The method of obtaining 18-carboxy-20-(carboxymethyl)-8-ethynyl-13 - ethyl-2,3-dihydro-3,7-12,17-tetramethyl-n, norfin-2 - propionic acid or its salts

 

(57) Abstract:

Usage: biotechnology, chemical and pharmaceutical industry. The inventive receiving 18 carboxy-20(carboxymethyl)-8-ethynyl-13 - ethyl-2,3-dihydro-3,7,12,17 - tetramethyl-N,N porphin-2 - propionic acid or its salts. Getting chlorin e6carry out when using as a raw material biomass of cyanobacteria, for example, of the genus Spirulina, obtained by cultivation in the conditions of artificial or solar light on a nutrient medium having a pH of 8.0 to 12.0, with pre-treatment of biomass alcoholic solution of alkali. The resulting alcoholic extract of chlorophyll treated with acid to obtain pheophytin a. Pheophytin washed with a nonpolar solvent such as hexane and subjected to saponification to the formation of the target product, which was precipitated by treatment with hydrochloric acid. 4 C.p. f-crystals.

The invention relates to organic chemistry, in particular to an improved process for the preparation of porphyrins, in particular to a method for producing chlorin e6(18-carboxy-20-(carboxymethyl)-8-ethynyl-13-ethyl-2,3-dihyd - ro-3,7,12,17 - tetramethyl-n, n. porphin-2-propionic acid) or its salts, which can find application in chemical farmacity the basis of chlorophyll . It is known that vegetable porphyrins, including chlorophyll and its derivatives are pharmacologically active compounds and can be used in medical practice, including as photosensitizers for photodynamic diagnosis and therapy of oncological diseases. Currently known methods for producing chlorin e6based on chemical transformations of chlorophyll in the target product.

In particular, the most common method of producing chlorin e6by processing the chlorophyll extract derived from soybeans, 30% hydrochloric acid with a mixture of pheophorbide and which is then treated with 0.5% methanol solution of alkali in a vacuum. The target product is removed from the reaction mixture with ether.

There is also known a method of producing chlorin e6from nettle leaves. For this nettle leaves pour ethanol. The result: an extract containing a mixture of chlorophylls and . The extract is treated with concentrated hydrochloric acid, resulting from each form of chlorophyll contained in the extract, is formed corresponding to the shape of pheophytin and . Subsequent alkaline saponification of different forms of pheophytin PR is osenia these products is 4:1, respectively. Along with these major products in the reaction mixture present their esters, as well as ficinia remains.

To highlight chlorin e6of a mixture of products, the reaction mixture is first neutralized with concentrated hydrochloric acid to pH 7.0, and then add additional concentrated hydrochloric acid in an amount corresponding hydrochloric number of chlorin e6. Thus from the reaction mixture extract 2/3 dissolved chlorine e6.

Consumption of concentrated hydrochloric acid for cleaning of chlorin e6120 ml per 1 l of the reaction mixture, and just to receive 1 g of chlorine consume 2.5 liters of concentrated hydrochloric acid.

At the specified way we obtain 1 g of chlorin e6from 1 kg of dried nettle leaves, with the purity of the product is from 30% to 60% of the basic substance. For more pure target product using the method of column chromatography of alumina (eluent-methylene chloride:benzene (2: 1)).

Thus, in known methods for producing chlorin e6in the first stage of the technological cycle are used extracts of chlorophyll obtained from chloropalladite parts of higher plants or Zelenyi the ratio of the individual forms of chlorophyll at different plants varies and depends on the adaptability of plants to different light conditions. "Sun-loving" plants the ratio of chlorophyll in the leaves is approximately 3:1, "shade" it is decreased.

When processing green plants organic solvents retrieves all forms of chlorophyll ( and ) and the subsequent processing of these extracts is formed or a mixture of pheophorbide and (first method), or a mixture of pheophytins and (as noted in the second method, which ultimately leads to the formation of a mixture of chlorin e6and the birthplace of the g7.

