Improved enzyme-mediated synthesis and supramolecular self-assembly of naturally occurring conjugates of ß-sitosterol.

Naturally occurring acylated ß-sitosteryl glucosides have been investigated for their novel properties. The synthetic protocol based on the literature data was improved and optimized. The main improvement consists in employing systems of ionic liquids combined with organic solvents in lipase-mediated esterification of (3ß)-stigmast-5-en-3-yl ß-d-glucopyranoside to get (3ß)-stigmast-5-en-3-yl 6-O-acyl-ß-d-glucopyranosides. Maximum yields of these products were achieved with Candida antarctica lipase B immobilized on Immobead 150, recombinant from yeast, in absolute THF and in the presence of either ionic liquid [1-butyl-3-methyl imidazolium tetrafluoroborate ([BMIM]BF4) or 1-butyl-3-methyl imidazolium hexafluorophosphate ([BMIM]PF6)] employed. Pharmacological activity of (3ß)-stigmast-5-en-3-yl 6-O-acyl-ß-d-glucopyranosides was studied in tests on MCF7 tumor cell lines; the compounds displayed moderate activity which was higher than the activity of ß-sitosterol. Supramolecular characteristics were discovered at (3ß)-stigmast-5-en-3-yl 6-O-dodecanoyl-ß-d-glucopyranoside that formed supramolecular polymer through multiple H-bonds in a methanol/water system (60/40). Its formation was confirmed by the independent UV-vis measurements during certain time period, by variable temperature DOSY-NMR measurement in deuteriochloroform, and visualized by transmission electron microscopy (TEM) and atomic force microscopy (AFM) showing chiral helical structures and complex superassembly systems based on fibrous supramolecular polymer. In contrary, no such properties have been observed for the other two (3ß)-stigmast-5-en-3-yl 6-O-acyl-ß-d-glucopyranosides under the given experimental conditions.

Isolation of melatonin by immunoaffinity chromatography.

A single-step, highly specific and easy-to-use method was developed for isolation and purification of melatonin from complex biological matrices. Polyclonal antibodies highly specific against melatonin (with cross-reactivities with related compounds below 0.02%, except for 6-hydroxymelatonin) were raised, characterised by enzyme-linked immunosorbent assay (ELISA) and used for preparation of immunoaffinity gel. Melatonin recovery by the immunoaffinity method was approximately 95%, allowing single-step processing of samples prior to electrospray HPLC-MS analysis (with detection limit 10 fmol). The method was successfully used for determining melatonin in human serum and turned out to be better than the non-specific solid-phase extraction published earlier.