JPH01124398A - Production of optically active hydantoins - Google Patents

Abstract

PURPOSE:To produce optically active hydantoins efficiently by converting racemic N-carbamoyl-alpha-amino acid into optically active hydantoin by stereoselective enzyme reaction and subjecting the product to optical resolution. CONSTITUTION:The enzyme produced by a microorganism in Aerobacter or Nocardia, capable of converting only one stereoisomer of an amino acid of formula I into the corresponding hydantoin formula II (R<1>, R<2> are alkyl, aryl; *is asymmetric carbon), is allowed to act on racemic N-carbamoyl-alpha-amino acid of formula I into optically active hydantoin of formula II. Then, the unreacting, optically active amino acid is separated, and the amino acid is subjected to thermal cyclization under acidic conditions to give optically active hydantoin of formula II.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a method for converting racemic N-carbamoyl-α-amino acids into optically active hydantoins using a stereoselective enzymatic reaction and optically resolving them. The purpose of this invention is to very advantageously produce an optically active compound or a synthetic intermediate thereof that exhibits physiological activity that is effective as a medicine.

[Prior art]

One of the objects of the present invention is to efficiently produce optically active hydantoins. Among the hydantoins, many are used as anticonvulsants or psychotropic drugs, and compounds that exhibit remarkable physiological activity 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 this point of view, 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 salt to form an N-carbamoyl α-amino acid, and then converting this into a mineral acid. 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 form hydantoins. 3) A method of adding optically active brucine to racemic hydantoins to form diastereomeric salts, and performing optical resolution using the difference in phase solubility in bath media (J, Med, Ch
em, 21 (12), 1294.4) A method of reacting a ketone with an optically active amine to obtain ketimine, asymmetrically adding hydrogen cyanide to this, and then reacting it with chlorosulfonyl isocyanate to obtain an optically active hydantoin (J
, Org.

Chem, 47.4081.1982) and other methods are 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. Generally, methods 1) and 2) require complicated operations in advance to obtain the 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 the method 4), which attempts to synthesize optically active substances at once by asymmetric synthesis, more than one mole of the corrosive optically active amine or chlorosulfonyl isocyanate is consumed, and unnecessary optically active sites derived from the amine are removed. It is extremely difficult to remove
It has many practical difficulties.

[Problem to be solved by the invention]

The present inventors focused on hydantoins or N-carbamoyl-α-amino acids that can be easily derived from α-amino acids, and developed a biochemical method for the purpose of developing an efficient synthetic method for optically active hydantoins. As a result of extensive research, we discovered that there are microorganisms that have the ability to produce optically active hydantoins through a stereoselective cyclic reaction using N-carbamoyl-α-amino acids as substrates, and we believe that this reaction is capable of producing optically active hydantoins. The present invention was completed by revealing that the present invention can be used as an extremely wide-applicable optical resolution method that is effective as a method for producing not only optically active hydantoin but also optically active amino acids.

[Means to solve the problem]

The outline of the present invention is expressed by the following formula.

The following margin NH= NH2 (VD (V) That is, the present invention enzymatically converts only one configuration of the racemic N-carbamoyl-a-amino acid (I) represented by the general formula (1) into hydantoins (It ), the unutilized enantiomer N-carbamoyl-6-amino acid 01D is fractionated and subjected to a heating cyclization reaction under acidic conditions to easily form optically active hydantoins Gv) while maintaining the steric configuration. The purpose is to chemically convert it into The method of the present invention can also be used as a method for optically resolving racemic N-carbamoyl-α-amino acids into optically active hydantoins (■) and (IV). Furthermore, optically active hydantoins (1) obtained by the method of the present invention
) and the enantiomer N-carbamoyl-α-amino acid (9)
are all alkali hydrolyzed to give the corresponding optical activity a
- Amino acids (VI, V) can be converted, so the method of the present invention has wide applicability, such as being able to be used as a method for optically resolving racemic N-carbamoyl-a-amino acids into optically active α-amino acids. .

The present invention will be explained in detail below.

The racemic N-carbamoyl-α-amino acid used as a raw material in the present invention is produced using a ketone as a starting compound.
- The well-known Strecker method or Buc Error method is a general method for synthesizing amino acids.
-Hα-amino acid is synthesized by the herer) method,
It can be easily synthesized by further reacting this with an alkali metal cyanate. In addition, hydantoins, which are synthetic intermediates of the Bluff error method, can be directly synthesized by hydrolyzing N-carbamoyl-α under controlled conditions.
- It is also possible to synthesize amino acids.

