Cannabinoids

PRIMARY COMPONENTS OF CANNABIS

 

What are cannabinoids?

 

Cannabinoids originally referred to a class of oxygen containing aromatic compounds which contained 21 carbons that were produced by Cannabis sativa. Now cannabinoids has a broader definition which refers to, phytocannabinoids which entail the original compounds but also anything that is similar in structure, as well as synthetic cannabinoids which are synthesized in a lab to have similar structure to natural cannabinoids. The structures of the common cannabinoids are similar to each other with a central aromatic ring containing two oxygens ortho to each other(substituted or not), a pentyl group, and the last group is what changes which is in between the oxygens. The only difference in the THCV, CBDV, etc. analogs is that the pentyl group is replace with a propyl, which comes about in the way it is made in nature.

 

Figure 1. Common cannabinoids that we test for.

 

How are cannabinoids made in nature?

 

The method molecules are made in plants is called biosynthesis, and for cannabinoids they start with cannabigerolic acid, CBGA, which is formed using a prenyltransferase enzyme[1], an enzyme is nature’s way of accelerating a reaction, in this case the combination of olivetolic acid and geranyl diphosphate (Figure 1). CBGA can then undergo a number of changes; Δ9-THCA synthase converts CBGA to Δ9-THCA, CBDA synthase converts CBGA to CBDA, heat or light causes a decarboxylation of CBGA which gives CBG (Figure 1). There are other transformations such as formation of CBCA which will not be discussed here.Figure 2. Biosynthesis of cannabinoids CBGA, Δ9-THCA, and CBDA starting from olivetolic acid and geranyl diphosphate. Decarboxylation of CBGA gives CBG (carboxylic acid shown in blue).

Synthase is a specific type of enzyme and there has been some work done on how these synthases work. Kuroki et al. obtained a crystal structure of the Δ9-THCA synthase and determined where CBGA interacts via hydrogen bonds to the synthase[2]. The mechanisms appear to slightly change from one paper to another but it appears that the FAD accepts an allylic hydride from CBGA which forms a resonance stabilized carbocation. It is important that the alkene is the E isomer to have the correct configuration for the next cascade step which can react in different ways depending on the synthase. For Δ9-THCA synthase it is a substitution type reaction where the phenolate oxygen attacks the tri-substituted alkene which then attacks the second alkene forming the two rings (Figure 2). In the case of the CBDA synthase an elimination reaction where the allylic hydrogen is deprotonated which then closes the ring forming CBDA. The FADH2 can be regenerated from reducing oxygen to hydrogen peroxide. The acid forms are what nature produces which can then be decarboxylated with heat, light, or acid.

Figure 2. Mechanism of the synthase of Δ9-THCA and CBDA.

 

How do we test for cannabinoids?

 

  • Homogenize the sample
  • Extract the cannabinoids from the plant material (matrix).
  • Centrifuge, cleanup, and dilute the sample.
  • Inject sample into UHPLC (Thermo Scientific Ultimate 3000, see Fig. X) which separates the cannabinoids and other organic molecules on a column based on their interactions with the stationary phase. Then they are pumped through a diode array detector, DAD, which looks at parts of the UV-vis spectrum where the organic compound adsorb light and depending on the intensity of the adsorption we can figure out the concentration for the given cannabinoids, this is called a chromatogram (Figure Y).

 

Figure X. UHPLC Thermo Scientific Ultimate 3000

Thermo Trace™ Ultimate 3000 HPLC system used to quantify cannabinoids.

 

 

Figure Y. Example chromatograph.

 

 

 

We use a UHPLC DAD method to quantify cannabinoids, we need 1  g of sample for the test.

 

[1]        M. Fellermeier, M. H. Zenk, FEBS Letters 1998, 427, 283-285.

[2]        Y. Shoyama, T. Tamada, K. Kurihara, A. Takeuchi, F. Taura, S. Arai, M. Blaber, Y. Shoyama, S. Morimoto, R. Kuroki, Journal of Molecular Biology 2012, 423, 96-105.