Method of obtaining monosialogangliosides

Abstract: A method of obtaining monosialoganglioside (GM1) from an aqueous solution containing a mixture of gangliosides, involves incubating the solution at a pH of from 5 to 6.5 in the presence of an neuroaminidase so as to hydrolyse di-, tri- and tetra- sialogangliosides therein to enrich the GM1 content of the mixture. The resultant solution is dried, the residue re-dissolved in a mixture of chloroform, methanol and water and is then subjected to exclusion chromatography. The appropriate fractions are collected and lyophilized.

Description: This invention relates to a method of obtaining monosialoganglioside -G M1 - (a glycosphingolipid that has a sialic acid (N-acetylneuraminic acid) moiety bound to monosaccharides as head groups, which gives a net charge to the polar head at neutral pH, and also has a non-polar carbohydrate moiety) from products containing a mixture of gangliosides. Glycosphingolipids are found in large quantities together with other lipids (e.g. phospholipids) in mammalian brains, mainly in the cortex. In bovine mammals, the content of glycosphingolipids is high enough for them to be extracted for commercial purpose. In a chemical composition of these compounds, units of monosaccharides, sialic acid and a non-polar fragment, the ceramide which is composed of a natural amino alcohol, sphingosine, forming an amide bond with fatty acids, are present. The units of monosaccharides are mainly galactose, glucose and an amino hexose, N-acetylgalactosamine. Furthermore, they contain N-acetyl (and/or glycolyl) neuraminic acid (sialic acid). All glycosphingolipids containing such acid belong to the group of acid glycosphingolipids, and they are classified under the specific name of gangliosides. More than sixty different ganglioside molecules containing different structures have been detected. According to the number of units of sialic acid, they are grouped as monosialogangliosides, disialogangliosides, trisialogangliosides and tetrasialogangliosides, which are mentioned for short as G M , G D , G T and G Q , respectively. The disappearance of the last residue of sialic acid produces G A1 , an asialosphingolipid of G M1 . A numerical suffix, N (equal to 5-n, where n indicates the number of units of neutral hexose) is added. The letters (generally a, b) indicate compounds which exhibit isomerism in relation to the positions of the sialic acid moieties with characteristic properties for those isomers eg, a different Rf in thin layer chromatography. Example: G D1b represents a disialoganglioside where n=4 and where the sialic acid moiety exhibits isomerism with reference to G D1a . Thus, G M1 is a compound formed by one unit of sialic acid and four units of hexose. This carbohydrate fragment is bound to a ceramide. It is composed of sphingosine forming an amide bond with a fatty acid fragment. More than one fatty acid has been identified by HPLC. They are mainly of C 18 and C 20 . (Hiroki NAKABAEYASHI, Masao IWAMORI and Yoshitaka NAGAI), J. Biochem., 96, 977-984 (1984). The structure of G M1 is illustrated in attached Fig. 1 and by the following schematic formula:- Gal = galactose; Gluc= glucose; NANA=N-acetylneuraminic acid; Gal Nac= N-acetyl-galactosamine. In the attached Fig.2, the IR spectrum of G M1 is shown, in which characteristic peaks corresponding to the C-C, C-OH, C-H, N-C and N-H bonds are indicated. CONSTANTS AND CHARACTERISTICS OF THE PRODUCT Molecular formula: C 73 H 131 O 31 N 3 (more abundant component). According to the suggested structure, MW is 1546.80. Melting point: 207°-232°(d). Appearance: white powder, slightly cream. Solubility: soluble in water. Soluble in mixtures of methanol-water, methanol-chloroform and methanol-chloroform-water. Practically insoluble in methanol, ethanol, acetone, chloroform, ethyl ether and light aliphatic hydrocarbons (petroleum ether). Infra red spectrum:- see the drawing. Specifications for finished product (lyophilized). a) Identity test 1) Infra red spectrum superimposable to the standard. 