Cannabinoids biosynthesis

Table 2 Properties of enzymes foimd in cannabinoid biosynthesis... 

Canadensolide H NMR, 4, 578 Cannabichromanone synthesis, 3, 726 Cannabichromene, 3, 675 photochemistry, 3, 721 synthesis, 3, 721, 746, 748, 782 Cannabichromenic acid thermolysis, 3, 721 Cannabifuran synthesis, 4, 698 Cannabinoids biosynthesis, 3, 877 structure, 4, 548 thermolysis, 3, 721 Cannabinol occurrence, 3, 665 as pharmaceutical, 1, 151 synthesis, 3, 721, 786 Cannabinol, hexahydro-synthesis, 3, 787 Cannabinol, A -tetrahydro-occurrence, 3, 718 thermolysis, 3, 721 Cannabinol, All6-tetrahydro-metabolism, 1, 239 Cannabinol, 3,4-cis-A1,2-tetrahydro-synthesis, 3, 782... 

Phlorisovalerophenone synthase (VPS) from flower cones of hop (Humulus lupulus L.) utilizes isovaleryl-CoA or isobutyryl-CoA as starter molecules [24] (Fig. 2). Three molecules of malonyl-CoA are added to these starters to form phlorisovalerophenone or phorisobuty-rophenone, respectively, precursors for the biosynthesis of hop bitter acids. The first committed step of cannabinoid biosynthesis in glandular trichomes of Cannabis sativa is catalyzed by a stilbene synthase carboxylate-like (STCSL) polyketide synthase using n-hexanoyl-CoA as starter molecule, yielding olivetolic acid [25]. 

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. 

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. 

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

The late cannabinoid pathway starts with the alkylation of ohvetolic acid (3.2 in Fig. 4) as polyketide by geranyl diphosphate (3.1) as the terpenoid unit. Terpenoids can be found in all organisms, and in plants two terpenoid pathways are known, the so called mevalonate (MEV) and non-mevalonate (DXP) pathway as described by Eisenrich, lichtenthaler and Rohdich [23,24,29,30]. The mevalonate pathway is located in the cytoplasm of the plant cells [30], whereas the DXP pathway as major pathway is located in the plastids of the plant cells [29] and delivers geranyl diphosphate as one important precursor in the biosynthesis. 

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. 

All the foregoing pharmacological effects of anandamide, in conjunction with the well-documented existence of specific systems for its biosynthesis, catabolism, and cellular reuptake to be discussed shortly, suggest that anandamide is indeed the endogenous cannabinoid ligand. The other two less studied A -acylethanolamide endocannabinoids and also 2-AG may serve similar functions. The differential roles of each of these four endocannabinoids are still unclear. 

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

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. 

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 resin secreted by Cannabis indica and Cannabis sativa, varieties of hemp, is known variously as marijuana, hashish or bhang and is abused as a hallucinogenic drug. It appears however to have some beneficial properties and is currently under test as an antiemetic in cancer therapy. The secretion contains a number of interrelated oxygen heterocycles, some of which are shown in Scheme 281, which attempts to indicate their biosynthetic relationships (70MI22401). The cannabinoids are probably derived from a monoterpene unit based on p-menthane and 5-n-pentylresorcinol (olivetol), acting the part of a polyketide. 2,2-Dimethylchromene biosynthesis also requires the intervention of an isoprene fragment. 

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). 

---