JPH01181797A - Production of d-threo-3-(3,4-dihydroxyphenyl)serine derivative - Google Patents

Abstract

PURPOSE:To industrially advantageously provide the title compound useful for drugs, etc., by stereospecifically decomposing the L isomer of a racemic- threo-3-(3,4-dihydroxyphenyl)serine derivative with microorganisms into protocathechuic aldehyde and glycine. CONSTITUTION:The L isomer of a racemic-threo-3-(3,4-dihydroxyphenyl)serine derivative of the formula (R<1> and R<2> are H, methyl or benzyl; R<1> and R<2> may be substitute with a methylene group to form a five-membered ring) is stereospecifically decomposed with microorganisms into the corresponding protocathechuic aldehyde derivative and glycine to provide a D-threo-3-(3,4- dihydroxyphenyl)serine derivative. The microorganisms are preferably the genuses Bacillus, Corynebacterium, Arthrobacter, Pseudomonas, Monascus, Neurospora, Fusarium, Gibberella, Byssochlamys, etc.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to the general formula (wherein Rl and R2 are the same or different and hydrogen,
It represents a methyl or benzyl group, and Rl and RZ may be substituted with a methylene group to form a five-membered ring. ) of the racemic-threo-3-(3,4-dihydroxyphenyl)serine derivative (hereinafter abbreviated as racemic-threo-doubes derivative) using the action of microbial enzymes. ．． The present invention relates to a method for producing only D-threodobus derivatives by stereospecifically decomposing corresponding protocatechualdehyde derivatives and glycine.

D-threodobus derivatives produced by the present invention are useful as pharmaceuticals.
No. 540) is an important intermediate in the synthesis.

(Prior art) D-Threodopus has conventionally been produced by chemically optically resolving a racemic threodoubes derivative obtained by condensing a protocatechualdehyde derivative and glycine in a strong alkali, and producing a 0-catechol. It was synthesized by chemically removing the protective group of the moiety (Japanese Unexamined Patent Publication No. 121-1989).
No. 258, Japanese Unexamined Patent Publication No. 58-216146). However, this method suffers from the following reasons: (1) the optically active amine used as the optical resolving agent is expensive, the operation is complicated and the yield is poor, and (2) the remaining optical antipode must be racemized. Therefore, it cannot be said to be an advantageous method for industrial production.

(Problems to be Solved by the Invention) Therefore, the present inventors believe that if only the L-form of the racemic threodopes derivative is stereospecifically decomposed into a protocatechualdehyde derivative and glycine, an expensive optical resolution method can be obtained. It was thought that D-threodobus derivatives, which are synthetic intermediates of D-threodobus, could be selectively obtained without the need for agents. A production method using asymmetric decomposition related to this method,
In particular, the method using microorganisms is completely unknown and new. In addition, the protocatechualdehyde derivative and glycine produced as a result of decomposition by microorganisms can be recovered from the reaction solution, so they can be condensed again into a racemic threodobus derivative using the chemical synthesis method described above and recycled. For example, wasteful (D-threodobus derivatives) can be obtained without complicated racemization.

(Means for Solving the Problems) Therefore, the present inventors conducted extensive research in order to establish a method for selectively synthesizing the racemic threondobus derivative using the microorganisms, and the L-form of the racemic threondobus derivative was We have searched widely in nature for microorganisms that specifically decompose protocatechualdehyde derivatives and glycine, and as a result, we have discovered that certain microorganisms have the ability to decompose them, and have arrived at the present invention.

That is, the present invention aims to decompose only the L-form of a racemic threodopus derivative using microorganisms to selectively obtain a D-threodopus derivative, and from this to produce one threodobus, which is useful as a pharmaceutical. That is.

The microorganisms used in the present invention are not particularly limited as long as they have the ability to decompose L-threodobus derivatives, but include, among others, Bacillus, Corynebacterium, Arthrobacter, Pseudomonas, Monascus, Good results can be obtained by using at least one microorganism selected from Group X consisting of microorganisms belonging to the genera Neurospora, Fusarium, Gibberella, and Bysochlamys. The culture medium for culturing these microorganisms varies somewhat depending on the strain used, but since the production of the enzymes involved in the method of the present invention does not require very complicated conditions, almost all culture substrates are suitable for culturing these microorganisms. Available for use.

- Examples of carbon sources include carbohydrates and sugar alcohols such as glucose, fructose, sucrose, maltose, starch, molasses, and glycerin, organic acids such as citric acid, acetic acid, and fumaric acid, ethanol, propatool, etc. Nitrogen sources include inorganic and organic ammonium compounds such as ammonium sulfate and ammonium chloride, nitrogen compounds such as urea, potassium nitrate, and aqueous ammonia, peptone, meat extract, casamino acids, corn steep liquor, yeast extract, and amino acids. Examples of the inorganic salts include nitrogen-containing organic compounds such as potassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, calcium carbonate, etc., but are not limited to these, of course. Furthermore, the decomposition activity may be further enhanced by adding threonine to the medium in some cases.

The bacterial strain is inoculated into this medium and cultured aerobically or anaerobically. The culture temperature is preferably 20°C to 40°C, and the pH is preferably 4 to 8. The bacteria are usually grown by culturing for 2 to 6 days.

