CS233088B1 - Trisododium salt of 6-(2-sulpho-4-(4-amino-3-sulpho-1-anthraquinone-amino)-aniline-4-(x-sulphoaniline)-1,3,5-triazine-2-yl-hydroxyet-hylcellulose and its preparation method - Google Patents

Description

(54) 6- [2-Sulfo-4- (4-amino-3-sulfo-1-anthraquinone-amino) -anilino-4- (x-sulfoanilino) -1,3,5'-triazine-2 trisodium salt -yl-hydroxyethylcellulose and a process for its preparation

233 088

The present invention relates to 6- [2-sulfo-4- (4-amino-3sulfo-1-anthraquinone-amino)] anilino-4- (x-sulfoanilino) -1,3,5-triazin-2-yl-hydroxyethylcellulose trisodium salt and how to prepare it.

Trisodium salt of 2-chloro-6- [2-sulfo-4- (4-amino-3-sulfo-lanthraquinone-amino)] anilino-4- (x-sulfoanilino) -1,3,5-triazine is a dye that reacts in an alkaline medium between 70 and 80 ° C with polysaccharides to form stable ether derivatives of fo.F. Kennedy: Advan. Carbohyd. Chem. Biochem. 29, 305 (1974)]. The dye thus bound to a suitable insoluble polysaccharide carrier serves as a ligand in the affinity chromatography of these enzymes, due to its interaction with some enzymes. from the soluble polysaccharides derivatized with this dye, the trisodium salt of 5- [2-sulfo-4- (4-amino-3-sulfo-1-anthraquinone-amino)] anilino-4- (x-sulfoanilino) -1,3,5 was prepared -triazin-2-yl-dextran, hydrolyzed starch, glycogen [p. Gemeiner, 0. Mislovic, 0. Zemek, L. Kuniak: Collect. Czech. Chem. Commun. 46, 419 (1981) J and hydroxyethyl starch.

All these polysaccharides are branched c-glucanes, unlike hydroxyethylcellulose, which is a β-glucan with a linear chain.

Conditions of reaction of hydroxyethylcellulose with trisodium salt of 2-chloro-6- [2-sulfo-4- (4-amino-3-sulfo-1-anthraquinone-amino)] anilino-4- (x-sulfoanilino) -1,3,5 -triazine differs from dextran staining conditions [p. Gemeiner et al., Collect. Czech. Chem. Commun, 46, 419 (1981)] by being milder but achieving the same degree of substitution. This is due to the greater reactivity of the hydroxyethylcellulose due to the presence of a greater amount of primary alcohol groups which react preferentially. In the isolation of the obtained trisodium salt of 6- [2-sulfo-4- (4-amino-sulfo-1-anthraquinone-amino)] anilino-4- (x-sulfoanilino) -1,3,5-triazin-2-yl - hydroxyethylcellulose is not possible with

- 3,233,088 molecular weights above 100,000 to use the gel chromatography method for the very high viscosity of the solution of this compound.

The present invention solves this problem. The present invention provides the trisodium salt of 6- [2-sulfo-4- (4-amino-sulfon-1-anthraquinonamino)] anilino-4- (x-sulfoanilino) -1,3,5-triazin-2-yl-hydroxyethylcellulose, wherein x is the position of substituent 2 or 4 of formula I

wherein R represents hydroxyethylcellulose with a degree of substitution

2.5 and average molecular weight 2.10 ⁴ to 5.4.10 ⁵ . The invention further provides a process for the preparation of these derivatives, characterized in that the individual fractions of hydroxyethylcellulose are reacted in an aqueous alkaline medium and in the presence of 1.7% sodium chloride at a temperature of 70 to 80 ° C and for 2 hours with trisodium salt. -6- [2-sulfo-4- (4-amino-3-sulfo-1-anthraquinone-amino)] anilino-4- (x-sulfoanilino) -1,3,5-triazine of formula II at weight ratios trisodium salt 2-chloro-6-

233 088

- [2-sulfo-4- (4-amino-3-sulfo-1-anthraquinone-amino)] anilino-4- (x-sulfoanilino) -1,3,5-triazine: hydroxyethylcellulose: sodium carbonate = 0.2 up to 0.32: 1: 0.54 to 0.59, whereupon the stained hydroxyethylcellulose is separated from the unbound dye by either flash chromatography (up to an average molecular weight Mw of 100,000) or by 6 to 7 fold precipitation followed by centrifugation from 0.5% aqueous sodium chloride solution (above Mw 100 000).

The advantage of the proposed process for the preparation of the trisodium salt of 6- [2-sulfo-4- (4-amino-3-sulfo-1-anthraquinone-amino)] anilino-4- (x-sulfoanilino) -1,3,5-triazine-2 -yl-hydroxyethylcellulose is that it provides colored derivatives of water-soluble polysaccharide with a linear structure ie hydroxyethylcelluloses which are useful for studying the interaction of a colored polysaccharide with an enzyme. A further advantage is the method of isolating this material from the reaction mixture because of its insolubility in the salt solution in 50 to 60% yield.