The enzyme used in the present invention can usually be obtained by culturing microorganisms, and the distribution of these microorganisms has the ability to stereoselectively cyclize N-carbamoyl-α-amino acids to produce optically active hydantoins. is extremely wide and covers a wide variety of species and genera. For example, as a species belonging to bacteria, the genus Aerobacter
, Agrobacterium spp.
), Bacillus , Corynebacterium , and the like. 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 temperature during culturing is 20 to 70"C, and the pH is 4 to 10.
range is used. 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 as the enzyme. 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. The cells can of course be used as live cells, but for convenience of storage and handling, they can also be used as dried cells such as freeze-dried cells. Furthermore, a bacterial cell grind product or a bacterial cell extract can be used as a microbial enzyme in this reaction.

The concentration of the reaction substrate N-carbamoyl-α-amino acid in the reaction solution can range from 0.1% to as high as 50%. A preferable reaction pH range is from 5 to 8. If the pH is less than 5, the enzyme is easily inactivated, and if the pH exceeds 8, the solubility of hydantoins increases, making it difficult to complete the reaction, making it impractical. optimal p
The pH of H varies depending on the reaction substrate and the enzyme used, but is generally within the pH range of 5 to 8. Since the pH gradually shifts to the alkaline side as the cyclization reaction progresses, it is desirable to maintain the optimum pH by adding a neutralizing agent from time to time.

As the neutralizing agent, mineral acids such as hydrochloric acid and sulfuric acid are suitable. The reaction temperature for this reaction is usually in the range of 30 to 60°C, but a temperature suitable for the enzyme used is employed. By selecting these reaction conditions and enzyme amounts, it is relatively easy to obtain a quantitative conversion rate, and even unreacted N-carbamoyl-α-amino acid can be converted into a single enantiomer with extremely high optical purity. It is possible to separate.

Known methods can be used to isolate the optically active hydantoins and the unused enantiomer N-carpamoino C-α-amino acid produced by this enzymatic cyclization reaction. In general, hydantoins that do not contain a hydrogen atom on the α-carbon atom, such as those produced by the method of the present invention, are extremely poorly soluble in aqueous media at pH 8 to 9 or below, and therefore are produced as the cyclization reaction progresses. Most of the hydantoins are usually precipitated as crystals. However, pH 1
When the amount is 1 or more, these hydantoins become alkali metal salts and exhibit easy solubility. On the other hand, the reaction substrate, N-carbamoyl-α-amino acid, becomes an alkali metal salt under the present reaction conditions (pH 5 to 8), exhibits extremely high solubility, and becomes completely dissolved, but at pH 4 or lower, the carboxyl group is It becomes a free state and shows extreme insolubility. Changes in the solubility of such hydantoins and N-carbamoyl-α-amino acids with respect to pH in an aqueous medium.

It is also possible to very easily separate and isolate the produced hydantoins and the unutilized enantiomer N-carbamoyl-α-amino acid using this method. 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 an organic acid such as acetic acid. By doing so, it is also possible to easily chemically cyclize and convert into optically active hydantoins. In addition, the carbamoyl group can be oxidized 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 acidic mineral acid. -Carbamoyl- It is also possible to convert α-amino acids into optically active α-amino acids.

In addition, undesired enantiomers, which are inevitably produced as a by-product of the present invention, can be obtained by using known methods such as alkaline hydrolysis, etc., to convert the α-amino acid into an α-amino acid, and then 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 explained below with reference to examples.

Example 1 A nutrient liquid medium having the following composition was prepared, and after dispensing 100ml each into a 50ON/capacity flask with a cotton stopper, 1
Steam sterilization was performed at 20"C for 20 minutes.

Medium composition: Meat extract 0.5% (wt%) yeast extract
0.5% Polypeptone 1.0% NaCl 0.15% Uracil 010% MnCl2-4H 2020 ppm (pH 7,0) The microorganisms shown in Table 1, which had been previously cultured on a bouillon agar slant at 30°C for 124 hours, were added to the above nutrient medium. One platinum loopful was inoculated and cultured at 33°C for 24 hours with shaking. Using 40 volumes of these culture solutions, the viable cells obtained by centrifugation were further washed with the same amount of 0.9% saline, centrifuged again to collect bacteria, and diluted with 10ml of the same saline. A bacterial cell suspension was prepared and used as the reaction component described below.

