Koenigs-Knorr Synthesis of Natural and Potential Metabolites of Cholesteryl Hexosides

The following glycosides of cholesterol have been synthesized: α -D-galactofuranoside, ß-D-glucopyranoside, bis-sulfo-3,6-β -D-glucopyranoside. 1-α -Bromides of protected sugars were used as glycosylation donors: tetra-O-benzoyl-D-galactofuranosyl and tetra-O-acetyl-D-glucopyranosyl. Peracylated sugars, i. e., precursors of sugar bromides, were synthesized in conditions that preferentially facilitated the envisaged ring: room temperature for pyranosic ring, high temperature for furanosic one. Constantly, cadmium carbonate was used as promotor. As a general scheme of work, glycosylation mixture was submitted to Zemplen saponification and the obtained glycolipids have been separated by characteristic methods. The glucoside of cholesterol was sulfated with chlorosulfonic acid in pyridine. Neutral or acidic glycolipids were characterized per se by chromatographic and chemical means as well as by IR spectroscopy. Sulfatide and galactocerebroside of rat brain and ganglioside GM4 of rooster testis were used as reference compounds. Alternatively, the synthesized glycosides were peracetylated and their 1 H and 13 C NMR spectra registered. New NMR spectroscopic data are presented for cholesteryl-α -D-galactofuranoside and -bis-sulfo-6- β -D- glucopyranoside

More blamed than praised, cholesterol is, nonetheless, the most investigated sterol, both in free form and as ester with fatty acids or sulfuric acid.However, the approaches dealing with cholesteryl glycosides are less numerous [1].Investigations concerning cholesteryl glycosides have been stimulated by a vast array of biochemical and physiological implications of steryl glycosides.As a rule, disclosing of these functions was preceded by the knowledge of their natural distribution.Surprisingly, in many cases the works linked with their widespread was preceded at its turn by steryl glycoside synthesis or closely intertwined with it [2].
The mechanism for avoiding desiccation in terrestrial vertebrates resides in a water barrier located in the outer portion of the integument [3].This water barrier is formed especially of lipids and at least in mammalian, avian, and reptilian skin these lipids are packaged into lamellar granules within the viable epidermal cells.Besides free sterols, four other sterols derivatives were found in chicken epidermis:β-D-glucosylsterols, 6-O-acyl-β-Dglucosylsterols, steryl esters and cholesteryl sulfate [4].Two types of glycolipids, acylglucosylsterol and glucosylsterol, have been described in snake epidermis by chemical, spectroscopic, and gas-liquid chromatographic methods [5].Lyme disease is an infectious disease relatively widespread in the United States, Europe, and Northern Asia, whose causing agent is the spirochete Borrelia burgdorferi and its subspecies.Lyme disease affects sequentially or simultaneously the skin, joints, nervous system, and heart [6].Glycolipids protruding outside B. burgdorferi cells are immnunoreactive i. e., they give positive reaction with sera from Lyme disease patients.Their structure elucidation led to the conclusion that they are cholesteryl 6-O-acyl-β-D-galactopyranoside, the acyl group being a variety of fatty acids [7,8].Other spectacular biochemical and physiological roles of steryl glycosides prove they are more than cellular membrane constituents.
Cholesteryl β-D-glucopyranoside is the molecule of stress for lower and higher organisms, being the first molecule biosynthesized in case of this (ab)normal state [9].Estradiol β-D-xylo(pyrano)side is a primer for glycosaminoglycan biosynthesis [10] and sitosteryl β-D-glucopyranoside is the primer molecule for cellulose biosynthesis in plants [11].Also in plants, steryl glucoside plays the role of special natural reagent, serving as a glucosyl donor to ceramides for glycosphingolipids biosynthesis [12].
In this paper, cholesteryl α-D-galactofuranoside, -β-Dglucopyranoside, -β-D-(bis-sulfo-3,6)glucopyranoside have been synthesized and characterized to be used as enzymatic substrates for furanosidases and glucosidases, respectively.The first and the third compounds are new and a new chemical condensing agent was used for the synthesis of cholesteryl β-D-glucopyranoside.Bis-sulfo derivative of cholesteryl glucoside is destined to investigate the influence of sulfate group(s) on glycosidases enzymatic activities acting upon steryl glycoside.At the same time, bis-sulfo glucoside structural motif is an excellent ligand for select in families.
NMR Spectra Registration. 1 H and 13 C NMR spectra of all compunds, i. e., glycosylated steroids and aglycones, were registered in peracylated form in CDCl 3 containing TMS. Constantly, the spectra of peracylated glycoside have been compared to the spectra of peracetylated aglycone.NMR experiments were performed on a Bruker Avance DRX 400 spectrometer using 400 and 100 MHz for 1 H and 13 C frequencies, respectively.The 1 H-1 H correlation spectroscopy (COSY) and 1 H- 13 C heteronuclear multiple quantum coherence (HMQC) experiments were carried out with an inverse probe. Cholesteryl-α-D-galactofuranoside.

