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. 2021 Feb 12;6(1):1–4. doi: 10.1089/can.2020.0027

Chirality in Cannabinoid Research

Crist N Filer 1,*
PMCID: PMC7891190  PMID: 33614946

Abstract

Mankind has long utilized Cannabis for diverse purposes. However, it has only been since the late 19th century that its individual cannabinoids began to be isolated, analyzed, and synthesized. By the mid-20th century it was discovered that many cannabinoids were asymmetric, with chirality often controlling their pharmacology. Increasingly accurate measurement and understanding of cannabinoid chirality will facilitate their synthesis and accelerate their medicinal applications.

Keywords: cannabinoid, cannabis, chiral HPLC, MicroED

The portfolio of cannabinoids in Cannabis is a unique pharmacopoeia. Since many of these substances contain asymmetric carbons, this perspective addresses the important subject of cannabinoid chirality, including current technical challenges and proposals to bridge analytical gaps. Opportunities in this area are indeed intriguing and compelling. For instance, based on the serendipitous affinity of (+)-cannabidiol and related compounds for the CB1 receptor,1 it would not be surprising to discover other cannabinoid unnatural (+)-enantiomers with unexpected and useful receptor binding. Also, given the precedent for differences in biological activity between enantiomers, regulatory agencies may require increasingly more rigorous measurement of cannabinoid enantiomeric purity. Clearly, the concept of chirality in cannabinoid research has evolved for more than a century with new instrumentation and methods. As background, a condensed history of it is useful to consider now.

Physician William O'Shaughnessy first introduced the science of Cannabis to Western medicine as early as 1839 while working for the British East India Company.2 At that time, he initiated careful animal toxicity studies before confidently treating various patients and their maladies with Cannabis. However, it was not until the end of the 19th century that chemists first began the methodical process of individual cannabinoid isolation, purification, and analysis. A brief review of a few specific substances is illustrative. Cannabinol was the first cannabinoid to be fully characterized, starting with the pioneering efforts of Wood et al. in 1899 and their discovery of its correct molecular formula.3 This structural problem then remained untouched for 30 years until Cahn extended the cannabinol investigation using colorimetric and derivatization reactions. Based on his meticulous investigations, Cahn even suggested a likely structure for cannabinol rather similar to 1 (Fig. 1) except for the incorrect positions of the resorcinol substituents.4 Finally, famed chemist Roger Adams in 1940 isolated and purified cannabinol as a crystalline solid from the red oil of Minnesota wild hemp and conclusively established its structure as 1 by a brilliant and convincing total synthesis.5 Clearly, this achievement was a chemistry tour de force for its time period, but the lack of any cannabinol stereochemistry challenges greatly simplified the task of its structure elucidation and synthesis.

FIG. 1.

FIG. 1.

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Structures of cannabinol (1), cannabidiol (2), delta-9-tetrahydrocannabinol (3), and cannabichromene (4).

Cannabinoid structural and synthetic chemistry then became far more complicated and Adams was again the first investigator to grapple with the enigma of cannabinoid stereochemistry and chirality in the context of cannabidiol. Compared with cannabinol, cannabidiol soon became a veritable Gordian knot. Purifying cannabidiol from Minnesota wild hemp, Adams isolated it as a crystalline solid and also determined its molecular formula. He then made two extraordinary observations about cannabidiol.6 Besides discovering that it was nonintoxicating, Adams also found that cannabidiol had a pronounced optical activity (ethanol) [α]27D of −1250. Therefore, in contrast to cannabinol, cannabidiol was a chiral molecule, possessing some element (center, axis, or plane) of asymmetry. This fact had profound implications for both the cannabidiol structure puzzle as well as its eventual and more difficult synthesis. Adams demonstrated that cannabidiol could be transformed to cannabinol,7 thereby confirming the basic carbon and oxygen cannabidiol framework.8 However, with only the limited benefit of UV and IR spectroscopy at his disposal, Adams was unable to decipher the exact stereochemistry of cannabidiol.

A hiatus of >20 years again elapsed before the cannabidiol stereochemistry conundrum was finally solved by mythic natural products chemist Raphael Mechoulam in his very first cannabinoid article.9 Mechoulam embraced the recent invention of nuclear magnetic resonance (NMR), quickly recognizing it as a transformational tool in organic structure determination. Using NMR chemical shift and coupling constant measurements, Mechoulam correctly identified the trans stereochemistry scaffold of cannabidiol as seen in structure 2. Employing analogous NMR studies, Mechoulam was also able to deduce the same trans stereochemistry arrangement for related cannabinoid delta-9-tetrahydrocannabinol as structure 310 and soon thereafter established the absolute chiral configurations of both 2 and 3.11 Eventually, the proposed structures for all three compounds as 1, 2, and 3 were corroborated by unequivocal X-ray crystallography.12–14 This crucial chirality information guided the clever stereoselective syntheses of the last two cannabinoids.15,16

Over the many decades since these early discoveries, the Cannabis chemovar has greatly expanded to a cornucopia of many hundreds of diverse substances, including >100 cannabinoids.17 New and exciting cannabinoids are continually being reported18 and two excellent accounts of this natural product growth have appeared. One of these articles by ElSohly19 chronicled the addition of cannabinoids from 1980 to 2005 and a recent magnum opus by Hanus serves as a widely cited comprehensive inventory of cannabinoids up to 2016.20 These publications also candidly revealed several instances of uncertainty with regard to cannabinoid stereochemistry and chirality. Concerning this gap, an examination of the literature was even more telling. The SciFinder® database contains ∼40,000 abstracted chemistry articles retrieved under the search term “cannabinoid.” However, when this astounding number of articles and patents was further refined and filtered by search terms such as “chiral” or “enantiomer” or “racemic,” less than about 1% of the abstracts contained these words. This would seem to indicate that the technical details of chirality and its rigorous measurement may not have kept pace with the discovery of new cannabinoids.