Reaction solution composition (1) (1)-N-carbamoyl-α-ferrylglycine or (几5)-4-carbamoylamino-6-
250 tt m of fluorochroman-4-carboxylic acid
Dissolve ole in 0.1M phosphate buffer at pH 7.2.
), substrate layer solution adjusted to a liquid volume of 2.5 ytl (2) 2.5 ml of the above bacterial cell suspension (1
) and (2) were placed in a small test tube with a stopper and allowed to react at 57°C for 48 hours while stirring with a stirrer using a magnetic rotor. 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 4-methyl-4-phenyl-imidazolidine-2,5'-dione or 6-fluoro-dione produced in the reaction system was analyzed by rapid liquid chromatography.
Spiro-[chroman-4,4'-imidazolidine) -
The amount of 2', s'-dione was quantified.

Measurement conditions: Column; Finepak S I L 0
n (4,6 IDX250wz) manufactured by JASCO ■, mobile phase; 60 mM phosphate buffer (p I-I 2.5)
/MeOH=83717 (V/V), flow rate; 2.
O shoulder l/min.

Detection: 210 nm, internal standard: 4-(2-methylthioethyl)imidazolidine-2,5-dione The results of measurements for each microorganism are shown in Table 1.

All of the imidazolidine derivatives produced below were optically active forms of the R form with respect to the carbon at the 4-position of the imidazolidine.

Example 2 A nutrient liquid medium having the following composition was prepared and placed in a 500#I shoulder shake flask with a cotton stopper for 150y. Dispense in portions and steam sterilize at 120°C for 20 minutes.

Medium composition: Meat extract 2.0% (w/v%) Glucose 0.8% NaCl 0.3% Uracil 0.1% pH 7.5 Pacillus sp. cultured in advance at 33°C in bouillon agar slant for 20 hours. (Eucillus sp.
cies) KNK 108 (Feikoken Joyori No. 88
No. 7 (FERM BP-887)) was inoculated into the above-mentioned nutrient liquid medium, and cultured under shaking at 33°C for 22 hours.

Using 200 ml of this culture solution, the viable cells obtained by centrifugation were further washed with the same amount of 09% saline, and then centrifuged again to collect the bacteria and used as a component of the reaction solution described below.

Reaction liquid composition: (1) (Its)-4-carbamoylamino-6-
Dissolve 1.20 f of fluorochroman-4-carboxylic acid as a sodium salt (pH 7.2) in a liquid volume of 20 ml.
Substrate solution adjusted to (2) The above culture [20 (live bacteria collected from lye
.

Suspend the bacterial cell suspension using 0.9% saline and adjust the liquid volume to 20 sl. Mix the above (1) and (2) and put it in a 100-l capacity four-sided round bottom flask, while stirring. The reaction was carried out at 37°C for 48 hours. During this reaction, the pH of the reaction system was continuously maintained at 7.2 using a 0.5 N-H (:!1 solution. After the reaction,
After the reaction solution was cooled and the pH was adjusted to 7.0, it was separated by centrifugation into an insoluble mixture of crystals and bacterial cells precipitated during the reaction and a clear solution. The insoluble mixture thus obtained was
After suspending in +/ water, acetic acid was added and extraction was performed twice using 1001111 to obtain an ethyl acetate extract. Ethyl acetate was distilled off from this extract under reduced pressure, and the resulting residue was recrystallized from ethanol to obtain 0.51 g of white crystals.

As a result of analysis, this compound was found to be ＠)-6-fluoro-spiro-[
Chroman-4,4'-imidazolidine]-z: 5'-
It was identified as Zeon.

NMRδppm (DMF-d1: 2.1 to 2.6
(2H, m, CH2), 4.1-4.8 (2H, m,
CH2), 6.8-7.2 (3H, m, arom
atic), 8.4 (IH.

s, NH), 1.0 (IH, br s, NH)I
Rcm (KBr-disk): 3340°32
05.1760.17M0,1490°1400.12
60.1160 (α'l 25--5 5.0° (cm 1.0
, MeOH)mp, 241-242°C On the other hand, the pH of the clear solution was adjusted to 1 using a 6N-H(:!l solution).
When adjusted to 0, a white precipitate was deposited. This precipitate was separated and recrystallized from ethanol to obtain 054 g of white crystals.

As a result of the analysis, this compound was identified as (S)-4-carbamoylamino-6-fluorochroman-4-carboxylic acid.

