Olivetol

Electrochemical Detection of Olivetol Based on Poly(L-Serine) Film Layered Copper Oxide Modified Carbon Paste Electrode (p-L-Serine/CuO/CPE)

Olivetol is an important polyphenol compound and intermediate in the synthesis of cannabinoids possessing many types of biological activities. A facile electrochemical sensor for olivetol was fabricated based on p-L-serine, and copper oxide (CuO) nanoparticles modified carbon paste electrode (p-L-serine/CuO/CPE). The proposed p-L-serine/CuO/CPE was applied to the electrochemical detection of olivetol by cyclic voltammetry (CV) and differential pulse voltammetric (DPV). Through the characterizations of materials and modified electrodes, the p-L-serine/CuO/CPE exhibited enhanced electrochemical signals for olivetol compared to bare CPE and CuO/CPE in both CV and DPV methods. Under the optimized conditions, the proposed p-L-serine/CuO/CPE showed a good quantitative analysis ability and a wide analysis range from 20 to 100 μmol L-1 of olivetol with a limit of detection of 1.04 μmol L-1. Based on the reproducibility, repeatability, and stability exhibited by this fabricated sensor and the cheap and accessible raw materials, the p-L-serine/CuO/CPE became a novel determination choice for olivetol in the electrochemical method with the advantages of being cost-effective and convenient.

Complete biosynthesis of cannabinoids and their unnatural analogues in yeast

Cannabis sativa L. has been cultivated and used around the globe for its medicinal properties for millennia¹. Some cannabinoids, the hallmark constituents of Cannabis, and their analogues have been investigated extensively for their potential medical applications². Certain cannabinoid formulations have been approved as prescription drugs in several countries for the treatment of a range of human ailments³. However, the study and medicinal use of cannabinoids has been hampered by the legal scheduling of Cannabis, the low in planta abundances of nearly all of the dozens of known cannabinoids⁴, and their structural complexity, which limits bulk chemical synthesis. Here we report the complete biosynthesis of the major cannabinoids cannabigerolic acid, Δ⁹-tetrahydrocannabinolic acid, cannabidiolic acid, Δ⁹-tetrahydrocannabivarinic acid and cannabidivarinic acid in Saccharomyces cerevisiae, from the simple sugar galactose. To accomplish this, we engineered the native mevalonate pathway to provide a high flux of geranyl pyrophosphate and introduced a heterologous, multi-organism-derived hexanoyl-CoA biosynthetic pathway⁵. We also introduced the Cannabis genes that encode the enzymes involved in the biosynthesis of olivetolic acid⁶, as well as the gene for a previously undiscovered enzyme with geranylpyrophosphate:olivetolate geranyltransferase activity and the genes for corresponding cannabinoid synthases^7,8. Furthermore, we established a biosynthetic approach that harnessed the promiscuity of several pathway genes to produce cannabinoid analogues. Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule that is known to alter receptor binding affinity and potency⁹. We also demonstrated that our biological system could be complemented by simple synthetic chemistry to further expand the accessible chemical space. Our work presents a platform for the production of natural and unnatural cannabinoids that will allow for more rigorous study of these compounds and could be used in the development of treatments for a variety of human health problems.

Synthetic bioactive olivetol-related amides: The influence of the phenolic group in cannabinoid receptor activity

Focusing on the importance of the free phenolic hydroxyl moiety, a family of 23 alkylresorcinol-based compounds were developed and evaluated for their cannabinoid receptor binding properties. The non-symmetrical hexylresorcinol derivative 29 turned out to be a CB2-selective competitive antagonist/inverse agonist endowed with good potency. Both the olivetol- and 5-(2-methyloctan-2-yl)resorcinol-based derivatives 23 and 24 exhibited a significant antinociceptive activity. Interestingly, compound 24 proved to be able to activate both cannabinoid and TRPV1 receptors. Even if cannabinoid receptor subtype selectivity remained a goal only partially achieved, results confirm the validity of the alkylresorcinol nucleus as skeleton for the identification of potent cannabinoid receptor modulators.

Biotransformation of olivetol by Syncephalastrum racemosum

A study of the biotransformation of olivetol by Syncephalastrum racemosum ATCC 18192 has led to the isolation of three metabolites, which were identified as 4'-hydroxy-olivetol, 3-(3,5-dihydroxyphenyl)-1-propanol, and 3-(3,5-dihydroxyphenyl)-1-propanoic acid. The structures of the isolated metabolites were deduced by comparison of their spectral properties (pmr, cmr, ms) with those of olivetol. The absolute configuration of 4'-hydroxy-olivetol was determined to be R by the Horeau partial resolution method. Biotransformation of olivetol therefore appears to occur by a subterminal oxidation process.

Structure of the Cannabis sativa olivetol-producing enzyme reveals cyclization plasticity in type III polyketide synthases

In the native pathway to therapeutic cannabinoid biosynthesis in Cannabis sativa, the three-step production of a key intermediate, olivetolic acid, is catalysed by the enzymes tetraketide synthase (TKS; linear tetraketide intermediate production in two stages) and olivetolic acid cyclase (OAC; final C2 → C7 aldol condensation). In the absence of OAC, a nonenzymatic C2 → C7 decarboxylative aldol condensation of the tetraketide intermediate occurs forming olivetol. TKS is a type III polyketide synthase, and the question arises why it is unable to form olivetolic acid directly, but instead forms this unwanted side product. We determined the TKS, CoA complex structure, and performed structurally guided mutagenesis studies to identify potential residues responsible for cyclization pathway discrimination in type III polyketide synthases. Prior studies suggested an 'aldol switch' is necessary to allow linear tetraketide intermediate release prior to cyclization, thereby enabling subsequent olivetolic acid production by OAC. However, our studies do not support the presence of a universal or predictable 'aldol switch' consensus sequence. Instead, we propose the mode of ordered active site water activation between type III polyketide synthases catalysing different cyclization mechanisms is subtle and homologue-specific. Our work indicates that subtle structural variations between homologous enzymes can have a major mechanistic impact on the catalytic outcome. This highlights the importance of embedding high-resolution structural analysis of multiple enzyme homologues with classical site-directed mutagenesis studies when investigating highly similar enzymes with different mechanistic pathway outcomes. ENZYMES: TKS, EC 2.3.1.206; OAC, EC 4.4.1.26; chalcone synthase, EC 2.3.1.74; stilbene synthase, EC 2.3.1.95; 2-PS, EC 2.3.1.-. ACCESSION NUMBERS: The atomic coordinates and structure factors for the crystal structure of TKS have been deposited in the Protein Data Bank with accession number 6GW3.