## 3 Biosynthetic Routes to Nitrogen-Containing and Phenolic Metabolites in Cannabis
### Hydroxcircinamic Acids and Lignanamides Biosynthesis  
Hydroxcircinamic acid amides (HCAAs) are obtained from the condensation of a cinnamic acid (or its analogues) with a polyamine or a phenylethylamine (Figure 5) [24; 49]. As such, HCAAs are considered polyamine end products, thus allowing their storage and regulation [49]. Corroborating this, a considerable portion of polyamines found in plant cells are either mono-, di-, or trisubstituted with phenolic acids [49]. Aromatic amino acids, agmatine as well as octopamine and the neurotransmitters dopamine and serotonin, can be condensed to form HCAAs [49]. There also are aromatic L-amino acid decarboxylases (E.C. 4.1.1.28) that are not substrate specific and are capable of decarboxylating multiple aromatic amines. These type II decarboxylases use a pyridoxal phosphate molecule (vitamin B\({}_{6}\)) as a cofactor [24]. The enzymes catalyzing the formation of HCAAs belong to the group of BAHD acyltransferases [46; 50]. Lignanamides are probably produced by the oxidative phenolic coupling of two HCAAs (e.g., \(N\)-p-coumaroyltyramine, \(N\)-caffeoyltyramine, etc.), but  
Figure 4: General phenylpropanolid pathway up to cinnamic acid thioesters’ biosynthesis. (* engaging step of the pathway). Red-dotted box: alternative pathway for \(p\)-coumaric acid biosynthesis. Arrow represents one biochemical reaction. PAL, phenylalanine ammonia-lyase. TPAL, tyrosine/phenylalanine ammonia-lyase. C4H, cinnmate 4-hydroxyase. 4 CL, 4-coumarate-CoA-ligase. Phenylalanine will undergo deamination by the enzyme phenylalanine ammonia-lyase (E.C. 4.3.1.24), which generates a molecule of NH\({}_{3}\) and trans-cinamic acid. Tyrosine/phenylalanine ammonia-lyase (E.C. 4.3.1.25) can realise the same reaction as PAL and can also convert tyrosine into \(p\)-coumaric acid. Cinnamate 4-hydroxyase (E.C. 1.14.19) adds a hydroxyl group to the _para_ position of trans-cinamic acid, generating \(p\)-coumaric acid. 4-coumarate-CoA-ligase (E.C. 6.2.1.12) uses an ATP molecule to convert \(p\)-coumaric acid to \(p\)-coumaroyl-CoA, releasing an AMP and a pyrophosphate molecule.  
no studies detailing their biosynthesis in _Cannabis_ have been found [29]. This underscores the lack of research efforts in this field, even though a fair number of papers describe their bioactivity in numerous species. In _Cannabis_ inflorescences of hemp varieties, the HCAA \(N\)-caffeoyltyramine is found in levels ranging from 0.1 to 76.2 mg/kg, with an average in the tens of mg/kg for four different cultivars [51]. Cannabisin A, B, and C are found in levels ranging from 0.003 to 2.85 mg/kg from the same samples previously described, with cannabis A generally found in higher concentrations and cannabis C in lower ones, with cannabis B right between the two [51].  
Figure 5: General biosynthesis of hydroxycinnamic acid amides from cinnamic acid thioesters. Blue-dotted boxes: some aminated molecules that can be condensed on a cinnamic acid moiety. Arrow represents one biochemical reaction. TYDC, tyrosine decarboxylase. ADC, arginine decarboxylase. PHT, putrescine N-hydroxycinnamoyltransferase. THT, hydroxycinnamoyl-CoA: tyramine N-(hydroxycinnamoyl)transferase. ACT, agmatine N4-counaroyltransferase. Tyrosine decarboxylase (E.C. 4.1.1.25) produces tyramine and CO\({}_{2}\) from tyrosine. Tryptamine decarboxylase (E.C. 4.1.1.105) produces tryptamine and CO\({}_{2}\) from tryptophan. Putrescine \(N\)-hydroxycinnamyotransferase (E.C. 2.3.1.138) produces a putrescine-derived hydroxycinnamic amide (e.g., \(N\)-\(p\)-coumarinoylputrescine). Hydroxycinnamoyl-CoA:tyramine \(N\)-(hydroxycinnamoyl) transferase (E.C. 2.3.1.110) produces a tyramine-derived hydroxycinnamoyl acid amide (e.g., \(N\)-\(p\)-coumarinoyltramine). Agmatine N4-coumaroyltrangerase (E.C. 2.3.1.64) produces an agmatine-derived hydroxycinnamic acid amide (e.g., \(N\)-\(p\)-coumarinoyltramine).