## 3 Biosynthetic Routes to Nitrogen-Containing and Phenolic Metabolites in Cannabis
### Flavonoids of Cannabis
Chalcone isomerase (E.C.5.5.1.6) produces a flavanone from a chalcone through an intramolecular cyclization of an oxygen of the malonyl portion of the chalcone with the double bond originating from coumaric acid (see Figure 10). Isoflavone synthase (E.C. 1.14.14.87) produces an isoflavrone from a flavanone by migration of their aryl group, from C-2 to C-3, combined with an oxidation of C-2, resulting in the double bond formation between C-2 and C-3. Flavone synthase I and II (E.C. 1.14.20.5) (E.C. 1.14.19.76) produce flavones from flavanone by oxidizing C-2 and C-3, forming a double bond. Flavanone 3-dioxygenase (E.C. 1.14.11.9) produces a dihydroflavonol from a flavanone by oxidizing C-3 using oxoglutate, resulting in a hydroxyl group. Flavonol synthase (E.C. 1.14.20.6) produces a flavonoid from a dihydroflavonol by oxoglutate-dependent oxidation of C-2 and C-3, resulting in a double bond. Dihydroflavonol 4-reductase (E.C. 1.1.1.219) produces a leucoanthocyanidin from a dihydroflavonol by reducing the ketone group to a hydroxyl. Leucanthocyanidin dioxygenase (E.C. 1.14.20.4) produces anthocyanindin from leucoanthocyanidin by oxidation of the C ring, resulting in loss of hydroxyl groups and positive charge acquisition in the heterocycle. UDP-glucose–flavonol \(O\)-glucoseyltransferase (E.C. 2.4.1.91) produces anthocyanin by addition of sugar moieties to anthocyanins. _Camnabis_ flavonoids are found primarily in the leaves and flowers, but have also been isolated and identified from the pollen and stem of the plant [12]. There is no evidence to date for the presence of flavonoids in _Camnabis_ roots. _Camnabis_ seeds are thought not to contain prenylflavonoids, but germination induces the production of cannflavin A and B in leaves of plants that are not yet producing cannabinoids. Flavonoid concentrations decrease as the plant grows [12]. Flavonoid biosynthesis has only been slightly investigated in _Camnabis_. The biosynthetic pathways are well described, but several enzymes remain to be confirmed in planta. However, it is likely that the biosynthetic pathway is closely related to that of plants already studied, since three flavones widespread in the plant kingdom are found in _Camnabis_, namely apigenin, luteolin, and chrysoreiol, and their glycosylated analogues [59]. The proposed pathway in _Camnabis_ would therefore correspond to that presented in Figure 11. The biosynthesis of cannflavins probably occurs from the flavone luteolin (Figure 12). [PERSON] et al. provided new evidence in 2019 that suggests that the sequential order of cannflavin biosynthesis would be as follows: 3\({}^{\prime}\)-methylation of luteolin, followed by prenylation. Indeed, they identified, by phylogeny, a candidate enzyme for the prenylation reaction (CsPT3) and a candidate for the methylation reaction (CsOMT21) of luteolin. The candidate for prenylation accommodates both dimethylallyl pyrophosphate (DMAPP) and geranyl pyrophosphate (GPP), with chrysoreiol as the preferred substrate, rather than luteolin. Furthermore, the fact that no reports of prenylated luteolin have been produced suggests that the first of the two enzymatic reactions is methylation, generating chrysoreiol [59]. The candidate enzyme CsOMT21 does not methylate flavonones lacking a 3\({}^{\prime}\)-hydroxyl group, suggesting specificity for this position. Additionally, the preferred substrate of this enzyme is luteolin, but it also methylates quercetin (a flavonoid) at the 3\({}^{\prime}\) position, with 57% of the activity of luteolin. Cannflavin A and B have been quantified in some hemp cultivars and show comparable levels. They are found in the range of 19.6-130.0 mg/kg and 11.9-215.5 mg/kg, with a mean of around 62 and 84 mg/kg, respectively.