JPH0555117B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to racemic N-carbamoyl-α-
An extremely advantageous method for producing optically active compounds or synthetic intermediates thereof that exhibit physiological activity useful as pharmaceuticals, etc., by stereoselectively cyclizing amino acids, biochemically converting them into optically active hydantoins, and optically resolving them. With the goal. [Prior Art] One of the objects of the present invention is to efficiently produce optically active hydantoins. Many hydantoins are used as anticonvulsants or psychotropic drugs, and compounds exhibiting various physiological activities are known. Usually used in racemic form, among hydantoins with asymmetric carbon atoms in the molecule, only those with a specific steric configuration are often involved in physiological activity, improving drug efficacy or reducing side effects. From these viewpoints, it is desirable to use optically active substances. Conventionally, methods for producing optically active hydantoins include: 1) Reacting an optically active α-amino acid with an alkali metal cyanate to form an N-carbamoyl-α-amino acid, which is then dissolved in a mineral acid. Method 2) Optically active cyanoacetic acid is induced into isocyanate, which is reacted with an amine to obtain N-carbamoylaminonitrile, and finally heated and cyclized in mineral acid to produce hydantoins. Method 3) Add optically active brucine to racemic hydantoins to form diastereomeric salts,
A method of optical resolution using the difference in solubility in solvents [Journal of Medical Chemistry (J.Med.Chem.21(12), 1294, 1978]) 4) Reacting a ketone with an optically active amine to obtain ketimine, A method such as a method of asymmetrically adding hydrogen cyanide to this and then reacting it with chlorosulfonyl isocyanate to form an optically active hydantoin [J.Org.Chem. 47, 4081, 1982]. It has been known. If a relatively easily available optically active form such as a natural amino acid can be used as a raw material, method 1) is advantageous, but if not, the racemic form may be induced in an acid or base and optically resolved. ,usually,
Methods 1) and 2) require complicated operations in advance to obtain raw materials. In addition, method 3), in which racemic hydantoins are directly optically resolved, requires brucine, which has extremely high toxicity, as a resolving agent. For these reasons, many problems can be expected when producing on an industrial scale. In method 4), which attempts to synthesize optically active substances at once by asymmetric synthesis, more than the equivalent mole of expensive optically active amines and chlorosulfonyl isocyanate are consumed, and unnecessary optically active sites derived from amines must be removed. It has many practical difficulties, such as being extremely difficult. [Problems to be Solved by the Invention] The present invention has solved the problem of N-carbamoyl-α, which can be easily derived from hydantoins or α-amino acids.
-Focusing on amino acids, we conducted intensive studies on biochemical methods with the aim of developing an efficient method for synthesizing optically active hydantoins. As a result, we found that some microorganisms use N-carbamoyl-α-amino acids as substrates and It was discovered that there are microorganisms that have the ability to produce optically active hydantoins through a selective cyclization reaction, and this reaction has an extremely wide range of applicability as it is effective as a method for producing not only optically active hydantoins but also optically active amino acids. The present invention has been completed by showing that it can be used as a wide optical separation means. [Means for Solving the Problems] The outline of the present invention is expressed by the following formula. That is, the present invention provides N represented by the general formula ()
- After converting only one configuration of the racemic form of carbamoyl-α-amino acid () to hydantoins () using the conversion ability of microorganisms, the unused enantiomer N-carbamoyl-α-amino acid () is separated. The purpose is to chemically convert the hydantoins into optically active hydantoins () while easily retaining the steric configuration by heating and cyclizing them under acidic conditions. The method of the present invention uses racemic N
-carbamoyl-alpha-amino acid ()
It can also be used as a method for optical resolution into optically active hydantoins such as and (). Furthermore, optically active hydantoins () and enantiomer N-carbamoyl-α-amino acids () obtained by the method of the present invention
can be converted into the corresponding optically active α-amino acid (,) by alkaline hydrolysis, so the method of the present invention can convert racemic N-carbamoyl-
It has wide applicability, as it can also be used as a method for optically resolving α-amino acids into optically active α-amino acids. The present invention will be explained in detail below. Racemic N- used as a raw material in the present invention
Carbamoyl-α-amino acids are produced by first synthesizing α-amino acids using a ketone as a starting compound by the well-known Strecker method or Bucherer method, which is a general method for synthesizing α-amino acids, and then further synthesizing the α-amino acids. It can be easily synthesized by reacting with an alkali metal cyanate. It is also possible to directly synthesize N-carbamoyl-α-amino acids by hydrolyzing hydantoins, which are synthetic intermediates of the Butfuerer method, under controlled conditions. These microorganisms used in the present invention, which have the ability to stereoselectively cyclize N-carbamoyl-α-amino acids to produce optically active hydantoins, are extremely widely distributed across various species. For example, examples of bacteria belonging to the genus include Aerobacter, Agrobacterium, Bacillus, and Corynebacterium. Also, the genus Nocardia belongs to actinomycetes. Cultivation of these microorganisms is usually carried out in liquid nutrient media. The culture medium usually contains assimilated carbon sources, nitrogen sources, and inorganic salt nutrients essential for the growth of each microorganism, but it also contains small amounts of various nucleobases, their derivatives, and other enzyme inducers. It is desirable to adaptively enhance enzymes. The culture temperature used is 20 to 70°C, and the pH range is 4 to 10. The growth of microorganisms can also be promoted by aeration and stirring. In the cyclization reaction of N-carbamoyl-α-amino acids to hydantoins, the culture solution, bacterial cells, or treated microbial cells of the microorganisms obtained as described above can be used. Although the culture solution of the microorganism can be used as it is, it is more preferable to use microbial cells separated from the culture solution. It goes without saying that the bacterial cells can be used as living cells, but for convenience of storage and handling, they can also be used as dried bacterial cells such as freeze-dried cells. Furthermore, even if the entire bacterial cell state is not maintained, any form of bacterial cell suspension, such as a bacterial cell grind product or a bacterial cell extract, can be used in this reaction as long as the conversion ability can be exhibited. The concentration of the reaction substrate N-carbamoyl-α-amino acid in the reaction solution can range from 0.1% to as high as about 30%. Preferred reaction PH
