JPH0587498B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to D-cysteine derivatives and methods for producing the same. The present inventors have discovered that a microorganism belonging to the genus Pseudomonas has D-cysteine synthase (3-Chloro-D).
-Alanine Chloride-lyase), and as a result of conducting various studies on the catalytic action of this enzyme, we have arrived at the present invention. That is, the gist of the present invention is the following general formula ()

[Chemical Formula] (In the formula, B represents an alkyl group substituted with one or more substituents A, A', A'', and A, A', A'' may be protected with a hydrogen atom or a protective group. -OH
or -COOH, and A, A', and A'' cannot be hydrogen atoms at the same time. It is also useful as an intermediate raw material for pharmaceuticals, etc. For example, S-carboxymethyl-D-cysteine and its ester derivatives are used as raw materials for semi-synthetic cephalosporin MT-141.The present invention will be described in detail below. First, in the general formula (), the alkyl group of B usually has about 1 to 8 carbon atoms. As the protecting group for -OH and -COOH in A, A', and A'', These include those that are commonly used. A method for obtaining the D-cysteine derivative according to the present invention using D-cysteine synthase will be explained. Microorganisms that produce D-cysteine synthase include microorganisms belonging to the genus Pseudomonas, such as Pseudomonas putida CR1-1 (Pseu-
domonas putida (Trevisan) Migula), Pseudomonas putida CR41-2, Pseudomonas
putida CR43-2, Pseudomonas fluo-rescens CR19-1
Migula) etc. All of these bacteria have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (Accession Numbers FERM-P No. 3458, 3459, 3460, and 3461, respectively). In addition, when identifying these bacteria, the mycological properties are described. Bergey's Manual of Determinatiye
Bacteriology) 8th edition, etc. were used. The mycological properties of these are described in Japanese Patent Publication No. 57-21986. The nutrients necessary for culturing these D-cysteine synthase-producing living microorganisms are not particularly limited, and various substances commonly used for culturing microorganisms can be used. For example, as the carbon source, carbohydrates such as glycose, sucrose, fructose, glycerol, sorbitol, molasses, and starch hydrolysates, and organic acids such as acetic acid and fumaric acid are used. Examples of nitrogen sources include nitrates, ammonium salts, corn stable liquor, yeast extract, meat extract, yeast powder, soybean hydrolyzate, cotton, seed flour, soybean flour, polypeptone, and peptone.
Potassium phosphate, sodium phosphate, magnesium sulfate, sodium chloride, etc. can be used as the inorganic salt. By adding β-chloro-D-alanine to these media, the productivity of D-cysteine synthase can be increased. The amount added is 0.1
~2% by weight is suitable. Culture temperature is 20-40℃.
A temperature of 27 to 37°C is particularly suitable. Cultivation is usually carried out aerobically for about 16 to 72 hours. The D-cysteine synthase obtained in this way is mainly present in the cells of microorganisms, and its separation and purification can be carried out using known methods such as ultrasonication, ammonium sulfate fractionation, ion exchange chromatography, and gel filtration. is applicable. That is, after the cells obtained after culturing are collected by centrifugation, the cells are disrupted by mechanical means such as ultrasonication, homogenizer, and grinding with glass beads, or by chemical means such as cell wall lytic enzymes. , obtain the supernatant by centrifugation. Next, this supernatant is subjected to ammonium sulfate fractionation, ion exchange chromatography such as "DEAE-Sephacel", adsorption chromatography using hydroxyapatite, etc., gel chromatography using "Sephacryl", "Sephadex", etc., and phenyl-chlorcephalose. It is purified by a combination of hydrophobic chromatography, etc. When producing D-cysteine derivatives using this cysteine synthase, β-substituted-alanine (D- or
DL−) is represented by the above general formula (),
Specific examples include β-chloro-D-alanine,
β-halogeno-D-alanine such as β-bromo-D-alanine, β-iodo-D-alanine; S-methyl-D-cysteine, S-ethyl-D-cysteine, S-hexyl-D-cysteine, etc. S-alkyl-D-cysteine and substituents thereof other than the cysteine moiety; S-aryl-D-cysteine such as S-phenyl-D-cysteine and substituents thereof other than the cysteine moiety; S-benzyl-D-cysteine S-aralkyl-D-cysteine and substituted substances thereof other than the cysteine moiety; S-alkenyl-D-cysteine such as S-allyl-D-cysteine and substituted substances thereof other than the cysteine moiety; and D-cysteine,
Examples include (D,D)-lanthionine and meso-lanthionine (these DL configurations can also be used). Moreover, pyruvic acid and ammonia can be used instead of these β-substituted alanines.
In this case, the ammonia may be a salt that generates ammonia, such as ammonium sulfate or ammonium chloride. Examples of the thiols represented by the general formula () include 2-mercabutoethanol, thioglycerol, thioglycolic acid, phenolyl mercaptan, and their esters and amide derivatives. In order to produce the D-cysteine derivative according to the present invention, the above β-substituted-alanine, or React pyruvic acid and ammonia with thiols.