NMRδppm CDMSO-ds): 2.3-2
．． 7 (2H, m, CH2), 3.9~4.5 (2
H, m, CH2), 5.7 (2H, s, Na2),
6.7 (IH, S, M), 6.8-7.4 (3H,
m, aromatic) ■几 art (KEr-
disk): 3470.

3380.1720, 1640.1560°1500.
1485,1425.1370゜(/2) o=+11
2.7° (c = 0.5, MeOH) mp,
196~l 97''C Example 3 (S)-Rubamoylamino-6-fluorochroman-4-carboxylic acid 400~ obtained in Example IM2 was converted into 2N-H
It was suspended in Cl 10 yxl and reacted at 80° C. for 3.5 hours while stirring with a magnetic cooler.

After the reaction, the mixture was left in a refrigerator overnight, and the precipitated crystals were collected and dried at 60°C under reduced pressure to obtain white crystals with a temperature of 340°C.

As a result of analysis, this compound was found to be (S)-6-fluoro-spiro-[chroman-4,4'-imidazolidine]-2: s
'-Dione was completely consistent with the standard sample.

(/Z)',;-+ 55.7° (cm1.0.M
eOH) mp, 24'1.5-242.5°C Example 4 Bacillus 5 B seeds obtained in the same manner as in Example 2 (
Bacillus 5pecies) KNK 10 g
(FERM BP-887 No. 887)
Add 15.0F of (RS-N-N-carbamoyl-α-methyl-3,4-methylenedioxyphenylalanine) to the culture solution 5QOrttl of )) and adjust the pH with 1ON-NaOH solution.
72 and reacted at 37°C for 48 hours. During this reaction, a 0.5N HCI solution was used to adjust the p I-
I was held continuously at 7.2. After the reaction, the reaction solution was cooled and the pH was adjusted to 70, and then separated by centrifugation into an unbathed mixture of crystals precipitated during the reaction and bacterial cells, and a supernatant.

When the pH of the thus obtained clear liquid was adjusted to 6N-I (1.0 using C1 solution, a white precipitate was deposited. This precipitate was collected by filtration, thoroughly washed with water, and then dried under reduced pressure.) -
7.31 of white crystals of N-carbamoyl-α-methyl-3,4-methylenedioxyphenylalanine were obtained.

NMRδppm CDMSO-da): 1.
3 (3H.

S, Q-CH3), 2.9-5.3 (2H, m, α
-CH2), 5,6 (2H,s, NFI2),
5.9 (2H,s, CH2), 7,0 (IH,s
, NH), 6.4-18 (3H, m.

aromatic) IRcyt (KBr-disk): 3500°3400.3330.2$00, 1695°1600.
1570.1490.1440゜1390.1280.
1245 [α piece = +92.0° (cm0.5.0.INlN-N
a0H), 190-192°C On the other hand, the insoluble matter obtained by centrifugation was heated with water at 1000 m? was added, the pH was adjusted to 11.5 with a 10N-NaOH solution, the mixture was stirred for 1 hour to dissolve mixed crystals, and then centrifuged again to remove the bacterial cells. Then, the pH of this supernatant liquid was readjusted to 7.0 using 6N-HC1 solution, and a white precipitate was deposited. This precipitate was separated and recrystallized from ethanol to give 2)-4'-methyl-4'-(3,4-methylenedioxyphenylmethyl)-imidazolidine-2: s'-
6.71 of white crystals of dione were obtained.

NMII δppm (DMSO-ds):
1.5 (3H.

s, a-CBr4), 2.8 (2f(, q, /2-
CHt), 5.9 (2I-1,s, CH2), 6.
5-6.8 (3FI, m.

aromatic), 7.9 (IH, s, NH
), 10.3 (IH, s, N1-I) IRCm (KBr-disk): 3 34
0°3190, 3070. 2895. 1760°1710, 1505. 1490. +440°1
405, 1300. 1280. 1245(/Z'
l 25 = +63.6° (c = 0.5. 0.lN
-Na0H)mp, 22.6-229°C Example 5 A nutrient liquid medium with the following composition was prepared and dispensed in 100 ml portions into 50 (1+7 capacity shoulder shake flasks) with cotton plugs, and incubated at 120"C. Steam sterilization was performed for 20 minutes.

Medium composition: Meat extract 0.5% (w/v%) Yeast extract 0.5% Polypeptone 1.0% NaC 10.3% Uracil 01% Manganese chloride tetrahydrate 20 ppmp I-I 7
．． 0 Corynebacterium cepedonicum (Ooryne
bacterium sepedonicum ) I
A loopful of F 013259 was inoculated into the above nutrient liquid medium, and cultured at 33°C for 24 hours with shaking.