The range of 5 to 8 is used. If the pH is less than 5, the ability is likely to be deactivated, and if the pH exceeds 8, the solubility of hydantoins increases and the reaction becomes difficult to complete, making it impractical. The optimum PH varies depending on the reaction substrate and the microorganism from which the ability is derived, but is generally within the range of PH5 to 8.
As the cyclization reaction progresses, the pH gradually shifts to the alkaline side, so it is desirable to maintain the optimum pH by adding a neutralizing agent from time to time. As a neutralizing agent, hydrochloric acid,
Mineral acids such as sulfuric acid are suitable. The reaction temperature for this reaction is usually in the range of 30 to 60°C, but
The temperature appropriate for each individual case is adopted. Under these reaction conditions, it is relatively easy to obtain an almost quantitative conversion rate by selecting the degree of activity of the ability, and even unreacted N-carbamoyl-α-amino acid can be converted into a compound with extremely high optical purity. It is possible to isolate it as a chiral body. The optically active hydantoins and the unused enantiomer N-carbamoyl- produced by this cyclization reaction
Known methods can be used to isolate α-amino acids. Generally, α-
Hydantoins, which do not contain hydrogen atoms in their carbon atoms, are extremely dissolvable in aqueous media at pH levels below 8 to 9, so most of the hydantoins produced as the cyclization reaction progresses usually precipitate as crystals. It becomes a state. However, at pH levels above 11, these hydantoins turn into alkali metal salts and are easily soluble. On the other hand, the reaction substrate N-
Carbamoyl-α-amino acid was prepared under the present reaction conditions (PH
5 to 8), it becomes an alkali metal salt, exhibits extremely high solubility, and is in a completely dissolved state.
At pH 4 or lower, the carboxyl group becomes free and exhibits extreme insolubility. Taking advantage of the change in solubility of hydantoins and N-carbamoyl-α-amino acids with respect to pH in an aqueous medium, it is extremely easy to generate hydantoins and the unused enantiomer N.
It is also possible to fractionate and isolate -carbamoyl-α-amino acids. Of course, it can also be easily separated and isolated by an extraction method that takes advantage of its high solubility in organic solvents such as ethyl acetate. The unused enantiomer N-carbamoyl-α-amino acid is prepared by a known method such as heating and stirring at 70 to 100°C under mineral acid acidity such as hydrochloric acid or sulfuric acid, or in organic acid such as acetic acid. By this,
It is also possible to easily chemically cyclize and convert it into optically active hydantoins. In addition, by hydrolysis using an alkali metal salt such as caustic soda, caustic potash, or barium hydroxide, or by oxidizing the carbamoyl group using an equivalent amount of sodium nitrite under mineral acid acidity, This N-
It is also possible to convert carbamoyl-α-amino acids to optically active α-amino acids. In addition, undesired enantiomers that are inevitably produced by the present invention can be converted into α-amino acids through alkaline hydrolysis or the like, and then converted into α-amino acids using known methods such as using an oxidizing agent such as hypohalous acid. It goes without saying that the efficiency of the present invention can be further improved by recovering the ketone by a method and recycling it as a new racemic N-carbamyl-α-amino acid. [Example] The present invention will be described below with reference to Examples. Example 1 A nutrient liquid medium having the following composition was prepared, and 100 ml each was dispensed into 500 ml shoulder flasks with cotton stoppers, followed by steam sterilization at 120° C. for 20 minutes. Medium composition: Meat extract 0.5% (wt%) Yeast extract 0.5% Polypeptone 1.0% NaCl 0.15% Uracil 0.10% MnCl _{2.4H 2} O 20 ppm (PH7.0) Cultured in advance at 30℃ for 24 hours in bouillon agar Slaton One platinum loopful of the microorganisms shown in Table 1 was inoculated into the above nutrient medium and cultured at 33°C for 24 hours with shaking. Using 40 ml of these culture solutions, centrifuge the viable cells, wash them with the same amount of 0.9% saline, centrifuge again to collect the bacteria, and reduce the volume to 10 ml with the same saline. A cell suspension was prepared and used as the reaction component described below. Reaction solution composition (1) (RS)-N-carbamoyl-α-ferrylglycine or (RS)-4-carbamoylamino-6-fluorochroman-4-carboxylic acid
Dissolve 250μmoles in 0.1M phosphate buffer (PH
7.2), Substrate solution adjusted to a volume of 2.5 ml (2) 2.5 ml of the above bacterial cell suspension Place the above (1) and (2) in a small test tube with a stopper, and stir with a stirrer using a magnetic rotor. 48 at 37℃
Allowed time to react. As a control, a substrate solution (1) without viable cells was placed under the same reaction conditions. After the reaction, a part of the reaction solution was sampled and the 4-methyl-4-phenyl-imidazolidine produced in the reaction system was analyzed by high performance liquid chromatography.
2,5'-dione or 6-fluoro-spiro-
[Chroman-4,4′-imidazolidine]-2′,5′-
The amount of dione was determined. Measurement conditions: Column; Finepak SILC ₁₈ (4.6mm
ID×250mm) manufactured by JASCO Corporation, mobile phase: 60mM phosphate buffer (PH2.5)/MeOH=83/17 (v/
v), Flow rate: 2.0ml/min, Detection: 210nm, Internal standard: 4-(2-methylthioethyl)imidazolidine-2,5-dione The results of measurements for each microorganism are shown in Table 1. .

