Cannabis through the looking glass: chemo- and enantio-selective separation of phytocannabinoids by enantioselective ultra high performance supercritical fluid chromatography.

By using the Inverted Chirality Columns Approach (ICCA) we have developed an enantioselective UHPSFC method to determine the enantiomeric excess (ee) of (-)-Δ9-THC in medicinal marijuana (Bedrocan®). The ee was high (99.73%), but the concentration of the (+)-enantiomer (0.135%) was not negligible, and it is worth a systematic evaluation of bioactivity.

Evaluation of the macrocyclic glycopeptide A-40,926 as a high-performance liquid chromatographic chiral selector and comparison with teicoplanin chiral stationary phase.

A new macrocyclic antibiotic of the vancomycin family, referred to by its industrial designation as A-40,926, was bonded to 5 microm silica particles and utilised as a chiral stationary phase (CSP). Since A-40,926 is structurally related to teicoplanin, the A-40,926 CSP was compared to a commercially available teicoplanin CSP. A set of 28 chiral compounds, including amino-acids and related compounds, compounds with a ring containing the stereogenic centre, compounds bearing aromatic structures near their stereogenic centres and alcohols, was tested for enantioseparation on the two CSPs. The results are compared and discussed in terms of enantioselective Gibbs energy difference. The A-40,926 CSP was able to resolve one compound that was not resolved by the teicoplanin CSP. However, it could not separate four compounds that the teicoplanin CSP did separate. It is shown that the A-40,926 CSP is complementary to the teicoplanin CSP, thereby enlarging the number of enantiomers that can be separated by the macrocyclic glycopeptide based CSPs.

Design and realization of a tailor-made enzyme to modify the molecular recognition of 2-arylpropionic esters by Candida rugosa lipase.

Within a research project aimed at probing the substrate specificity and the enantioselectivity of Candida rugosa lipase (CRL), computer modeling studies of the interactions between CRL and methyl (+/-)-2-(3-benzoylphenyl)propionate (Ketoprofen methyl ester) have been carried out in order to identify which amino acids are essential to the enzyme/substrate interaction. Different binding models of the substrate enantiomers to the active site of CRL were investigated by applying a computational protocol based on molecular docking, conformational analysis, and energy minimization procedures. The structural models of the computer generated complexes between CRL and the substrates enabled us to propose that Phe344 and Phe345, in addition to the residues constituting the catalytic triad and the oxyanion hole, are the amino acids mainly involved in the enzyme-ligand interactions. To test the importance of these residues for the enzymatic activity, site-directed mutagenesis of the selected amino acids has been performed, and the mutated enzymes have been evaluated for their conversion and selectivity capabilities toward different substrates. The experimental results obtained in these biotransformation reactions indicate that Phe344 and especially Phe345 influence CRL activity, supporting the findings of our theoretical simulations.

New synthetic strategies for the preparation of novel chiral stationary phases for high-performance liquid chromatography containing natural pool selectors.

Twelve new chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC) were generally prepared starting from the macrocyclic glycopeptide antibiotic teicoplanin, according to novel and efficient 'one-pot' synthetic strategies. Their chiral recognition abilities were evaluated under polar-organic mode HPLC, towards a variety of biopharmacological interesting racemates, such as beta-amino acids and quaternary ammonium salts (e.g. carnitine and its derivatives). All materials were prepared by two different synthetic strategies, both leading to the formation of one or two stable ureidic functions on the CSP structure. The influence of the different spacers and of the silica matrix nature on the chiral performances was investigated. The obtained results suggested that the optimal synthetic strategy was that leading to the formation of two ureidic functions on the CSP structure, spaced-out by a six-carbon atoms aliphatic chain; the best chromatographic results were reached with the use of the spherical LiChrospher silica gel. Enantioselectivity factors (alpha) particularly high and short-time analyses characterised the analytical procedures; in addition, analytes lacking in chromophore groups were easily detected by evaporative light scattering (ELS) with no need of preliminary derivatization.

Role of the carbohydrate moieties in chiral recognition on teicoplanin-based LC stationary phases.

For this study, we used the macrocyclic antibiotic teicoplanin, a molecule consisting of an aglycone peptide "basket" with three attached carbohydrate (sugar) moieties. The sugar units were removed and the aglycone was purified. Two chiral stationary phases (CSPs) were prepared in a similar way, one with the native teicoplanin molecule and the other with the aglycone. Twenty-six compounds were evaluated on the two CSPs with seven RPLC mobile phases and two polar organic mobile phases. The compounds were 13 amino acids or structurally related compounds (including DOPA, folinic acid, etc.) and 13 other compounds (such as carnitine, bromacil, etc.). The chromatographic results are given as the retention, selectivity, and resolution factors along with the peak efficiency and the enantioselective free energy difference corresponding to the separation of the two enantiomers. The polarities of the two CSPs are similar. It is clearly established that the aglycone is responsible for the enantioseparation of amino acids. The difference in enantioselective free energy between the aglycone CSP and the teicoplanin CSP was between 0.3 and 1 kcal/mol for amino acid enantioseparations. This produced resolution factors 2-5 times higher with the aglycone CSP. Four non amino acid compounds were separated only on the teicoplanin CSP. Six and five compounds were better separated on the teicoplanin and aglycone CSPs, respectively. Although the sugar units decrease the resolution of alpha-amino acid enantiomers, they can contribute significantly to the resolution of a number of non amino acid enantiomeric pairs.

Synthesis and applications of novel, highly efficient HPLC chiral stationary phases: a chiral dimension in drug research analysis.

This review provides an overview of the synthesis and application of stable and versatile HPLC chiral stationary phases (CSPs), with emphasis placed on the binding strategies developed to anchor several structurally different chiral selectors to silica-gel microparticles. In addition, selected applications relating to the use of these CSPs for the direct resolution of racemates of biological and pharmaceutical relevance will be described. This review discusses enantioselective molecular recognition and dynamic stereochemistry of stereolabile compounds with reference to receptor-based chiral stationary phases (CSPs) and dynamic HPLC on CSPs, respectively.

Direct chromatographic resolution of carnitine and O-acylcarnitine enantiomers on a teicoplanin-bonded chiral stationary phase.

R-(-)-Carnitine (vitamin B(T)) plays an important role in human energy metabolism, by facilitating the transport of long-chained fatty acids across the mitochondrial membranes. Its (S)-enantiomer acts as a competitive inhibitor of carnitine acetyltransferase, causing depletion of the body R-(-)-carnitine stock. Consequently, the separation of carnitine enantiomers is very important both to study their biological activities and to control the enantiomeric purity of pharmaceutical formulations. In the present paper we describe an easy, fast and convenient procedure for the separation of the enantiomers of carnitine and O-acylcarnitines by enantioselective HPLC on a laboratory-made chiral column containing covalently bonded teicoplanin as selector. High enantioselectivity factors (alpha values ranging from 1.31 to 3.02) and short-time analyses characterize the analytical procedure; in addition, analytes are easily detected by evaporative light scattering with no need for preliminary derivatization. The effects of pH and ionic strength of the mobile phase and of the nature of the organic modifier on the enantioselective separations were also investigated.

Direct HPLC separation of beta-aminoester enantiomers on totally synthetic chiral stationary phases.

The direct separation of beta-aminoester enantiomers by HPLC on synthetic chiral stationary phases based on a pi-acidic derivative of trans 1,2-diaminocyclohexane as selector is described. The application of different columns containing the stationary phase with opposite configurations and in the racemic form to the determination of enantiomeric excess in chemically impure samples is demonstrated.