2) There must be a coincidence of R f with an authentic sample of it using thin layer chromatography in the different systems of this patent. b) Humidity The lyophilized product must contain not more than 0.5% of water. c) Residual solvents It must contain not more than 0.1% d) Melting point The melting point must not be depressed when the substance is mixed with an authentic sample of it. DESCRIPTION OF THE INVENTION The monosialoganglioside G M1 is found in the natural mixture at concentrations of 20% by weight. Therefore, the obtaining of G M1 by isolation is not economically attractive. The transformation of the mixture of gangliosides G M1 by enzymatic or chemical hydrolysis is the most viable industrial method. In DE-A-3624816, a chemical method for obtaining G M1 is described wherein the sialic acid is first separated in units hydrolyzing di- and tri-sialogangliosides present in the natural mixture with dilute inorganic or organic acids; secondly, the portions thus obtained are chromatographically separated and purified in silica gel columns. However, there is available literature about the use of enzymes in this transformation. Both the enzymatic and chemical methods are based on the kinetic control of the reaction. The rate of transformation of G T into C D and C D into G M1 is high. In contrast, the hydrolysis rate of G M1 into G A1 is low. If the experimental conditions are controlled, it is possible to hydrolyze the mixture selectively and obtain G M1 in high yield. The enzymatic hydrolysis using neuraminidase has been described in several publications, eg 1) Richard Kuhn et al., Chem. Ber., 96 , 866 (1963), 2) Keiko Nohara-Uchida et al., J.Biochem., 103 , 840/2 (1988), 3) Myers Metal, Biochem. 23 , 1442/8 (1984), 4) Drzernick R. et al., Histochem. Journal , 5 , 271/90 (1973), 5) Schauer R. et al., Hoppe-Seyler's Z. Physiol. Chem. 349 961/8 (1968), 6) S. Hakonori et al., Biochem. , 8 , 5082/88 (1969), 7) Welger D.A. et al., J. Neurochem. , 20 , 607/12 (1973), and 8) Lipovak V. et al., J. Biol. Chem. , 246 (24), 7642 (1971). EP-A-0451267 discloses a process for preparing G M1 from gangliosides using neuraminidase isozyme S derived from bacteria of the genus Arthrobacter, wherein an aqueous solution containing the gangliosides and a buffer (pH about 3-6) is incubated with an aqueous solution of the neuraminidase followed by extraction of G M1 with a mixture of chloroform and methanol, and lyophilisation. Hoppe-Seyler's Z. Physiol. Chem, vol 358. no. 7,1977, pages 789-795 discloses the immobilization of neuraminidase derived from Clostridium perfringens , the increased stability under various conditions of the immobilized enzymes relative to the soluble enzyme, and the use of the immobilized enzymes to cleave various substrates including G D1a micelles and human erythrocytes. According to the present invention, there is provided a method of obtaining monosialoganglioside (G M1 ) from an aqueous solution containing a mixture of gangliosides by incubating the solution in the presence of neuroaminidase so as to hydrolyse di-, tri- and/or tetra-sialogangliosides therein to enrich the G M1 content of the mixture, extracting G M1 from the reaction solution by a procedure involving the use of a solvent mixture containing an aliphatic alcohol and a haloalkane, and lyophilising, characterised in that the incubating is effected at a pH of from 5 to 5.5 in the presence of an immobilized neuroaminidase obtained from Clostridium perfringens, the resultant solution is dried, the residue is re-dissolved in a solvent mixture comprising an aliphatic alcohol, water and a haloalkane, the resultant solution is subjected to exclusion chromatography, the fraction(s) containing the G M1 are collected, and such fraction(s) are lyophilized. In the present application, the hydrolysis of the natural mixture of gangliosides to G M1 is effected using an immobilized neuraminidase obtained from Clostridium perfringens at a pH of 5 to 5.5. This method allows G M1 to be obtained in very high yield and purity. Considering that during the transformation of the gangliosides with more than one unit of sialic acid into monosialo, the free sialic acid is formed, it is necessary to eliminate it as well as the traces of G D1 present when the enzymatic reaction is stopped. Under the conditions described in this invention, no G A1 is present. This purification is carried out by filtration chromatography through gels (exclusion chromatography), eluting with mixtures of chloroform:methanol: water, more preferably with a volume ratio 60:30:4.5. The passage of the transformation product through Sephadex® G50 (Sephadex® G25 may also be used) allows G M1 of high purity to be obtained. Sephadex® G50 is made of dextran crosslinked with epichlorhydrin. The pH range is chosen to obtain a high rate of transformation in a favourable time. The main advantage of this method is that the immobilized enzyme can be generated and used again. In this way, the inconvenience of its high price is overcome and our method is industrially viable. The enzymatic activity depends on the pH as will be apparent from the following Examples. EXAMPLES : Example 1: Experiment at