereoselective fashion which are relevant aspects in many areas of chemical industry (such as pharma-, food-, cosmetic-, agro-industry etc.). During the course of this project a biocatalytic concept for the regioselective CO2-fixation (para-carboxylation) of electron-rich aromatic compounds as attractive alternative to traditional chemical methods have been established, the latter requiring rather harsh reaction conditions and suffer from poor yields/selectivities. The applied enzymes (para-decarboxylases) depend on an only recently discovered cofactor (prenylated flavin). Based on mutational studies and quantum mechanical calculations we could gain a more detailed insight in the catalytic mechanism of this novel enzyme-catalyzed reaction. Another focus was based on the promiscuous activity of phenolic acid decarboxylases, hence able to catalyze the asymmetric addition of non- natural nucleophiles (NuHs) across C=C bonds which displays an elegant and sustainable method towards the synthesis of chiral synthons (sec-alcohols, sec-amines etc.) the latter important building blocks in organic synthesis. Overall, we were able to successfully extend the portfolio of biocatalytic reactions (para-carboxylation, asymmetric addition of NuH across C=C bonds) leading to numerous high impact publications.