Novel Biocatalytic D-D Bond Formation for Organic Synthesis

Carbon-carbon (C-C) forming reactions represent the most important transformations in organic synthesis to set up the carbon backbone of organic compounds. Surprisingly, only a very limited number of enzymatic C-C bond forming methods are applicable for organic synthesis until to date. The berberine bridge enzyme (BBE) catalyses an outstanding intra-molecular C-C bond forming reaction, for which no chemical equivalent exists. The only reagent required for this transformation is molecular oxygen. Since very recently this enzyme can be produced in significant quantities, which allows now to perform a thorough investigation of the scopes and limitations of the enzyme for organic synthesis. Optimisation of the reaction conditions (O2 -pressure, catalase, light) will enable an upscaling of the remarkable C-C bond forming transformation to a preparative scale. Changing the substitution pattern from N-methyl to N-ethyl will clarify a possible chiral induction on a novel chiral carbon centre. Finally, instead of an intra-molecular C-C formation, the possibilities of inter-molecular C-C bond formations e.g. iso-chinolin derivatives with phenol will be evaluated. Hints for a revised enzymatic mechanism of this novel enzyme will be obtained by performing structure function studies. This study will stimulate novel efforts to exploit biochemical C-C bond forming reactions for organic synthesis. Especially the cheap reagent required for this outstanding reaction, namely molecular oxygen shows the incredible possibilities Nature provides us for reactions, which mankind is not able to do with standard chemical means.

The project was dealing with new ways to synthesise natural and non-natural isoquinoline alkaloids using an enzyme called the Berberine Bridge Enzyme from California poppy. Isoquinoline alkaloids are substances that are widely spread in nature - mainly occurring in plants - and which have several applications in medical treatments. The isolation of these substances from natural sources requires often large amounts of biomass and yields of pure substance are rather poor. Also the synthesis employing organic chemistry to access to these substances comprises many steps and is economically unfavourable. The enzyme employed in the project, originating from poppy, is able to perform a unique reaction unknown in organic chemistry, and opens new synthetic pathways to isoquinoline alkaloids. This enzyme is able to form a new bond between two carbon atoms by just consuming molecular oxygen as the only reagent required. We found that additional to the natural substrate also various non-natural substrates - synthesised in racemic form - can be converted and are accepted by the enzyme; this allowed to prepare novel isoquinoline alkaloids. To characterise the enzyme in detail, optimisation studies concerning the optimal reaction conditions including for example temperature, pH-optimum or co-solvents were performed. For the studies a model substrate was designed and synthesised. Tests with different organic solvents showed that the enzyme is remarkable stable. Especially plant enzymes are normally quite sensitive to organic solvents. Even in the presence of high concentrations of organic solvent (for example 70% v/v of toluene) full activity was retained. When 99.4% v/v of toluene were used there was still a significant activity measurable. The organic solvent allowed to dissolve large quantities of substrate and to perform transformations at a concentration of 20 g/L. The enzyme acted enantioselectively, thus only the (S)-enantiomer is converted to the corresponding product. This allowed the isolation of the (S)-product and the remaining (R)-substrate both in enantiomerically pure form. In this project, 10 non-natural substrates were synthesised to test the substrate scope of the enzyme. The four best accepted substrates were chosen for a successful upscaling to a 500 mg scale. Furthermore the products and substrates were characterised in detail using different analytical methods (HPLC, GC, GC-MS, HR-MS, NMR).