[ka]

[C] Enzymes are not limited to purified enzymes, but can also be used in the form of bacterial cells, extracts, and crudely purified enzymes. Reaction temperature is 10-80
℃, preferably 20-50℃. The reaction time varies depending on the amount of enzyme used, the concentration and type of substrate, and the reaction temperature, but is selected from the range of 10 minutes to 100 hours, usually 30 minutes to 48 hours. The appropriate concentrations of the substrates β-substituted alanine, pyruvic acid and ammonia, and thiols are each 0.1 to 40% by weight. After completion of the reaction, the produced D-cysteine derivative is isolated according to a conventional method, for example, by treatment with an ion exchange resin. Hereinafter, the present invention will be explained in more detail with reference to Examples. Reference example 2 containing Pseudomonas putida CR41-2 in 500 ml of a medium consisting of 0.1% β-chlor-DL-alanine, 0.5% polypeptone, 0.5% yeast extract, and 0.2% NaCl (PH7.0). Inoculated 70 flasks,
Incubate with shaking at 28°C for 17 hours. The cells obtained from culture solution 35 were suspended in a standard buffer (potassium phosphate buffer PH7.0 containing 0.01mM pyridoxal phosphate, 0.1mM EDTA, and 0.5mM 2-mercaptoethanol), and then treated with ultrasound. , Obtain a bacterial cell extract by centrifugation. This bacterial cell extract is subjected to ammonium sulfate fractionation, and the D-cysteine synthase activity fraction (30 to 60% saturation) is dialyzed and then passed through a "DEAE-Sefacel" column (6 x 90 cm) to adsorb the enzyme. After washing with 3150ml of 0.05M standard buffer,
Perform elution with 0.1M standard buffer. The obtained D-cysteine synthase-containing eluate was subjected to ammonium sulfate fractionation, and the fraction saturated with ammonium sulfate of 40 to 50% was collected, and after dialysis, it was purified using a hydroxyapatite column (3 x 35
cm), the active fraction passes through and unnecessary proteins are removed. D-cysteine synthase activity was determined using a “Sephacryl” S-200 column (1.5×
90cm) to perform gel filtration. The obtained D-
The cysteine synthase-containing fraction was adsorbed onto a column of phenylchlorsepharose 4B equilibrated with a standard buffer containing 20% ammonium sulfate, and after washing, 15%
20 each of standard buffers containing 10% and 5% ammonium sulfate
Wash with ml. Next, elute with 0.01M standard buffer that does not contain ammonium sulfate. The obtained D-cysteine synthase showed a single band in polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis, and ultracentrifugation analysis also showed that it was a single protein. The specific activity of purified D-cysteine synthase is 265 units/
mg protein. The above results are shown in Table-1.

[Table] Activity measurement method 0.2M potassium phosphate buffer (PH8.0) 500μ
, 1mM pyridoxal phosphate 100μ, lactate dehydrogenase 5μ, NADH (2mg/ml) 80μ,
β-Chlor-D-alanine (1mg/ml) 200μ
The enzyme solution was added to the solution, the volume was made up to 2 ml with H ₂ O, and the mixture was reacted at 30° C. The amount of pyruvic acid produced was measured using a spectrophotometer as a change in OD (334 nm) due to a decrease in NADH. 1 unit of enzyme activity per minute
Defined as the enzyme activity that produces 1 μmole of pyruvate. Example 1 100 units of protein-pure purified D-cysteine synthase was added to 100 ml of 0.1M potassium phosphate buffer (PH7.5) containing 10 mmol of β-chloro-D-alanine and 50 mmole of thioglycolic acid ethyl ester.
was added and reacted at 30℃ for 2 hours, then 20ml of 30% trichloroacetic acid solution was added to the reaction solution to stop the reaction.
Centrifuge to obtain supernatant. S-ethoxycarbonylmethyl-D-cysteine produced in the reaction solution is detected by thin layer chromatography and an amino acid analyzer. Add ammonia solution to this and leave at room temperature for 24 hours to make 0.4M.
This treatment results in S-ethoxycarbonylmethyl-
D-cysteine can be hydrolyzed to S-carboxymethyl-D-cysteine. This solution was diluted 10 times with distilled water and applied to a "Dowex 1 x 2" column (4 x 30 cm, OH ^-1 type). After washing this column thoroughly with distilled water,
Elution was performed with 0.7M acetic acid. Fractions containing S-carboxymethyl-D-cysteine were detected by thin layer chromatography, and these fractions were collected, concentrated to dryness, dissolved in a small amount of distilled water, cooled on ice, and added with a small amount of ethanol. , left overnight. White powdery crystals were obtained, and the crystals were collected and recrystallized. As a result, 1467 mg of S-carboxymethyl-D-cysteine was obtained, and the yield based on β-chloro-D-alanine was 82%. It was shown to have a D-type configuration because it was degraded by D-amino acid oxidase in the presence of FAD and catalase. HOOCCH ₂ -S-CH ₂ CH (NH ₂ ) COOH Elemental analysis C H N S calcd; 33.51 5.06 7.82 17.89 found; 33.18 5.17 7.80 17.88 Optical rotation [α] ²⁵ _D °=-1.0 (C=3.65) mp197℃ Conducted Example 2 Pseudomonas putida CR1-1 is cultured aerobically at 28°C for 20 hours under the conditions shown in Table 2. Then, centrifuge 10 ml of the culture solution and collect the bacterial cells. This was added to 10 ml of 0.1 M potassium phosphate buffer (PH 8.0) containing 1 mmol of β-chloro-D-alanine, 5 mmol of thioglycolic acid ethyl ester, and 1 μmol of pyridoxal phosphate, and reacted at 30° C. for 2 hours. As a result of analysis using an amino acid analyzer, 700 μmol of S-ethoxycarbonylmethyl-D-cysteine was produced in the reaction solution. By hydrolyzing this with ammonia and purifying it according to the method of Example 1, crystals of S-carboxymethyl-D-cysteine can be obtained.

