## 3 Biosynthetic Routes to Nitrogen-Containing and Phenolic Metabolites in Cannabis
### Biosynthesis of Spermidine and Spermidine Alkaloids  
Given their vital function within organisms, the biosynthetic pathways of polyamines are similar from one kingdom of life to another [24]. Polyamine anabolism begins with either the non-proteinogenic amino acid L-ornithine or with the amino acid L-arginine, which are decarboxylated by ornithine decarboxylase (or arginine decarboxylase, respectively) (Figure 6). The production of putrescine from arginine requires two additional steps, compared to the direct ornithine pathway. The biosynthesis of spermidine requires an aminopropyl group donor, obtained through _S_-adenosylmethionineamine. Figure 6: Spermidine biosynthetic pathway. **(A)** General biosynthesis of putrescine. ODC, ornithine decarboxylase. Arrow represents one biochemical reaction. ADC, arginine decarboxylase. AIH, agmatine minohydrolase. CPA, _N_-carbamoylplutrescine amidase. Ornithine decarboxylase (E.C. 4.1.1.17) produces putrescine and CO\({}_{2}\) from L-ornithine. Argin decarboxylase (E.C. 4.1.1.19) produces agmatine and CO\({}_{2}\) from L-arginine. Agmatine minohydrolase (E.C. 3.5.3.12) produces \(N\)-carbamoylplutrescine and NH\({}_{3}\) from agmatine. _N_-carbamoylplutrescine amidase (E.C. 3.5.1.53) produces putrescine through hydrolysis, generating NH\({}_{3}\) and CO\({}_{2}\) as well. **(B)** General biosynthesis of spermidine. SAMDC, _S_-adenosylmethionine� decarboxylase. SPDS, spermidine synthase. _S_-adenosylmethionine� decarboxylase (E.C. 4.1.1.50) produces \(S\)-adenosylmethionineamine and CO\({}_{2}\) through decarboxylation of _S_-adenosyl-L-methionine. Spermidine synthase (E.C. 2.5.1.16) utilizes the aforementioned _S_-adenosylmethionineamine to transfer a propylamine group to putrescine, generating spermidine and S-methyl-5\({}^{\prime}\)-thioadenosine. Cannabisativine was first reported in 1975, and its structure was elucidated by X-ray crystallography [39]. Little is known about its biosynthesis, other than the two terminal nitrogens of spermidine, which are thought to be condensed onto another molecule, most likely a 14-carbon fatty acid, generating a dihydropyriphyllin-type skeleton [29]. Given the immense diversity of natural products, and with a flabbergastingly low number of enzymes, two mechanisms are laid out to explain their advent. The first is that organisms tend to make use of metabolic branches common to other species, forming shared molecular skeletons and founding specialized metabolic pathways where the skeletons are tailored by various enzymes belonging to a specific phylogenetic group (scaffolding principle). The second one is the use by homologous enzymes of substrates that are more or less similar, thus allowing molecular constructions related through the enzyme catalyzing the reaction, but nevertheless molecularly different [52]. In the case of cannabisativine and its dehydration product, anhydroranchabisativine, the second explanation is likely to be the most plausible. Indeed, very similar alkaloids have been reported in several species of the _Equisetum_ genus, including palustrine and palustridine, characterized by a 13-atom macrocycle, formed, according to the hypothesis, by the enantioselective condensation of the terminal nitrogens of spermidine, on a 10-carbon fatty acid (Figure 7) [29]. This suggests that a similar enzymatic reaction occurs in _Cannabis_, as in _Equisetum_ species, but using a 14-carbon fatty acid. A notable difference between the dihydropyriphyllin alkaloids of _Equisetum_ species and _Cannabis_ is the position of the double bond in the six-atom heterocycle. Indeed, the double bond is between C5 and C6 of the fatty acid for cannabisativines, as opposed to C4 and C5 for palustrine and palustridine (C1 being the carbon bearing the amide function). Two possibilities then seem conceivable: either the fatty acid has its double bond before the spermidine condensation, and since the fatty acid  
Figure 7: Dihydropyriphyllin skeleton and similar _Cannabis_ and _Equisetum_ sp. alkaloids.