The disadvantages of the known methods for producing chlorin e6are the complexity of the technological process, the use of large volumes of concentrated hydrochloric acid (2.5 l per 1 g of chlorin e6), a small yield and low purity of the target product (from 1 kg of dried nettle leaves receive 1 g of chlorin e6, the purity of which is not higher than 60%), the use of chromatographic method for purification of the target product. In addition, given the complex environmental conditions, does not exclude the possibility of contamination of vegetable raw materials of pesticides and other toxic chemicals, and the use of spontaneously grown biomass of green plants as a source of chlorophyll can lead ultimately to the pollution of Mecca, significant reduction of corrosive hydrochloric acid and obtain the desired product with a purity not less than 90% without the use of chromatographic purification.

This is achieved by the proposed method of producing chlorin e6implying that as a source of raw materials use biomass of cyanobacteria usually of the genus Spirulina, mainly grown in the light in controlled conditions on an artificial nutrient medium having a pH ranging from 8.0 to 12.0. The cyanobacteria biomass treated with an aqueous-alcoholic solution of alkali, mainly when the ratio of water:alcohol of 1 to 2-3 and the concentration of alkali 10-2-10-3N. filtered and extracted chlorophyll "a" ethyl alcohol. The combined organic extracts are treated or hydrochloric acid formed pheophytin filtered off, washed with a nonpolar organic solvent, usually hexane.

Alkaline hydrolysis of pheophytin carried out in the absence of oxygen in the alcohol medium when heated. The formation of the alkali salt of chlorin. After cooling, the reaction mixture was adjusted with hydrochloric acid to a pH of 6.0-7.0 (consumption of acid 50-55 ml per 1 l of the reaction mass, obrazovanii transferred to salt conventional techniques.

The use of the proposed method can improve the yield of the target product at least 3 times to obtain a product with a purity of not lower than 90% in Addition, the consumption of concentrated hydrochloric acid was reduced approximately 10 times.

To remove water-soluble proteins, hydrocarbons and other impurities, the cyanobacteria biomass pre-treated with an aqueous-alcoholic solution of alkali, for example, when the ratio of water and alcohol of 1 to 2-3 and the alkali concentration of 10-2-10-3N. To increase the yield of the target product, it is expedient according to the claimed invention to use the biomass of cyanobacteria containing (dry residue) of 2.0 to 4.5 wt. chlorophyll forms , 46-62 wt. proteins, 4-9 wt. lipids, 10-20 wt. carbohydrates, 10-12 wt. ash elements.

Further objectives and advantages of the invention will become apparent from the subsequent detailed description of the method of producing chlorin e6and specific examples of implementation of this method.

In accordance with the claimed invention receive 18-carboxy-20-(carboxymethyl)-8-ethynyl-13-ethyl-2,3-dihydro-3,7,12, 17 - tetramethyl-n.N. porphin-2-propionic acid, called chlorin e6carry out from a substance containing x is lnyh environments in terms of solar or artificial light.

Monoculture of cyanobacteria grown in a special fermenters closed, eliminating the possibility of green algae, and therefore getting into the biomass of chlorophyll forms .

Use chloropalladite plant materials standard composition grown in algological and clean environment ensures high purity of chlorin e6free from traces of heavy metals and other impurities caused by pollution. This fact is especially important in the manufacture of medical products.

The biomass of cyanobacteria, it is advisable to handle hot water-alcohol solution of alkali, for example, at a temperature of 40-60aboutC, the ratio of water and alcohol of 1 to 2-3 and the alkali concentration of 10-2-10-3N. When this treatment is the destruction of cell membranes, removed low molecular weight fractions of carbohydrates and soluble proteins, which makes it difficult for subsequent extraction of chlorophyll and contaminate the extract, and therefore, the target product. Biomass after processing is extracted with ethyl alcohol, extracting the chlorophyll . On polucheniya in the reaction mass pheophytin , which is marked by filtration.

Received pheophytin , the output of which is 10 g of 1 kg of biomass of cyanobacteria, washed with a nonpolar solvent such as hexane, to remove carotenoids and lipids from the surface of the crystals pheophytin .

Then cleaned pheophytin dissolved and subjected to saponification with alkali in the absence of oxygen, for example, in a stream of nitrogen. In the reaction mass is formed target product chlorin e6. To highlight chlorin e6the reaction mixture is neutralized with hydrochloric acid, taken according to the invention in quantities of 50-55 ml per 1 l of the reaction mixture until crystals deposition of chlorin e6that is separated by filtration.