pH: 5. a) Insoluble neuraminidase (Sigma type VI A), of Clostridium perfringens, bound to agarose: 5.3 units per gram of gangliosides to be hydrolyzed. b) Sodium acetate/acetic acid buffer, at pH 5.0, 23.7 ml per gram of gangliosides to be hydrolyzed. Incubation temperature of the mixture: 37°C. Hydrolysis profile:- % NANA evolved with reference Hydrolysis time (Hours) to the theoretical amount 0.5 10.81 1 19.00 2 22.30 3 32.63 4 32.61 19.5 42.40 20 43.48 21 44.90 22 52.20 23 53.30 24 53.30 25 54.60 Example 2 : Experiment at pH: 5.3 a) Insoluble neuraminidase (Sigma type VI A) of Clostridium perfringens bound to agarose: 5.3 units per gram of gangliosides to be hydrolyzed. b) Total gangliosides (natural mixture). c) Sodium acetate/acetic acid buffer, at pH 5.3, 23.7 ml per gram of gangliosides. Incubation temperature of the mixture: 37°C. Hydrolysis profile:- % NANA evolved with reference Hydrolysis time (Hours) to the theoretical amount 0.5 8.65 1 18.42 2 24.60 3 32.43 4 49.00 5 51.34 15.5 64.00 16 66.50 23 93.00 24 103.12 25 103.70 Example 3: Experiment at pH: 5.5 a) Insoluble neuraminidase (Sigma type VI A) of Clostridium perfringens bound to agarose: 5.3 units per gram of gangliosides. b) Total gangliosides (natural mixture). c) Sodium acetate/acetic acid buffer, at pH 5.5, 23.7 ml per gram of gangliosides. Incubation temperature: 37°C. The hydrolysis profile is as follows:- % NANA evolved with reference Hydrolysis time (Hours) to the theoretical amount 0.5 7.40 1 19.00 2 29.35 3 54.00 4 60.00 5 76.00 6 81.74 6.5 82.00 24 88.00 25 91.30 26 95.70 27 98.00 28 99.00 28.5 99.24 29 102.20 30 103.30 The working method with the immobilized enzyme can be batchwise. Once the reaction is finished, we proceed according to the following Example: Example 4 : The enzyme immobilized in agarose is centrifuged, the supernatant containing G M1 is separated and is kept till it is purified. The residue is washed twice with the buffer solution at the working pH, and twice with a solution 2.0 M (NH 4 ) 2 .SO 4 , at pH 7.0. It is kept in this medium and can be used again more than five times. Example 5 : A solution of G M1 obtained according to Examples 1 to 3, from 1 g of total gangliosides (natural mixture), is taken. It is dried in a rotary evaporator at 40-50°C. The residue is taken up in a mixture of chloroform:methanol:water in a respective volume ratio of 60:30:4.5, and the solution obtained is passed through a Sephadex column G50 containing 10 g of Sephadex in the above mentioned mixture of solvents. The fractions are analyzed by thin layer chromatography (J. Neurochem. 10 , 613 (1963), J. Chromatog. 106 , 425 (1975) revealing either with resorcinol reagent (Biochem. Biophys. Acta, 24 , 604 (1957) or with iodine, and those containing pure G M1 are put together. The assembled fractions are lyophilized.

Claims: A method of obtaining monosialoganglioside (G M1 ) from an aqueous solution containing a mixture of gangliosides by incubating the solution in the presence of neuroaminidase so as to hydrolyse di-, tri- and/or tetra-sialogangliosides therein to enrich the G M1 content of the mixture, extracting G M1 from the reaction solution by a procedure involving the use of a solvent mixture containing an aliphatic alcohol and a haloalkane, and lyophilising, characterised in that the incubating is effected at a pH of from 5 to 5.5 in the presence of an immobilized neuroaminidase obtained from Clostridium perfringens, the resultant solution is dried, the residue is re-dissolved in a solvent mixture comprising an aliphatic alcohol, water and a haloalkane, the resultant solution is subjected to exclusion chromatography, the fraction(s) containing the G M1 are collected, and such fraction(s) are lyophilized. A method according to claim 1, characterized in that said exclusion chromatography is effected using a column of dextran crosslinked with epichlorhydrin. A method according to claim 1 or 2, characterized in that said solvent mixture is a mixture of chloroform, methanol and water. A method according to claim 3, characterized in that the chloroform, methanol and water are in a volume ratio 60:30:4.5, respectively. A method according to any preceding claim, characterized by the further steps of washing the immobilized enzyme after the hydrolysis, and reusing the washed immobilized enzyme in the incubating step.

Inventors: CUMAR FEDERICO A, MAMMARELLA CARLOS, RODRIGUEZ PABLO, CUMAR, FEDERICO A., MAMMARELLA, CARLOS, RODRIGUEZ, PABLO
Applicants: TECHNOCHEMICAL CORP S A, TECHNOCHEMICAL CORPORATION S.A.
Application year: 1991
Main classification: C12P19
IPC codes: C12N9, C12P19