JPS63245695A - Production of optically active sulfur-containing carboxylic acid ester and antipodal sulfur-containing carboxylic acid thereof - Google Patents

Abstract

PURPOSE:To readily obtain the titled carboxylic acid useful for synthesizing various physiologically active substances, etc., in good yield, by reacting a specific carboxylic acid with a microorganism having the ability to asymmetrically esterify sulfur-containing carboxylic acids, etc., in an aerobic aqueous solvent. CONSTITUTION:A strain, such as the genus Candida, capable of asymmetrically esterify sulfur-containing carboxylic acids is cultivated in a desired synthetic culture medium at pH3-11 and 20-50 deg.C to provide microbial cells, which are then treated to afford a cell-free extract. The resultant extract is subsequently suspended in an aqueous solvent, such as phosphoric acid based buffer solution, or nonaqueous solvent, such as alcohol, and 0.01-50wt.% sulfur-containing carboxylic acid expressed by the formula [R1 is H, (ar)alkyl or aryl; R2 is alkyl; n is 1-20] to aerobically carry out asymmetric esterification reaction. The reaction is continued until the d-isomer of the carboxylic acid is completely esterified to afford a reaction solution, which is then extracted, separated and purified to recover the aimed optically active sulfur-containing carboxylic acid ester and antipodal carboxylic acid thereof.

Description

Detailed Description of the Invention Industrial Field of Application R3 R, -8-(CI(2)n-CH-Cα (1) (wherein, 几 is hydrogen, an alkyl group, an aralkyl group or an aryl group, R3 is an alkyl group, n is an integer from 20 to 20.

This optically active carboxylic acid ester and its counterpart octacarboxylic acid are used as raw materials for synthesizing various optically active physiologically active substances.

Conventionally, the method for producing optically active carboxylic acid esters has been to synthesize a racemic form of the above carboxylic acid ester using an organic synthesis method and then separate it using an optical resolving agent. A method is used to separate them into ordinary substances.

However, this method requires a large amount of expensive resolving agent at the stage of obtaining the optically active carboxylic acid ester;
Furthermore, this method has drawbacks such as the resolving agent being easily mixed into the product as an impurity and the resolving process being complicated, so that it has not always been a satisfactory method for producing optically active carboxylic acid esters on an industrial scale.

Furthermore, a method for producing optically active fused carmene ester and 70 enantiomer fused carboxylic acid represented by the above formula (1) using the Kit biological method has not yet been known.

This modification is based on Japanese Patent Application No. 61-268540 (filing date: 1982).
(November 13, 1999) This is a further development of the invention related to K.

In order to solve the problem, the present inventors asymmetrically extracted either the 6 carboxylic acids or the 1 carboxylic acid from a mixture of 6 carboxylic acids and 1 carboxylic acid represented by formula (1). We discovered a method of esterification.

That is, a combined carboxylic acid represented by the general formula % () where R, , is hydrogen, an alkyl group, an aralkyl group, or an aryl group, 几 is an alkyl group, and n is an integer from 1 to 20. A microorganism or a cell-free extract of a microorganism having the ability to asymmetrically esterify is subjected to an asymmetric esterification reaction aerobically in an aqueous or non-aqueous solvent in the presence of alcohol to obtain the above formula (1). The present invention relates to a method for producing an optically active fused carboxylic acid ester and its enantiomer fused carboxylic acid, which is characterized by obtaining one carboxylic acid and its 4-mer ester represented by the following.

The alkyl group as the substituent R of the fused calyl ester represented by formula (1) is an alkyl group having 1 to 5 carbon atoms, such as a methyl group, an ethyl group, etc., and the aralkyl group is, for example, a benzyl group or an aryl group. For example, 7
Examples include E=, x group, etc.

Examples of the alkyl group include methyl group, ethyl group,
Examples include butyl group.

Examples of microorganisms and cell-free extracts of microorganisms having the ability to asymmetrically esterify the combined carboxylic acids used in the present invention include No. 151! These include yeast, enzymes, and bacteria listed in .

These microorganisms are used as a culture solution containing them, isolated bacterial cells, or treated bacterial cells.

In addition, if the enzymes used in this l1hW14 have the ability to perform asymmetric esterification, the origin of these enzymes,
Purity etc. are not particularly limited.

Table 1 Bacteria (genus) that can be used in the present invention: Candjda, Rhigopus, Aspergillus! Mucor lJl'o%/<If Liu A Chromobac
terium Table 1 (Uruki) 1 Amano'10 (Mucor javanicu
s) Asymmetric esterification is carried out using a general aerobic medium, a culture solution cultured in a synthetic medium, bacterial cells, or a single cell-treated product as a medium.

The temperature is 50°C.

