Biocatalytic C-H activation via alpha-Ketoglutarate dependent Dioxygenases

Biocatalysts are the tools in nature that build up and transform chemical compounds. These tools can be produced in the laboratory by microorganisms and unfold their activity under physiological conditions to prepare chemical compounds. In many cases, these biocatalytic transformations are superior to conventional chemical methods for one specific task. One example for such a catalyst from nature is involved in the biosynthesis of podophyllotoxin a compound isolated from Podophyllum leaves. This natural product is the precursor to etoposide an anticancer agent, which is used in everyday clinical chemotherapeutic treatments and is on the World Health Organizations list of essential medicines. This project aims at the investigation of this key step in the podophyllotoxin biosynthesis. The goal is to find out the possibile variations of the structure in order to come up with more potent and selective medicines. In addition, the structural motif, which is created by this transformation, is not unique to podophyllotoxin, but exists in a number of other pharmaceuticals too. The developed tools could be of benefit for the preparation of these drugs too. Another important aspect can be found in the biocatalysts relatives a subfamily of dioxygenase enzymes who catalyse steps in the biosynthesis of extremely relevant natural products, such as fungicides, painkillers and antibiotics. The knowledge acquired in the course of this project could give a more detailed picture of these tools from nature and give a better understanding about their mode of action.

Enzymes depict the catalysts - or more colloquial the motors - of biological systems and thus enable chemical transformations that are hard to perform with conventional methods. In this research project the emphasis was addressed to one enzyme class, which has been believed to be too instable for its exploitation as catalysts for designed processes. However, the enzyme class is responsible for the biological production of anticancer molecules in plants and thus its chemical transformations mark key steps in the synthesis of these drug molecules. Our efforts have been dedicated to the isolation of the enzyme, it's production on a large laboratory scale and on the upscale of this highly important chemical transformation which yields the drug molecule in the end. A highly stable, high yielding and scalable process has been elaborated in our laboratories and the final process delivered gram quantities of the target molecule (usual methods delivered the molecule in milligram quantities). These results can be used for the development of a large-scale production of the target molecule, may deliver appropriate quantities for further drug development endeavors and promote additional research on these highly active anticancer agents. Two molecules - etoposide and teniposide - of this class are already frequently used in chemotherapeutic regimen for the clinical treatment of cancer patients (approved drugs) and are part of the WHO's list of essential medicines. In addition, our research has proven this class of catalysts to be much more stable and usable for designed processes and may open the door to large-scale transformations, which lack a counterpart among conventional tools. The key feature may be to choose the appropriate milieu for the catalyst and on the other hand to protect the key enzyme from highly reactive species, which are formed during the process by the addition of other protective enzymes. This process may be best compared to sun cream, which protects the skin and lets it act as initially intended. Last but not least, another key step in the synthesis of the drug molecule was investigated in order to learn how this methodology is working and which screws can be tweaked in order to get a better process. In depth computer calculations were used to mimic the process in the digital world and important insights were gained, which allowed the optimization of the process, the expansion of the methodology to the preparation of other structural motifs and the identification of the chemical reactivity behind this artificially designed catalyst.