Biocatalytic dealkylation toolbox for organic synthesis

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Complex organic synthesis generally requires the use of orthogonal protecting groups. For this, methyl ethers and related alkyl derivatives are highly relevant. They are easily introduced, cheap, chemically robust and fulfill almost all criteria for a perfect protection of hydroxy groups. However, their removal requires strongly acidic and/or nucleophilic conditions, substantially limiting their actual usability in the synthesis of complex molecules. We aim to establish a generic biocatalytic solution based on redox-active enzymes for O dealkylation. They catalyze this transformation under mild conditions utilizing molecular oxygen as oxidant. The aim is to create a versatile toolbox for organic chemists to remove O-alkyl side chains in a regio-, and chemoselective fashion. We will address two major research questions: (1) “How much chemical space can be covered by enzymatic O-dealkylation?” and (2) “Can biocatalysts be identified and engineered towards a promiscuous, chemo- and regioselective efficient dealkylation tool for the organic chemist?” Dealkylation will be catalyzed by P450-monooxygenases, which upon hydroxylation of alkyl ethers yield unstable hemi-acetals, decomposing into the free hydroxy group and an aldehyde. We will use established tools for in silico-based enzyme discovery (i.e, BLAST search), a wide range of protein engineering methods (e.g. structure-guided rational design, directed evolution) in combination with adapted in-house- developed high throughput assays (HTA) to develop and improve the biocatalysts for synthetic applications. A smart synthetic library of compounds will be established and biocatalysis will be followed by organic analysis, purification and structural elucidation (prep. & anal. HPLC-MS, 1D/2D-NMR spectroscopy, HRMS-analysis). We aim to establish novel O-dealkylating enzymes as a general means for the cleavage of alkyl ethers and closely related stable O-protecting groups. We will provide biochemical data of yet unexplored proteins and focus on the scope and limitations of cytochrome P450s from the BM3 family and the enzymes recently discovered by the Bornscheuer group in marine bacteria which naturally catalyze the demethylation of 6-O-methyl-D-galactose. As the X-ray structure of one P450 enzyme has been solved, rational protein design approaches are highly feasible to alter their substrate scope. We will explore and study further candidate enzymes to enlarge the toolbox of suitable P450s with extended substrate scopes. The core team combines expertise from different scientific disciplines, namely synthetic chemistry (Christian Stanetty, TU Wien), biocatalysis (Florian Rudroff, TU Wien) and enzyme discovery, protein engineering & biocatalysis (Uwe Bornscheuer, University of Greifswald) in Germany. This scientific symbiosis complements the necessary know-how to accomplish the planned research.