Biosynthesis of Cannabinoids

In this section, the latest developments and recent publications on the biosynthesis of A9-THC and related cannabinoids as precursors are discussed. Special points of interests are the genetic aspects, enzyme regulation, and the environmental factors that have an influence on the cannabinoid content in the plant. Because of new and innovative developments in biotechnology we will give a short overview of new strategies for cannabinoid production in plant cell cultures and in heterologous organisms. 

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After identification of A9-THC as the major active compound in Cannabis and its structural elucidation by Mechoulam and Gaoni in 1964 [66], a lot of work was invested in chemical synthesis of this substance. Analogous to the biosynthesis of cannabinoids, the central step in most of the A9-THC syntheses routes is the reaction of a terpene with a resorcin derivate (e.g., olivetol). Many different compounds were employed as terpenoid compounds, for example citral [67], verbenol [68], or chrysanthenol [69]. The employment of optically pure precursors is inevitable to get the desired (-)-trans-A9-THC. 

Shoyama Y, Yagi M, Nishioka I, Yamauchi T, Cannabis. 8. Biosynthesis of cannabinoid acids. Phytochemistry 14 2189—2192, 1975. 

In the 1970s the biosynthesis of cannabinoids was investigated with radiolabeling experiments. 14C-labeled mevalonate and malonate were shown to be incorporated into tetrahydrocannabinolic acid and cannabichromenic acid at very low rates (< 0.02%). Until 1990 the precursors of all terpenoids, isopentenyl diphosphate and dimethyl-allyl diphosphate were believed to be biosynthesized via the mevalonate pathway. Subsequent studies, however, proved that many plant terpenoids are biosynthesized via the recently discovered deoxyxylulose phosphate pathway (Eisenreich et al., 1998 Rohmer, 1999). It was shown that the Cio-terpenoid moiety of cannabinoids is biosynthesized entirely or predominantly (>98%) via this pathway (Fellermeister et al., 2001). The phenolic moiety is generated by a polyketide-type reaction sequence. 

The formation of both compounds is accompanied by the biosynthesis of cannabinoid-inactive or weakly active congeners, which have been suggested to exert an enhancement of AEA and 2-AG actions via various mechanisms collectively referred to as entourage effects (Ben-Shabat et al. 1998 Mechoulam et al. 1998b for review). 

Seventy cannabinoids from C. sativa have been described up to 2005 [2j. Mostly they appear in low quantities, but some of them shall be mentioned in the following overview - especially because of their fimctions in the biosynthesis of A9-THC and their use in medicinal applications. 

The lUPAC name of cannabidiol is 2-[(lS, 6iI)-3-methyl-6-prop-l-en-2-yl-l-cyclohex-2-enyl]-5-pentyl-benzene-1,3-diol. Cannabidiol (CBD, 2.9) in its acidic form cannabidiolic acid (CBDA, 2.10) is the second major cannabinoid in C. sativa besides A9-THC. As already mentioned for A9-THC, variations in the length of the side chain are also possible for CBD. Important in this context are the propyl side chain-substituted CBD, named cannabidivarin (CBDV, 2.11), and CBD-C4 (2.12), the homologous compound with a butyl side chain. Related to the synthesis starting from CBD to A9-THC as described in Sect. 3.1, it was accepted that CBDA serves as a precursor for THCA in the biosynthesis. Recent publications indicate that CBDA and THCA are formed from the same precursor, cannabigerolic acid (CBGA), and that it is unlikely that the biosynthesis of THCA from CBDA takes place in C. sativa. 

Fig. 4 Biosynthesis of THC and related cannabinoids a GOT, b THCs, c CBDs, d CBCs...

Cadas H, di Tomaso E, Piomelli D. Occurrence and biosynthesis of endogenous cannabinoid precursor, A-arachidonyl phosphatidylethanolamine, in rat brain. J Neurosci 1997 17 1226-1242. 

Cadas H, Gaillet S, Beltramo M, Venance L, Piomelli D. Biosynthesis of an endogenous cannabinoid precursor in neurons and its control by calcium and cAMP. J Neurosci 1996 16 3934-3942. 

In their next study, Shoyama and Nishioka isolated new spirocom-pounds cannabispirol and acetyl cannabispirol. This is in addition to the already known cannabispirone and cannabispirenone from a Japanese hemp variety. The two scientists included them in their biogenetic schema alongside the cannabinoid acids. In a further study, Shoyama et al. dealt with the biosynthesis of propylcannabinoid acids by in vitro incubation with raw enzyme solution from three species of Cannabis sativa KL. A biogenetic schema is presented illustrating the relationship between methyl, propyl and pentyl cannabinoid acids. 

Shoyama Y, Hirano H, Nishioka I, Cannabis. Part 16. Biosynthesis of propyl cannabinoid acid and its biosynthetic relationship with pentyl and methyl cannabinoid acids, Phytochemistry 25 1909—1912, 1984. 

Extensive studies on the endocannabinoid system have revealed a number of cannabinergic proteins involved in the inactivation and biosynthesis of endocannabinoids. These include fatty acid amide hydrolase (FAAH) (Di Marzo et al. 1994 Gaetani et al. 2003 Piomelli et al. 1999), monoglyceride lipase (MAG) (Dinh et al. 2002), and the anandamide transporter (ANT) (Beltramo et al. 1997 Di Marzo et al. 1994 Fegley et al. 2004 Hillard et al. 1997). The above three proteins and the two cannabinoid receptors have received considerable attention and show great promise as potential targets for the development of novel medications for various conditions, including pain, immunosuppression, peripheral vascular disease, appetite enhancement or suppression, and motor disorders. 

Kuwae, T., Shiota, Y., Schmid, PC., Krebsbach, R., Schmid, H.Fl.O., 1999. Biosynthesis and turnover of anandamide and other A-acylethanolamines in peritoneal macrophages. FEBS Lett. 459, 123-127. Lau, A.H. and Chow, S.S., 2003. Effects of cannabinoid receptor agonists on immunologically induced histamine release from rat peritoneal mast cells. Eur. J. Pharmacol. 464, 229-235. 

Gerwick WH, Wise ML, Sonderstrom K, Murray TF. (1997) Biosynthesis and cannabinoid receptor affinity of the novel eicosanoid, conjuncated triene anandamide. Advances in Experimental Medicine and Biology 407 329-334. 

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