Various glycolipids were synthesized using thiolactosides as scaffolds for glycosylation in animal cells. The basic building blocks,
Sulfur-containing compounds are gaining wide attention due to their potential application in the pharmaceutical industry as carbohydrate-based therapeutics or diagnostic agents. Thioglycosides, in which the glycosidic oxygen has been replaced by sulfur, are valuable stable glycoside analogues. The S-analogues of natural glucosides are resistant to enzymatic hydrolysis and have been proven to be useful glycosidase inhibitors with potential as therapeutics [
Suzuki and his coworkers reported that S-glycoside analogues of gangliosides possess inhibitory activity for the influenza virus sialidase [
The priming of glycosphingolipid synthesis by lactosyl ceramide analogues having nondegradable thioglucosidic linkages inhibited the cell surface expression of endogenous GM3 in B16 cells [
Thioglycosides are used as glycosyl donors in the synthesis of biologically important oligosaccharides. They are among the widely used glycosyl donors considering the ease of preparation and shelf stability. Thioglycosides are compatible with numerous protection and deprotection steps required in oligosaccharide synthesis [
In this research,
Chemical synthesis of
1H and 13C NMR spectra were recorded at a 600 MHz JEOL spectrometer in Me2SO-
Dulbecco Modified Eagle’s Medium (DMEM) and Ham F12 (1 : 1), trypsin, and insulin-transferrin-selenium (ITS-X) solution were from Gibco. The lactoside primers were dissolved in sterile Me2SO to an initial concentration of 50 mM. SepPak C18 was from Waters. HPTLC plates were from E. Merck, Darmstadt, Germany. CHCl3 : MeOH : 0.25% KCl(aq) = 5 : 4 : 1 (v/v/v) was used as developing solvent for HPTLC. HPTLC plates were sprayed with resorcinol and heated to detect the separated glycolipids. The mass spectrum was recorded on a Bruker Esquire HCT Ultra ESI LC MS using MeOH : acetonitrile (1 : 1, v/v).
To a solution of
A 50 mM stock solution of
Mouse B16 melanoma cells (2 × 106 cells per 10 cm dish) were cultured in 10 mL 1 : 1 DMEM-F12 supplemented with 10% fetal bovine serum (FBS). When the cells were about 70%–80% confluent, the culture medium was changed to 1 : 1 DMEM-F12 (without FBS) containing 50
Lipids from the culture media were purified using SepPak C18 column (Waters Cat. no. WAT020515) preconditioned with methanol. 2 mL of the collected medium was loaded to the column, washed with water and the desired products were eluted with 3 mL methanol. Evaporation of the eluent was accomplished using a centrifuge evaporator. Lipids from the culture medium fractions were analyzed by HPTLC with CHCl3 : MeOH : 0.2% aq KCl (5 : 4 : 1, v/v) as developing solvent. HPTLC plates were sprayed with resorcinol HCl reagent and heated for 10 min at 120°C in an oven to detect the glycolipids. The putative bands corresponding to glycosylated thiolactosides were analyzed to determine the structure of the products. The HPTLC plate was scanned, and the amount of elongated product was measured using SORBFIL TLC Video densitometer software.
Mass production of GM3-type oligosaccharide using
The purified thiolactosides were introduced to mouse melanoma B16 cells, Madin-Darby canine kidney (MDCK) cells, and African green monkey kidney (Vero) cells.
After 15 minutes from the addition of the thiolactoside primers, a remarkable change on B16 cell morphology was observed. Although B16 cells are epidermoid, the cells became elongated and acquired a slender shape similar to fibroblast cells as shown in Figure
Effect on B16 cells after incubation in the absence (Control) and in the presence of
Cell proliferation of B16 cells was the fastest in the presence of
As shown in Figure
Culture medium collected after 48-hour incubation of B16 cells in the presence of
HPTLC results confirmed the chain elongation of the thiolactoside primers as shown in Figure
Samples | Quant. on plate (ug) |
---|---|
Alpha 1st | 3.06 |
Beta 1st | 2.44 |
Alpha 2nd | 2.02 |
Beta 2nd | 2.83 |
HPTLC profile of lipids from the culture medium fraction obtained after incubation of B16 cells in the absence of thiolactosides (C) and in the presence of the
On the other hand, incubation of Vero cells in the presence of
HPTLC profile of lipids from the culture medium fraction obtained after incubation of MDCK cells or Vero cells in the presence of the
Vero cells
MDCK cells
Administration of
Products of cellular enzyme-catalyzed glycosylation of thiolactoside.
HPTLC profile of lipids from the culture medium fraction obtained after incubation of B16 cells in the presence of the
Mass spectra of (a) dodecyl thiolactoside, (b) GM3-type glycolipid, and (c) Gb3-type glycolipid.
Dodecyl thiolactoside
GM3-type glycolipid
Gb3-type glycolipid
Lactosyl ceramide is the common intermediate in glycolipid biosynthesis. An amphiphilic thiolactoside, an analogue of lactosyl ceramide, was examined in this study for the ability to prime glycolipid synthesis in various animal cells. Simpler in structure than the natural precursor, the thiolactoside employed has a lactose moiety linked to a hydrophobic dodecyl aglycone unit via S-glycosidic bond. The thiolactosides were chemically prepared and were obtained in fairly good yield via conventional methods of glycosylation using a Lewis acid as catalyst and deprotection under Zemplen conditions.
Incubation of cells for 72 h in the presence of the
Glycosylation was cellular specific. B16 cells gave GM3-type ganglioside, the predominantly expressed glycolipid in the cell surface. MDCK cells also gave mainly GM3-type glycolipid. On the other hand, African green monkey kidney (Vero) cells gave Gb3- and Gb4-type glycolipids aside from GM3-type ganglioside. The results also agree with a related work reported in the literature [
The
According to the literature, S-glycosides are relatively stable and the lipid moiety is hardly affected by hydrolases (13). Although the natural biosynthetic process in the production of glycolipids involves O-glycosides, it was surmised that employing the
Mass production of GM3-type oligosaccharide using
Various glycolipids could be obtained using thiolactosides and animal cells. Like the natural precursor with an O-glycoside linkage, the thiolactosides could also be taken up by cells and glycosylated to afford glycolipids having the same saccharide structures as the cells produce. It is envisioned that incorporation of the thiolactosides in a variety of cells could generate a library of biologically important glycolipids. Glycolipids obtained by cellular glycosylation of thiolactosides could be further employed for the synthesis of more complex oligosaccharides. This strategy will even shorten the rather tedious preparation of oligosaccharides. Moreover, the glycolipids generated from thiolactosides have potential application as therapeutics by virtue of the presence of the sulfur atom.
This work was supported by a research grant from the New Energy and Industrial Technology Development Organization (NEDO).