Generation of Cannabigerolic Acid Derivatives and Their Precursors by Using the Promiscuity of the Aromatic Prenyltransferase NphB

NphB is an aromatic prenyltransferase with high promiscuity for phenolics including flavonoids, isoflavonoids, and plant polyketides. It has been demonstrated that cannabigerolic acid is successfully formed by the reaction catalysed by NphB using geranyl diphosphate and olivetolic acid as substrates. In this study, the substrate specificity of NphB was further determined by using olivetolic acid derivatives as potential substrates for the formation of new synthetic cannabinoids. The derivatives differ in the hydrocarbon chain attached to C6 of the core structure. We performed in silico experiments, including docking of olivetolic acid derivatives, to identify differences in their binding modes. Substrate acceptance was predicted. Based on these results, a library of olivetolic acid derivatives was constructed and synthesized by using different organic synthetic routes. Conversion was monitored in in vitro assays with purified NphB versions. For the substrates leading to a high conversion olivetolic acid-C8, olivetolic acid-C2 and 2-benzyl-4,6-dihydroxybenzoic acid, the products were further elucidated and identified as cannbigerolic acid derivatives. Therefore, these substrates show potential to be adapted in cannabinoid biosynthesis. The conversion of novel olivetolic acid derivatives with the highly promiscuous prenyltransferase NphB is analyzed as a tool for the creation of synthetic cannabinoid libraries. By using in silico and in vitro experiments CBGA derivatives were synthesized and characterized as products of enzyme catalysis.image

Automated summary

This study utilized the highly promiscuous enzyme NphB to synthesize a library of synthetic cannabinoids by converting novel olivetolic acid derivatives. The substrates were characterized using in silico experiments and were further synthesized and analyzed in vitro. The resulting products were identified as CBGA derivatives, demonstrating the potential of these substrates in cannabinoid biosynthesis.