Results and Discussion
Galactofuranosylation of cholesterol (0.608 g, 1.58 mmol), by using 1.25 g (1.89 mmol) tetra-O-benzoyl-α-Dgalactofuranosyl bromide, produced 0.5940 g of a mixture consisting of two compounds, in the ratio 10:1.The major one proved to be cholesteryl β-D-galactofuranoside [15].The minor one appeared as a waxy solid, after Zemplen saponification, and contained D-galactose and cholesterol in the ratio 1:1, as was proved by quantitative determination of its constituents after acidic hydrolysis.It clearly separated from cholester yl-β-D-galactofuranoside and -β-Dgalactopyranoside by TLC (fig.2).Cholesteryl hexoside nature of our steryl glycoside was confirmed by ESI MS data, in the presence of ammonium, an ion at m/z 735 (M + NH 4 + ) being evidenced.This cholesteryl galactoside produced formaldehyde by periodic acid oxidation, this reaction indicating a furanosic ring for D-galactose.Its 1 H and 13 C NMR spectra proved that the isolated compound identified, based on our results and others [13,14,20,21], as being cholesteryl-β-D-(6-sulfo)glucopyranoside.
The major one (R F 0.31) was easily separated by the unreacted neutral glycoside, the difference between their R F values being appreciable (0.45, fig.5).Its chromatographical behaviour by TLC in comparison with neutral and acidic glycosphingolipids suggested two negative charges on the compound, rather than one: although cholesteryl β-D-glucopyranoside migrated very similar to galactocerebroside, the synthetic sulfate ester migrated much slower than cerebroside 3-sulfate (sulfatide) from rat brain (fig.6).visualization by mostain Glucosylation reaction of cholesterol could be followed by TLC, by comparing the image of glucosylation mixture before and after Zemplen hydrolysis (fig.3).4).This way, amounts of reaction products of reasonable magnitude for physico-chemical analyses as well as for biochemical [19] and biological investigations have been obtained.A new chemical condensing agent, cadmium carbonate, was used in this paper for the Koenigs-Knorr synthesis of cholesteryl β-D-glucopyranoside; reaction product was chromatographically separated (fig.4) and completely characterized.
Chemical sulfation of cholesteryl β-D-glucopyranoside with chlorosulfonic acid in pyridine produced at least two sulfation product, besides unreacted cholesteryl β-Dglucopyranoside (R F 0.76) (fig.5).The minor one (R F 0.49), being in trace amounts, could not be separated in order to be characterized.The compound was only tentatively Cholesteryl α-D-galactofuranoside was compared, in terms of chemical, physical and spectral properties with two diastereoisomeric glycosides, cholesteryl β-Dgalactofuranoside and -β-D-galactopyranoside [15,18].The compound described in this paper clearly separated of the latter two compounds by TLC (fig.2).Colesteryl α-Dgalactopyranoside was ruled out by two arguments: 1 H and 13 C spectral properties as well as production of formaldehyde by periodate oxidation.
There have been a good agreement between our results, concerning 1 H and 13 C NMR spectra, and the results of [22] about decyl α-D-galactofuranoside as well as the results of [23] about n-pentenyl α-D-galactofuranoside.
Glucosides of cholesterol and sitosterol were amongst the first synthesized glycosterol [2] and the synthetic compounds were used for the first identification of steryl glycosides in plants, i. e., in the tall morning-glory (Ipomoea purpurea Roth) [32].
In some preliminary experiments we have proved that a crude enzymic extract of digestive tract of snail (Helix pomatia) hydrolyzed cholesteryl β-D-glucopyranoside that had been emulsified with biliary acids [19] and the action of the enzyme(s) on sulfated steryl glycoside follows to be tested.
Cadmium carbonate is an excellent chemical condensing agent for glucosylation of cholesterol to cholesteryl β-D-glucopyranoside.