Example 1 mg of hydroxyethylcellulose (HEC) with an average molecular weight (Mw) of 350,000 is dissolved in 2 ml of distilled water at 60 ° C. 7 mg of 2-chloro-6- (2-sulfo-4- (4-amino-3-sulfo-1-anthroquinone-amino)] anilino-4- (x-sulfoanilino) -1,3 trisodium salt is then added. 5-triazine (Cibacron Blue 3G-A) dissolved in 0.2 mL of distilled water The reaction mixture is stirred at 60 ° C for 30 min, then 43.75 mg of sodium chloride is added and stirring is continued After raising the temperature to 80 ° C, 15 mg of sodium carbonate dissolved in 0.05 ml of distilled water are added and the reaction mixture is stirred for a further 2 h. After cooling, the reaction mixture is diluted with 20 ml of distilled water and neutralized with dilute water. hydrochloric acid to pH 7. The colored hydroxyethylcellulose is precipitated by adding 20 ml of 1% aqueous sodium chloride solution and cooling to 4 DEG C. The precipitate is centrifuged at 4.10 rpm and the supernatant is separated and the precipitate is dissolved in 20 ml of distilled water at 50 DEG C. Cibacron Blue-HEC is reprecipitated from the solution with 20 ml of 1% aq of sodium chloride at 4 ° C and centrifuged again. This is repeated 6 times until the supernatant is colorless. The obtained Cibacron Blue-HEC is dissolved in 20 ml of distilled water and dialyzed for 24 h against

233 088 distilled water. After separation of the insolubles by filtration, the resulting blue solution is evaporated to dryness in a vacuum rotary evaporator at 5 kPa * after addition and re-evaporation of ethanol and then acetone to give a dry Cibacron Blue-HEC (15.2 mg) containing 68 yumole of Cibacron Blue- HEC / g.

Example 2

The procedure of Example 1 except that HEC (Mw = 41,000) and 6.6 mg of Cibacron Blue 3G-A was used. . The column volume is 100 ml, 21.5 cm high and 2.5 cm diameter. Elute with distilled water, eluting with a column flow of 30 to 35 ml / h to obtain dry Cibacron Blue-HEC (21 mg) containing 79.3 yumole Cibacron Blue / g.

Example 3

The procedure of Example 1 except that HEC (Mw = 540,000) and 6.6 mg of Cibacron Blue 3G-A was used. Dry Cibacron Blue-HEC (17.8 mg) was obtained containing 60.6 yuroole of Cibacron Blue / g.

Example 4

The procedure of Example 1 was followed except that HEC (Mw 350,000, 8 mg of Cibacron Blue 3G-A and 70 ° C) was obtained to obtain a dry Cibacron Blue-HEC (16.5 mg) containing 65.4%. umol Cibacron Blue / g.

The invention has utility in enzyme engineering and in biochemistry for the isolation and purification of enzymes or as substrates and inhibitors of enzyme-catalyzed reactions.

OBJECT OF THE INVENTION

Claims (2)

233 088 A 6- 2-sulfo-4- (4-amino-3-sulfo-1-anthraquinonamino) anilino-4- (x-sulfoanilino) -1,3,5-triazin-2-yl-hydroxyethylcellulose trisodium salt, wherein: x is the position of substituent 2 or 4 of formula I wherein R represents hydroxyethylcellulose with a degree of substitution A 5 2.5 and an average molecular weight of 2.10 to 5.4.10. 2. A process according to claim 1 wherein the individual fractions of hydroxyethylcellulose are reacted in an aqueous medium in the presence of 1.7% sodium chloride at 70-80 [deg.] C. and for 2 hours with trisodium salt of 2-chloro-6- 2. -sulfo-4- (4-araino-3-sulfo-1-anthraquinone-amino) anilino-4- (x-sulfoanilino) -1,3,5-triazine of formula II at weight ratios trisodium salt 50; Na (II) 7,233,088 2-chloro-6- 2-sulfo-4- (4-amino-3-sulfo-1-anthroquinone-amino) anilino-4- (x-sulfoanilino) -1,3,5-triazine: hydroxyethylcellulose: Sodium carbonate = 0.2 to 0.32: 1: 0.58 to 0.69, whereupon the stained hydroxyethylcellulose is separated from the unbound dye by either flash chromatography (up to an average molecular weight Mw of 100,000) or by 6-7 fold precipitation followed by centrifugation from 0.5% aqueous sodium chloride solution (over Mw 100,000). Correction in printed descriptions of the invention In the description of the invention, fe autonMau n & all x 233 233 < 4026 * 83 > Correct i Xnt «Olz β 06 8 l> / 0® OJ C O B 13/06