[Table] Example 3 β-chloro-D-alanine 10 mmol, 2-mercaptoethanol or thioglycerol 50 mmol
Contains 0.1M potassium phosphate buffer (PH7.5)
Add 100 units of protein-purified purified D-cysteine synthase to 100 ml and react at 30°C for 3 hours.
Add 20 ml of 30% trichloroacetic acid solution to the reaction mixture to stop the reaction, and centrifuge to obtain a supernatant. This was applied to a "Dowex 50 x 8" column (4 x 30 cm). After washing this column thoroughly with distilled water,
Elution was performed with 1M aqueous ammonia. generated S
Fractions containing -substituted-D-cysteine derivatives were detected by thin layer chromatography, and these fractions were collected and concentrated to dryness, then dissolved in a small amount of distilled water, cooled on ice, and left to stand after adding a small amount of ethanol. did. White powdery crystals were obtained, and the crystals were collected and recrystallized. The physicochemical properties of the product are shown below. S-(2-hydroxyethyl)-D-cysteine Molecular weight 165.21 Elemental analysis C H N S calcd; 36.35 6.71 8.48 19.41 found; 35.69 6.74 8.41 − Optical rotation [α] ²⁵ _D °＋1.8° (C = 4.0) mp195 °C S-(2,3-dihydroxypropyl)-D-cysteine Molecular weight 195.24 Elemental analysis C H N S calcd; 36.91 6.71 7.17 16.42 found; 36.50 6.75 7.25 16.02 Optical rotation [α] ²⁵ _D °−1.0° (C = 4.0 ) mp192℃ Example 4 β-chloro-D-alanine 4 mmol, thiols shown in Table 3 2 mmol, pyridoxal phosphate
Proteinically pure purified D-
Add 18 units of cysteine synthase, react at 30°C for 30 minutes, and then add 2 ml of 30% trichloroacetic acid. Amino acids produced in the reaction solution were quantified using an amino acid analyzer. The results are shown in Table-3.

[Chemical] Example 5 4 mmol of various D-amino acids shown in Table 4, 2-
Mercaptoethanol or methyl thioglycolate 2 mmol, pyridoxal phosphate 2 μmol,
Add 20 units of D-cysteine synthase to 10 ml of 0.5 M ammonia buffer (PH8.5) containing 2 μmol of EDTA.
After reacting at 30°C for 1 hour, add 2 ml of 30% trichloroacetic acid. The results are shown in Table 4.

【table】

[Table] Example 6 Pyruvate 0.2 mmol, (NH ₄ ) ₂ SO ₄ 0.3 mmol,
EDTA 3μmol, pyridoxal phosphate 0.2μmol,
0.2M _NH4 containing 20 units of D-cysteine synthase
Add 0.5 mmol of the thiols shown in Table 5 to 3 ml of OH-NH ₄ Cl buffer and react at 30°C for 2 hours. The results are shown in Table-5.

【table】

【table】

【table】

Claims (1)

[Claims] 1. A D-cysteine derivative represented by the following general formula (). [Formula, B represents an alkyl group substituted with one or more substituents A, A′, A″, and A, A′, A″ are
May be protected with a hydrogen atom or a protecting group -
It represents OH or -COOH, and A, A', A'' cannot be hydrogen atoms at the same time.) 2 (i) The following general formula () [Chemical formula] (In the formula, X is a halogen atom or [Formula] A, A′, A″, and B have the same meanings as in the general formula (). However, A,
A′ and A″ may both be hydrogen atoms at the same time.) β-substituted alanine represented by () or () pyruvate and ammonia are combined with the general formula () in the presence of D-cysteine synthase. A method for producing a D-cysteine derivative, which comprises reacting it with thiols represented by (A, A', A'', B are the same as in the general formula ()).