## 3 Biosynthetic Routes to Nitrogen-Containing and Phenolic Metabolites in Cannabis
### Flavonoids of Cannabis  
Flavanones are the dominant precursors for the biosynthesis of the different subclasses of flavonoids. It is noteworthy that the intramolecular cyclization of chalcones occurs spontaneously at room temperature, whereas stereospecific cyclization requires the enzyme (Figure 10) [56]. Flavanones are generally modified according to three types of reaction: oxidation, hydroxylation, and displacement of the aryl group (Figure 11) [56]. Figure 9: Hypothetical biosynthetic pathway of cannabisativine and anhydrocannabisativine. ACL, acyl-CoA ligase. Arrow represents one biochemical reaction and dotted arrow represents unknown (7) putative reaction. NMT, \(N\)-myristoyltransferase. First step is thought to be a condensation between spermidine and a fatty acid, presumably myristic acid. The open-end nitrogen atom of spermidine would then be fixed on C-3 and C-7 of the myristoyl moiety. A series of oxidation would generate the end products cannabisativine and anhydrocannabisativine. The order in which those oxidation reactions occur still is unknown. Naringenin is such a flavanone, which can be transformed into different subclasses of flavonoids in _Cannabis._ [PERSON] et al. (2020) quantified the molecule in the plant's inflorescences, with levels neighboring 0.5-1.0 mg/kg [51]. Such a low concentration compared to other flavonoids could be explained by naringenin's role as an upstream precursor these compounds. Cannflavin A and B were quantified by [PERSON] et al. (2017); their concentration in pre-flowering leaves ranges from 21 to 280 mg/kg for cannflavin A and 9 to 106 mg/kg for cannflavin B, with cannflavin A being the most abundant [57]. In _Cannabis_ inflorescences of the hemp cultivar, cannflavin A ranges from 20 to 130 mg/kg, and cannflavin B from 12 to 215 mg/kg, with cannflavin B being the most abundant [51]. It is unknown if this seeming discrepancy in cannflavin levels is due to the cultivar, stage of development, tissue, environmental growth conditions, or something else. Flavone synthase I is a dioxygenase requiring a molecule of 2-oxoglutarate to function, while FNS II is an enzyme of the cytochrome P450 family [56]. Flavones in _Cannabis_ are namely exemplified by luteolin and apigenin. Luteolin seems to be produced in higher concentrations than apigenin across multiple cultivars. Indeed, mean levels of luteolin are reaching 20 mg/kg depending on the cultivar, with lower ones reaching 10 mg/kg. Apigenin, on the other hand, has mean levels of around 6 mg/kg [51]. 7-O-glycoslated forms of those flavones have been identified, with levels comparable to luteolin and apigenin. Flavanone 3-dioxygenase is important in the regulation of flavonoid metabolism, concomitant with flavone synthases I and II [56; 58]. Dihydroflavonols serve as precursors for the biosynthesis of anthocyanins and flavonoids [58]. Catechin and epicatechin are dihydroflavonols found in _Cannabis_, with levels ranging from 0.1 to 334.0 mg/kg, with a mean of around 50 mg/kg for catechin and ranging 1.6 to 194.6 mg/kg with a mean of around 60 mg/kg for epicatechin [51]. Flavonols quercetin and kaempfer are, respectively, found in ranges of 6.2-58.5 mg/kg, with a mean of around 16 mg/kg, and 0.3-13.6 mg/kg, with a mean around 5 mg/kg. Figure 10: Intramolecular cyclization of chalcones and flavanone numbering. Figure 11: General biosynthetic pathway of flavonoids from \(p\)-coumaroyl-CoA. Flavanone is in bold, to emphasize its precursor role to other flavonoids. Arrow represents one biochemical reaction. CHS, chalcone synthase. CHI, chalcone isomerase. IFS, isoflavue synthase. FNS, flavone synthase I and II. F3H, flavanone 3-dioxygenase. FLS, flavonoid synthase. DFR, dihydroflavonol 4-reductase. LDOX, leucoanthocyanidin dioxygenase. UFGT, UDP-glucose–flavonol O-glucosyltransferase. Chalcone synthase (E.C.2.3.1.74) produces a chalcone from a phenylpropanol thioester (e.g., \(p\)-coumaroyl-CoA) by addition and subsequent cyclization of 3 molecules of malonyl-CoA.