Asymmetric esterification can be carried out by adding a carboxylic acid as a starting material to the culture solution, or by using the bacterial cells or a processed product of the bacterial cells as they are or by immobilizing them.

If this reaction is carried out in an aqueous solvent, deionized water,
It is carried out in a buffer such as phosphate or Tris. pH2～
12. The temperature is 20-90°C.

When carried out in a non-aqueous solvent, alcohols such as methanol, ethanol, propatool, butanol, etc., acetone, cellosolve, hydrocarbons such as benzene, isooctane, etc. are used. A solvent that dissolves the raw material carboxylic acid is preferred.

For convenience of handling, bacterial cells may be dried, such as freeze-dried cells, spray-dried cells, cells treated with organic solvents such as acetone, toluene, etc., or cell treatments such as cell destruction products or cell extracts. You can also use objects. The concentration of raw material ester in the reaction medium or culture solution is 0.01 to 50% by weight
is preferred.

As the esterification reaction progresses, the pH of the reaction solution decreases due to the generated carboxylic acid, but in this case, it is preferable to maintain the optimum pH using a suitable neutralizing agent.

It is preferable to continue the reaction until the six starting carboxylic acids are completely esterified.

Separation and purification of the product from the reaction solution or culture solution can be carried out by conventional methods such as extraction, distillation, column chromatography, etc.

Examples Examples 1 to 8 Contents 50-(±)-3-mercapto-2 in Erlenmeyer flask
- Add 2.5 g of methylpropionic acid and 27.5 g of normal octatool saturated with water, then add the enzyme shown in Table 2 at a concentration of 6000 units per 1 g of acid, and place in a stirrer at 30°C for 7 days. The reaction was carried out by stirring. After the reaction is complete, add an equal amount of water to the reaction solution and hydroxylate) IJ
The pH was adjusted to 7.0 using aqueous solution, and the unreacted acid was transferred to the water layer, and the unreacted normal octatool and the synthesized ester were transferred to the oil layer.

The pH of the aqueous layer was lowered to 3.0 or less with sulfuric acid, and 3-mercapto-2-methylpropionic acid was extracted with chloroform.

This extract was concentrated under reduced pressure to obtain an oily substance.

Dissolve this in methanol and use JASCO's Shokoko, J[(
The luminous intensity was measured using DII'-360). The oil layer was fractionated by vacuum distillation, and the obtained 3-mercapto-2-methyl-propionic acid ester was dissolved in methanol, and the brightness was measured using the brightness meter described above. In addition, extracted 1-3-mercapto-2-methyl-propionic acid and fractionated d-
The synthesis rate was determined from the weight of 3-mercapto-2-methyl-propionate, and the results are shown in Table 2.

Example 9.10 Contents 500-Erlenmeyer flask K(±)-3-Mercapto-
Add 10 g of 2-methylpropionic acid and 110 g of normal octatool, and add the fermentation mixture shown in Table 3 to 1 g of acid.
6000 units were added to the solution, and the mixture was reacted at 40° C. for 7 days using a rotary shaker.

10 dK twice the amount of water was added to the reaction solution, the pH was adjusted to 7.0 with sodium hydroxide, the unreacted acid was transferred to the water layer, and the unreacted normal octatool and the synthesized ester were transferred to the oil layer.

The pH of the aqueous layer was lowered to 3.0 or less with sulfuric acid, and 3-mercapto-2-propionic acid was extracted with chloroform. When this extract was concentrated under reduced pressure, an oily substance was obtained. This was dissolved in meta/-#iC and
P-360) was used to measure the brightness.

The oil layer was fractionated by vacuum distillation, and the obtained 3-mercapto-2-methylpropionic acid ester was dissolved in methanol, and the brightness was measured using the above-mentioned brightness meter. The synthesis rate was determined from the weight of methylpropionic acid and the fractionated d-3-mercapto-2-methylpropionic ester.

The time course of the reaction was measured by the method described above, and the results are shown in Table 3.

Table 93: Effects of the Invention The microbiological method of the present invention allows optically active d-carboxylic acid esters or their enantiomers to be produced from a combined carboxylic acid in a relatively simple manner. - Carboxylic acid could be produced with good yield.

Claims (1)

[Claims] General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (1) (In the formula, R_1 is hydrogen, an alkyl group, an aralkyl group, or an aryl group, R_2 is an alkyl group, and n is an integer from 1 to 20. A microorganism or a cell-free extract of a microorganism having the ability to asymmetrically esterify a sulfur-containing carboxylic acid represented by A method for producing an optically active sulfur-containing carboxylic acid ester and its enantiomer sulfur-containing carboxylic acid, which comprises carrying out a reaction to obtain an l-form of the carboxylic acid represented by the above formula (1) and a d-form ester thereof.