Leaves from 10 different strains of fiber hemp were collected before anthesis or, in one case, also during and after anthesis. All samples were subjected to thermal decarboxylation of the biomass and ethanol extraction; then the crude ethanol extract was analyzed quantitatively by reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) (see Figure S1 in the Supporting Information for linearity, LOD, and LOQ values, and Figure S2 and Table S3 for chromatograms of standard mixture and resolutions values, respectively). The samples contained CBD (2) or CBG (4) as the dominant phytocannabinoid and low amounts of Δ⁹-trans-THC (3) (from below the detection threshold up to 0.16%). Figure 2 shows examples of chromatographic profiles of crude plant ethanol extracts of CBG-rich and CBD-rich Cannabis strains (C37 and Finola, respectively).

Major cannabinoids were quantified, and their concentrations as percentage (% w/w) values are reported in Table 1. In all samples, CBC (1a) was a relatively minor constituent, in the range of 0.02–0.67% of dry weight, with titers overall higher in the CBG varieties than in the CBD-rich strain.

Figure 3 reports the CBC content (% w/w) for the cultivars examined, and, in the single strain where leaves were collected at different development stages (i.e., entries 7a–f), the contents of CBC decreased during and after anthesis, in accordance with previous observations of higher CBC concentrations in juvenile tissues.¹⁸

After RP-UHPLC analysis, a crude CBC-enriched fraction was obtained by preparative TLC and analyzed by normal-phase enantioselective UHPLC using the ICCA strategy previously validated for the chiral analysis of phytocannabinoids.¹⁴ By calculation of the relative (%) area of the two enantiomers, the enantiomeric excess (ee) of CBC (1a) was calculated in all samples. Values ranging from 3% to 48% were observed, with the nature of the dominant enantiomer differing within the samples analyzed (Table 2).

The ICCA approach made it possible to avoid the coelution of impurities with retention times similar to those of the enantiomers under relative quantification, resulting in a more precise and reliable quantification of the ee, despite small variations between the values recorded from one column to another. In this work, the two enantiomers of CBC (1a) were discriminated based on the sign they showed on the ECD trace set at 280 nm (Figure S4 in the Supporting Information). No relationship between the dominance of a specific phytocannabinoid (CBD or CBG) and that of a specific enantiomer of CBC was observed, and no significant variation of the ee was observed during plant development (see Figure 4).

The optical purities of chiral chromenes are scarcely affected by heat.¹⁷ Still, they are significantly eroded by UV exposure,¹⁷ and there is the possibility that racemization could, to a certain extent, potentially affect the enantiomeric composition of CBC (1a) when data from different laboratories are compared. Nevertheless, the discovery of opposite enantiomeric dominance in different strains strongly suggests an enzymatic origin for the variation of the scalemate composition. It is not uncommon that the composition of natural scalemates significantly differs in related plants or even in different organs of the same plant.¹⁹ For mono- and sesquiterpenoids, this has been associated with the different expression of synthases with complementary enantioselectivity,^20,21 while the chirality status of scalemic lignans has been shown to depend on the expression of enantiomeric directing proteins.¹⁹ CBCA (1b), the native form of CBC (1a), is derived from CBGA (4) by the agency of a specific oxidase (CBCA-synthase), a member of the berberine bridge enzyme (BBE)-like gene family.²²⁻²⁴ The enzyme oxidizes the resorcinol core of CBGA (4) to an achiral quinone methide (5a), which next spontaneously electrocyclizes to CBCA (1b) (Scheme 1). Early studies on a partially purified enzyme suggested the formation of a 5:1 scalemate of CBCA,²² but additional confirmation was not reported, and nor was the dominant enantiomer identified or the presence of the directing proteins investigated. Oxidation to a quinone methide is also involved in the dimerization of phenylpropane alcohols (monolignols) to lignans, where an oxidative enzymatic phase is followed by a nonenzymatic coupling assisted by enantiospecific directing proteins.¹⁹ The enantiomeric status of CBC therefore could result from the involvement of synthases or of directing proteins with antipodal enantioselectivity. Further studies will be necessary to clarify this issue, which is of general relevance for the whole class of chromene and chromene-derived scalemates.²⁵ Remarkably, a similar tandem oxidation–electrocyclization of CBGA (6) might be involved in the scalemic state of cis-THC (8b),²⁶ of which the acidic precursor (8a) could derive from the E-Δ^1′,6 isomer 7b of the quinone methide involved in the biogenesis of CBCA (1b) (Scheme 1).