【table】

〔effect〕

The present invention is an optically active hydantoin, (S)-6-, which has recently attracted attention as a preventive or therapeutic agent for specific chronic symptoms of diabetes (cataracts, neurological diseases, etc.).
Fluoro-spiro-[chroman-4,4'-imidazolidine]-2',5'-dione (USAN; Sorbinil) and optically active α-amino acids (S) widely used as antihypertensive agents. -α-methyl-3,4-dihydroxyphenylalanine (L-
Considering that it can be advantageously applied as an efficient optical resolution means to the synthesis of methyldopa, etc., the present invention, which uses inexpensive biological reactions such as microorganisms, provides an extremely practical and effective method for producing these optically active compounds. It can be said that this is something that is provided. Furthermore, the present invention provides a completely new finding that N-carbamoyl-α-amino acids, which do not have a hydrogen atom on the α-carbon, can be biochemically converted into hydantoins through a cyclization reaction, which was previously unanticipated. It provides:

Claims (1)

[Claims] 1 General formula () [In the formula, R ¹ and R ² are each independently a different alkyl group, aralkyl group, aryl group, or a substituted product thereof, or R ¹ and R ² form one asymmetric cyclic compound. ] General formula () corresponding to only one configuration to the racemic N-carbamoyl-α-amino acid represented by [In the formula, R ¹ and R ² are the same as above (). * represents an asymmetric carbon. ] By the action of the conversion ability of a microorganism belonging to the genus Aerobacter, Agrobacterium, Bacillus, Corynebacterium or Nocardia, which has the ability to convert into hydantoin shown by the general formula () After converting into active hydantoin and separating unreacted optically active N-carbamoyl-α-amino acid from the generated hydantoin, the optically active N-carbamoyl-α-amino acid is heated under acidic cyclization to obtain the corresponding optically active hydantoin. 1. A method for producing optically active hydantoins, characterized by producing hydantoins. 2 R ¹ and R ² are [X represents a halogen atom, and Ra and Rb each represent a hydrogen atom or a methyl group. The manufacturing method according to claim 1, which uses 4-carbamoylaminochroman-4-carboxylic acids represented by the following. 3. The production method according to claim 1 or 2, wherein the optically active N-carbamoyl-α-amino acid is in the S form with respect to the asymmetric carbon in formula ().