This conclusion is further supported by another examination of this same large SciFinder database for articles relating to the chiral high performance liquid chromatography (HPLC) of cannabinoids. Remarkably, no more than a few dozen chiral HPLC publications could be located and most of these articles addressed synthetic cannabinoid analogues21 rather than those in the Cannabis chemovar. Anecdotally, in reviewing articles announcing newly discovered cannabinoids, if chirality is mentioned at all it is often measured by the early 19th century method of optical rotation. However, one recent publication by Italian investigators22 stands out in technical contrast and these authors largely hold the same perspective noted in this study. Commenting on Cannabis natural product isolation they remarked that “analytical approaches to crude plant extracts have been developed by just taking into account the single cannabinoids, irrespective of their stereochemistry.”

Using a novel supercritical fluid chiral HPLC technique termed an inverted chirality columns approach (ICCA) they made some interesting stereochemical discoveries, one of which regarding the intriguing cannabinoid cannabichromene (4). Cannabichromene, the only major fluorescent cannabinoid,20 has been reported to be racemic. However, in re-evaluating the chirality of natural cannabichromene with ICCA, this more recent Italian study discovered that it was a scalemic mixture with an enantiomeric excess of about 25% of the (+)-enantiomer. As commented on by a reviewer of this article, the Italian authors also employed ICCA for the first time to detect a small amount of the (+)-delta-9-tetrahydrocannabinol enantiomer in medicinal marijuana. As a further resource to compliment the refereed literature, several useful application notes on cannabinoid chiral HPLC analysis by well-known manufacturers can easily be located with Internet searching.

Besides chiral HPLC analysis, there are several more useful technologies to consider in exploring cannabinoid chirality. Circular dichroism (CD) is a well-documented spectroscopic technique for the determination of natural product absolute configuration. There are many hundreds of literature references employing CD, but it has only infrequently been applied to cannabinoids.23 A more recent and powerful adaptation of CD is vibrational circular dichroism (VCD), extending the spectroscopy into the infrared and near-infrared regions. Instrumentation and newer developments for VCD have been recently reviewed.24 The literature contains only a few references to this valuable method in the cannabinoid area and most of these are in the context of synthetic cannabinoid characterization.25

An exhaustive literature search revealed several more useful techniques that have not yet been employed in cannabinoid research and may also facilitate chiral determinations. The first of these is an ingenious NMR method, chiral lanthanide shift reagent (CLSR) analysis, developed by George Whitesides at MIT in the early 1970s.26,27 This is a well-documented NMR technique that has been utilized on diverse compounds in scores of literature publications. In brief, when an asymmetric lanthanide (such as europium) reagent is added directly to an NMR tube of a racemic or scalemic compound mixture, the NMR peaks of each enantiomer can often be separated by virtue of the differential shielding/deshielding effect imparted by the complexed lanthanide metal geometry. Integration of the separated enantiomer NMRs can conveniently allow calculation of enantiomeric excess values. Most cannabinoids contain enough oxygen functional groups to accommodate lanthanide metals with coordination sites as well as sharp NMR peaks (especially aromatic) for integration. Also, this NMR technique has been demonstrated to work very effectively on lipophilic compounds.28 Having direct experience with this powerful method, we provided the Whitesides group with a very lipophilic diene (compound 17 of reference 27) for their successful CLSR analysis of it.

A second and more recent technical breakthrough may well represent a sea change in rapid organic structure determination. As we have discussed, X-ray crystallography has always been viewed as the ultimate tool for organic compound structure determination, including stereochemistry and chirality. A revolutionary new approach, termed microcrystal electron diffraction (MicroED), first successfully applied to proteins, has been recently demonstrated on small molecules as well.29 The significant advantage of this method is that it obviates the need to grow large crystals required for X-ray analysis, often difficult or impossible for many low melting cannabinoids. Instead, using a weaker electron beam (to prevent microcrystal damage) from a transmission electron cryo-microscope with improved data collection, MicroED allows analysis of compound powders that are themselves composed of microcrystals.

This perspective has provided a brief history, current state, and potential future direction for chiral analysis of cannabinoids. Nature has bestowed us with a treasure trove of diverse substances, artfully assembled in the prolific Cannabis trichome laboratories. Our increasingly accurate understanding of cannabinoid chirality will accelerate their synthesis and harness their full potential for the benefit of human health.

Abbreviations Used

CD

circular dichroism

CLSR

chiral lanthanide shift reagent

HPLC

high performance liquid chromatography

ICCA

inverted chirality columns approach

MicroED

microcrystal electron diffraction

NMR

nuclear magnetic resonance

VCD

vibrational circular dichroism

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

Cite this article as: Filer CN (2021) Chirality in cannabinoid research, Cannabis and Cannabinoid Research 6:1, 1–4, DOI: 10.1089/can.2020.0027.

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