UBC Theses and Dissertations

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UBC Theses and Dissertations

Exploration of strategic approaches towards the novel biocatalytic conversion of tetrahydrocannabinol (THC) to cannabinol (CBN) Khalifa, Charfeddine


Clinical studies on cannabis reveal its importance in alleviating inflammation and counteracting certain neurodegenerative disorders. However, these benefits carry side effects induced by the hallucinogenic property of ∆⁹-tetrahydrocannabinol (THC). Investigation into cannabinoids has led to the discovery of cannabinol (CBN), a degradation product of THC that provides multiple benefits without effect on the perception, cognition, or behavior of the patient. Current methods of production rely on traditional farming and degradation of THC to produce CBN which are less sustainable and efficient than fermentation-based cannabinoid production. CBN formation is the result of oxidative degradation of THC, and currently, there’s no enzyme associated with this reaction. To create a biosynthetic route towards CBN, a multistep biosynthetic mechanism has been prepared utilizing CYP450 monooxygenases and dehydratases to facilitate the double bond formation on THC. Cytochrome P450 19A1, 2C9, and 3A4 were in-vitro assayed with THC and THCA to examine their metabolite products and provide a solution to achieving the first step in the proposed mechanism. THC and THCA were also docked to these enzymes to create predictions on expected metabolites. An alternative approach was to use the consensus sequence motif of Cytochrome P450 19A1 and BLAST search the C. sativa genome for candidate enzymes that could potentially convert ∆⁹-tetrahydrocannabinolic acid (THCA) to cannabinolic acid (CBNA) or present a candidate suitable for the formation of the first intermediate. The candidates were then modeled and docked with THCA to obtain the five models with the highest binding affinity. These candidates were further docked with THC. Out of these, the docking of THC and THCA with CsCYP84A1 provides the best models that allow the formation of the first intermediate. Similarly, CYP19A1 docking with THC and THCA, and CYP2C9 docking with THCA provided models that predict the formation of the first intermediate. Only the in-vitro assays of CYP2C9 show catalytic activity against THC to produce 11-OH-THC, while the assay with THCA produces 11-OH-THC, 11-OH-THCA, and an unknown metabolite that needs to be identified. The assays need to be re-analyzed with a GC-MS to identify finite metabolites that were not observed when THC and THCA decreased over time.

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