Thus, the proposed method allows to obtain 1 kg of biomass of cyanobacteria 3.5 g of chlorin e6with a purity of at least 90% at a flow rate of concentrated hydrochloric acid in the amount of 220 ml per 1 g of the target product, which is 10 times less than the amount of acid used in the method-analogue.

P R I m e R 1. The biomass of cyanobacteria of the genus Spirulina is grown in a closed glass fermenter under conditions of sunlight or artificial lighting on liquid nutrient medium, representing Vodniy. The culture medium has a pH ranging from 8.0 to 12.0. The carbon source is carbon dioxide, periodically introduced into the nutrient medium. After reaching the suspension of certain cell concentration (2.5-3.0 g/l) biomass of cyanobacteria is separated from the nutrient medium by filtration. 1 kg of the thus obtained biomass of cyanobacteria with a humidity of 75-80% pour 2.5 liters heated to 60aboutWith alkaline ethanol solution obtained by dissolving 4 g of NaHCO3in 1 l of distilled water and subsequent mixing with ethanol in the ratio 1:3, respectively.

The mixture of biomass with the specified solution is stirred for 3-5 min, and then filtered. Biomass on the filter is shaken out three times with ethyl alcohol, extracts containing chlorophyll , summarize and treated with glacial acetic acid at the rate of 1.5 ml per 1 l of the extract. The resulting crystals pheophytin filtered and washed with hexane. The output of pheophytin is 10,

Pheophytin in an amount of 10 g was dissolved in 3 l of alcohol by boiling in a stream of nitrogen, add 400 g of KOH and carry out the saponification reaction for 10 minutes then the mixture is neutralized with a solution containing 45 ml of concentrated soles filtered. The crystals are dried in the air without light. The purity of the obtained product 92%

P R I m m e R 2. Use the cyanobacteria biomass obtained in conditions similar to those specified in example 1.

1 kg of the above biomass of cyanobacteria pour 1.5 liters of alkaline ethanol solution, which is obtained by dissolving 40 ml of 1 N. NaOH in 1 l of distilled water and subsequent mixing with ethanol in a ratio of 1 to 2, respectively.

The mixture of biomass with the specified solution is stirred for 3-5 min, filtered, biomass extracted three times with ethyl alcohol to extract the chlorophyll . The extracts are combined and add 1.2 ml of concentrated HCl per liter of extract. The resulting crystals pheophytin filtered and washed on the filter with hexane. The output of pheophytin is 10 g 10 g pheophytin dissolved in 3.3 liters of ethyl alcohol boiling, then add 400 g of KOH and carry out the saponification reaction for 10 min in vacuum. After that the reaction mixture is neutralized with hydrochloric acid to pH 6-7. Consumption of concentrated hydrochloric acid is 55 ml.

Crystals of chlorin e6from the reaction mixture is filtered and dried. Weight obtained Karlovich, such as the one outlined in example 1, when the cultivation of the strain of Spirulina species Fr-A 127.

Alcohol extract of chlorophyll receive, as described in example 1. To 1 liter of the extract add 2 ml of 1.0 N. oxalic acid. The resulting crystals pheophytin filtered and washed on the filter with hexane. The output of teofilina of 9.7 g

Received pheophytin omelet, as described in examples 1 and 2. The output of chlorin e6is 3.3 g with a purity of 90%

1. The METHOD of OBTAINING 18-CARBOXY-20-(CARBOXYMETHYL)-8-ETHYNYL-13-ETHYL-2,3-DIHYDRO-3,7-12,17-TETRAMETHYL-N, NORFIN-2-PROPIONIC ACID OR ITS SALTS, including extraction of chlorophyll from plant materials, treatment of the extract with acid, followed by alkaline hydrolysis and isolation of the target product in the form of acids or salts, characterized in that the feedstock used the biomass of cyanobacteria, which is pre-treated with an aqueous-alcoholic solution of alkali, after treatment of the extract chlorophyll acid product obtained is filtered off and washed with a nonpolar organic solvent and subjected to alkaline hydrolysis in a vacuum.

2. The method according to p. 1, characterized in that is used as raw material of biobattery of the genus Spirulina, grown on the light under controlled conditions on a nutrient medium having a pH of 8.0 to 12.0.

4. The method according to p. 1, characterized in that as the use of alkali hydroxides or carbonates of alkali metals.

5. The method according to p. 1, characterized in that as a non-polar organic solvent used hexane.

 

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