Using 200m/ml of this culture solution, the viable cells obtained by centrifugation were further washed with the same amount of 0.9% saline, centrifuged again to collect the bacteria, and used as a component of the reaction solution below. did.

Reaction solution composition: (D (R8)-N-carbamoyl-α-methylleucine 2.4Of was dissolved as a sodium salt (-pl
17.2), substrate solution (2) adjusted to a liquid volume of 40 tttl Live bacteria collected from H fertilizer culture solution 200 t.
0.09% saline solution and adjusted the liquid volume to 40yst. Mix (1) and (2) above, put into a 200ml four-bottle round bottom flask, and heat at 37°C without stirring. The reaction was carried out for 20 hours. During this reaction, the pH of the reaction system was continuously maintained at 7.2 using a 0.5 N-H0I solution. After the reaction, the reaction solution was centrifuged to remove bacterial cells. The pH of the obtained supernatant was readjusted to 7.0, and 100% of ethyl acetate was added.
Extraction was performed twice with M/, and ethyl acetate in the extract was distilled off under reduced pressure to obtain 10.42 y of white crystals of -4-methyl-4-isobutylimidazolidine-2,5-dione.

NM⇠ δppm (DMSO-de): 1.8
(6H.

t, CHs), 1.3 (5H, s, a-CH3),
1.4~L 8 (5H, m, CH2CH2), Otsu 9
(I H+s.

NH), 10.6 (IH, s, NH) R, cm
(K]3r-disk): 3200.

3050.2950,1760,1705°1570,
1430. 1370. 1280°1230, 1
200 (α)25=+0.8° (cm0.5, 0.I
N-NaOH)mp, 131-134°C After adjusting the pH of the aqueous layer extracted with ethyl acetate to 1.0,
Extraction was performed again twice with 100 rttl of ethyl acetate, and the ethyl acetate in the extract was distilled off under reduced pressure to yield (S)-N=carbamoyl-a-methylleucine 1.10! 7 was obtained as white crystals [[α')25=+8.0° (cm0.5
゜O,IN-NaOH)). Thus obtained (S)
-N-carbamoyl-α-methylleucine 1.001 was added to 25 ml of 2N-HC1 solution and cyclized by heating at 80°C for 2 hours, (S)-4-methyl-4-isobutylimidazolidine-2,5- Dione 0.851 was obtained as white crystals.

(α) 25 = o, so (c = 0.5, 0.
I N-Na0H) mp, 131-134°C Example 6 Nocardia corallina, which had been previously cultured on a bouillon agar slant at 30°C for 124 hours, was added to a nutrient liquid medium prepared in the same manner as in Example 5.
) Inoculated with one loopful of I FO3358 and incubated at 30°C for 2 hours.
Culture was performed under shaking for 4 hours.

Using 200 m/ml of this culture solution, the viable cells obtained by centrifugation were further washed with the same amount of 09% saline, and then centrifuged again to collect the bacteria and used as a component of the reaction solution described below.

Reaction solution composition: (1) Substrate solution in which 240 g of (几5)-N-carbamoyl-α-methylphenylglycine was dissolved as a sodium salt (pH 7.2) and the liquid volume was adjusted to 40 yttl. Mix (1) and (2) with the bacterial cell suspension suspended in 0.09% saline and adjusted to a volume of 40 ml, put into a 200 ml four-eye round bottom flask, and stir without stirring. The reaction was carried out for 20 hours at "C". During this reaction, the pH of the reaction system was continuously maintained at 7.2 using a 0.5 N-HG!1 solution. After the reaction,
After cooling the reaction solution and adjusting the pH to 0, it was separated by centrifugation into an insoluble mixture of crystals precipitated during the reaction and bacterial cells, and a supernatant. 200ml of water was added to the insoluble matter obtained by centrifugation, and the pH was adjusted to 11.0 with a 10 N-NaOH solution.
5 and stirred for 1 hour to dissolve mixed crystals,
The cells were removed by centrifugation again. Then 6N-HOI
When the pH of this supernatant liquid was readjusted to 0 using the solution, a white precipitate was deposited. This precipitate was separated and recrystallized from ethanol to obtain 1.031i' of white crystals of -4-methyl-4-phenylimidazolidine-2,5-dione.

