Synthesis of oligosaccharides of the linkage region of proteoglycans using regioselective glycosylation.

Proteoglycans (PGs) are complex macromolecules that are composed of glycosaminoglycan (GAG) chains covalently attached to a core protein through a tetrasaccharide linker. Biosynthesis of PGs is complex and involves a large number of glycosyltranferases. We report herein for the first time the synthesis of a collection of various sulfoforms of the disaccharide GlcA-1,3-ß-d-Gal and trisaccharides GlcNAc-1,4-α-d-GlcA-1,3-ß-d-Gal and GalNAc-1,4-ß-d-GlcA-1,3-ß-d-Gal using a regioselective glycosylation. Preliminary results on the impact of sulfation of these disaccharides upon recombinant chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1) involved in chondroitin sulfate chain initiation is also reported.

Effect of modified di- and trisaccharides on hyaluronidase activity assessed by capillary electrophoresis-based enzymatic assay.

The activity of eukaryote hydrolase-type of hyaluronidases was studied using a miniaturized capillary electrophoresis (CE) assay developed in our laboratory. Few nanoliters of reagents are sufficient and no labeling is required for this assay. The effect of natural and original synthetic effectors of hyaluronidase was evaluated. These di- and trisaccharides from linkage region of proteoglycans were synthesized in 30-40 steps from monomeric units using classical protection, deprotection, glycosylation and deoxygenation reactions. The influence of the chain length (di/trisaccharide), the modification type (methoxy/deoxy) and its position (2/4/6) was studied. The inhibition and/or activation percentages were determined at two concentrations of effectors; 0.2 mM and 2 mM. The half maximal effective concentration (EC50) values were evaluated (n = 2) for the most effective inhibitors (∼1 mM) and activators (∼0.2 mM). Results showed that hyaluronidase was mostly inhibited in a concentration-dependent fashion by a deoxy modification and activated by a methoxy modification. Trisaccharides were found to be more effective on hyaluronidase activity than disaccharides. Position 4 was found to be more favorable for hyaluronidase activity than position 6 and the activity in position 2 was negligible. For a better understanding of the enzyme function mode, the inhibition constant (Ki) was also evaluated by CE (Ki ∼ 2 mM). These results are of great interest especially as few activators of hyaluronidase are presented in the literature.

Hyaluronidase reaction kinetics evaluated by capillary electrophoresis with UV and high-resolution mass spectrometry (HRMS) detection.

The biology of hyaluronidase activity on age related turnover of the hyaluronic acid (HA) in skin dermis and epidermis has not been established. Elucidation of this phenomenon enables discovery of novel compounds for skin health. As a simple and green technique, capillary electrophoresis (CE) was used for the first time for the determination of the kinetic constants (Km, Vmax and IC50) of the enzymatic degradation of HA. Reaction products were identified using CE/high-resolution mass spectrometry (HRMS) after appropriate optimization. Best results in terms of signal sensitivity were obtained using 10 mM ammonium acetate (pH 9.0) BGE, a sheath liquid composed of methanol-water (80:20, v/v) with 0.02% (v/v) formic acid at 10 µL min-1 and an ESI voltage at -4 kV. Km and Vmax were determined (n = 3) using CE/UV at 200 nm as 0.24 ± 0.02 mg mL-1 and 150.4 ± 0.1 nM s-1, respectively. They were also successfully obtained by CE/HRMS (n = 3) with Km of 0.49 ± 0.02 mg mL-1 and Vmax of 155.7 ± 0.2 nM s-1. IC50 of a standard natural inhibitor, epigallocatechin gallate, was also determined by CE-UV/HRMS. Kinetic constant values obtained by CE compared well with literature which validated the developed CE-based assay. In addition, the activity of homemade tetrasaccharides of biotinylated chondroitin sulfate CS-A or CS-C (4- or 6- sulfated in a homogeneous or heterogeneous way) on the hydrolysis reaction of hyaluronidase was evaluated. Hyaluronidase was mostly dose-dependently inhibited by CS-A tetrasaccharides sulfated in a homogeneous way. Two trisaccharides from truncated linkage region of proteoglycans were also tested as inhibitors or activators. CE-based assay showed that even a small modification of one hydroxyl group changes the influence on hyaluronidase activity. CE-based assay can be used for the screening of natural and synthetic inhibitors of hyaluronidase activity for cosmetic and therapeutic applications.

Revised structure, total synthesis, and absolute configuration of kopeolin and kopeolone.

An enantioselective total synthesis of two sesquiterpenoids, kopeolin and kopeolone, has been achieved. Using the diastereoselective addition of an organocerate as a key step, we controlled the absolute stereochemistry of a crucial stereocenter present in these natural products. This approach allowed us to confirm a structural revision that we previously proposed (Chem.-Eur. J. 2013, 19, 10632-10642) and to fully characterize these natural products while elucidating their absolute stereochemistry.