The racemic threondobus derivative as a substrate may be added to the medium from the early stage of culture, or a method of adding it in several portions may give good results. Furthermore, the bacterial cells taken out from the culture solution may be added to a reaction system containing a racemic threodobus derivative to react. It is also possible to use microbial cells whose cell membrane permeability to the substrate has been increased by pretreatment such as acetone powdering, ultrasonication, surfactant treatment, or toluene/ethanol treatment. A disrupted bacterial cell solution or immobilized bacterial cells may be used, and a surfactant of 0.1 to Q, 5 w may be used in the reaction system.
/v% addition may give good results in some cases. The reaction may be enhanced by adding pyridoxine, pyridoxal, or pyridoxal-5-phosphate to the reaction system.

The reaction is carried out at a pH of 5 to 9, preferably 7 to 8, and a temperature of 20 to 40.
C1 Preferably carried out at 25-30°C.

A suitable reaction time is 6 to 60 hours, but it is necessary to appropriately select these reaction conditions depending on the bacterial species.

Otherwise, there is a risk that the decomposition of the L-unit may be incomplete, or that not only the L-unit but also the D-threodopus derivative may be decomposed.

To isolate the D-threodoubes derivative remaining in the reaction mixture, conventional methods such as ion exchange resin and solvent extraction are used. After isolation, treatment with a Lewis acid such as aluminum chloride, aluminum bromide, or boron trichloride is performed. By doing so,
The desired D-Threadobes can be easily obtained.

Note that when RI and Rt of the D-threodoubes derivative represented by general formula (1) are both hydrogen, it is D-threodoubes itself, so the method of the present invention cannot be carried out using racemic threondoubes as a substrate. For example, D can be prepared directly from the reaction mixture without isolation or treatment with a Lewis acid.
-You can get Threador Doves.

(Example) The present invention will be explained in more detail with reference to Examples below.
The present invention is not limited by the examples.

Example 1 Polypeptone 0.5%, yeast extract 0.2%, KH.

PO, 0.1%, MgS Oa 0.05%, L-
One platinum loopful of Arthrobacter ureafaciens IFO12140 was inoculated from a slant medium into a pH 6 liquid medium containing 0.1% glutamic acid and 0.2% D-threonine, and cultured with shaking at 28°C for 3 days. did. DL-threo-3-(3,
4-methylenedioxyphenyl) serine 0.7%, pyridoxal-5-phosphate 0,0 O1%, Tween-
The suspension was suspended in 5.0 OmZ of 0.1M potassium phosphate buffer, pH 7.0, containing 800.1% and shaken at 28°C for 2 days. The remaining amount of substrate after removing the bacterial cells from the reaction solution by centrifugation was quantified by high performance liquid chromatography, and found to be 49% of the amount at the start of the reaction. Next, 50% of the reaction supernatant
0d was adjusted to pH 1 or less with hydrochloric acid, and the resulting precipitate was separated into a t-apar. 100 d of strongly acidic cation exchange resin DOWEX 50W-X8 (H type) was added to the furnace solution, and the mixture was stirred at room temperature for 1 hour to adsorb the remaining threo-3-(3,4-methylenedioxyphenyl)serine. I let it happen. Add 100 grams of IN ammonia water to the resin that has been washed with water in a separate furnace, and stir for 1 hour.
The furnace liquid was dried under reduced pressure, and the resulting dry matter was dissolved in an appropriate amount of IN hydrochloric acid and the specific optical rotation was measured.
It was found that (3,4-methylenedioxyphenyl)serine remained. ((r)M' = +28.7
(C=1.0, I N -HfJ), m, p, 158°
C0 In addition, D-threo-3- based on the raw material 0 body
The yield of (3,4-methylenedioxyphenyl)serine was 98%.

Examples 2 to 11 The same procedure as in Example 1 was carried out except that the inoculum shown in Table 1 was used, and the substrate remaining in the reaction supernatant was quantified by high performance liquid chromatography, and the results shown in Table 1 were obtained. I got it. From the results of specific rotation measurement, it was confirmed that there were no bodies in either case.

Table 1 Examples 12 to 14 Both R' and R'' in general formula (1) are hydrogen (a)
, both had a methyl group (b), and both had a benzyl group (c), and the reaction was carried out using the inoculum shown in Table 2. The reaction conditions and other treatments were the same as in Example 1, and the results shown in Table 2 were obtained.

Table 2 (Effects of the Invention) According to the present invention, an optically active one-threodobus derivative can be produced by a simple process of reacting a racemic threondobus derivative with a microorganism. Also,
By condensing the protocatechualdehyde derivative produced by the decomposition with glycine again, a racemic threodobus derivative, which is a substrate for the enzyme reaction according to the present invention, is obtained and can be reused. Conventionally, there has been a disadvantage that complicated operations such as optical resolution and racemization are required to obtain D-threodobes, but these are improved by the present method.

That is, the present invention provides a method for producing a novel 1-threodobus derivative using microorganisms, and by implementing the present invention industrially, an intermediate raw material for 1-threodobus that is useful as a pharmaceutical can be obtained at a low cost. .

Claims (2)

[Claims] (1) General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (I) (Here, R^1, R^2 are the same or different, hydrogen,
It represents a methyl or benzyl group, and R^1 and R^2 may be substituted with a methylene group to form a five-membered ring. ) is stereospecifically decomposed into the corresponding protocatechualdehyde derivative and glycine using microorganisms. D-threo-3-(3,4
-Production method of dihydroxyphenyl) serine derivative. (2) The microorganism is Bacillus genus, Corynebacterium
Genus Arthrobacter, Genus Pseudomonas, Genus Monascus, Genus Neurospora
urospora), Fusarium
The method according to claim (1), wherein the method is at least one microorganism selected from the group consisting of microorganisms belonging to the genus ), Gibberella, and Byssoch-lamys.