NM 几 899m (DMS 0-de): 1.7
(3H.

s,a-C! H3), 7.3-7.6 (5H, m,
aromatic), 8, 611H,S, NH), 1
0.8 (IH,s, NH)IRcm (KBr-
disk): 3280°3210.1765.17
10.1495°1440.1400.13G5,12
50°(ff)25=-14to(c=0.5,
0. I N-NaOH) mp, 240-242'
C On the other hand, the pH of the centrifuged supernatant after the bacterial cell reaction was adjusted to 8N.
-HC! White when adjusted to 1.0 using l solution.

A precipitate was deposited. This precipitate was washed thoroughly with water and then dried under reduced pressure to obtain (S) -N-carbamoyl-α-methylphenylglycine t0711 [[α]25;
+52° (cm 0.5, 0.IN-NaOH)
). (S)-N-carbamoyl-α- thus obtained
Methylphenylglycine 0.80F 2N-H2SO4
The solution was suspended at a temperature of 2° and heated for 3.5 hours at 80°C for cyclization reaction with stirring. After cooling, the precipitated crystals were collected and recrystallized from ethanol to obtain 0.68 g of (S)-4-methyl-4-phenylimidazolidine-2,5-dione.

(/2'l parable = +139.5° (cm0.5, 0.
I N -NaOH)mp, 240-242°C Example 7 4-Carbamoylamino-6-fluoro-2-methylchroman-4-carboxylic acid ((几5) (2S, 4R) and (2 liters,
4S)] as the reaction substrate, Example 2
Perform the same operation as above, and use the ethyl acetate extract (28
,4R)-6-fluoro-2-methyl-spiro-(chroman-4,4'-imidazolidine)-2: s'-
Obtained 049g of white crystals of dione ((12)" knee 2
24° (cm1.0, EtOH), mp.

250-252°C)). In addition, the white precipitate obtained from the centrifuged supernatant of the bacterial cell reaction solution was treated in the same manner as in Example 3.
(2L 48)-N-carbamoylamino-6-fluoro-2-methylchroman-4-carboxylic acid was heated and cyclized to form (2R,48)-6-fluoro-2-methyl-spiro-[chroman-4. 4'-imidazolidine) -
q: 0,45f white crystals of 5'-dione were obtained [[
α)”=+2 2 5° (cm1.0.

EtOH), mp. 250 ~25
2 °C].

Reference Example 1 (S)-N-carbamoyl-α-methyl-3,4-methylenedioxyphenylalanine obtained in Example 4 5,0
g, crystalline barium hydroxide 25f and water 125yptl
were mixed and boiled under reflux for 60 hours. After the reaction is complete, add 1 more water
25 and neutralized to pI-It8 with dilute sulfuric acid. The resulting barium sulfate precipitate was separated from P, and the P solution was decolorized with hot decolorizing charcoal and concentrated under reduced pressure. 80% 20% hydrochloric acid and 4 g of phenol were added to the residue, and the mixture was refluxed for 19 hours with stirring. After the reaction, an oily substance was separated and removed from the reaction solution, and excess hydrochloric acid was distilled off under reduced pressure. After adding 10 ml of water to the obtained residue and dissolving it under heating, and decolorizing with decolorizing charcoal, the pH was adjusted to 60 with 5N ammonia water containing a small amount of sodium bisulfite, and the mixture was left in a refrigerator overnight. After washing the precipitated crystals with a small amount of cold water and drying them, (S)-α-methyl-3,4-dihydroxyphenylalanine 3/2
3.2y of hydrate was obtained.

[α]25--55° (C=2.0.N-HGI!l
) mp, 306-307°C (decomposition) [Effect] The present invention is directed to optically active hydantoin, (S)-, which has recently attracted attention as a preventive or therapeutic agent for specific chronic symptoms of diabetes (cataracts, neurological diseases, etc.). 6-fluoro-spiro-[chroman-4,4'-imidazolidine)-2s'
-dione (USAN; 5orbinil
)) and the optically active α-amino acid (S)-α-methyl-3,4-dihydroxyphenylalanine (L-methyldopa), which is widely used as an antihypertensive agent. Considering that the present invention can be applied to enzymes such as inexpensive microorganisms, it can be said that the present invention provides an extremely practical and effective method for producing these optically active compounds. Furthermore, the present invention
This new finding shows that N-carbamoyl-α-amino acids, which do not have a hydrogen atom on the α-carbon, can be biochemically converted into hydantoins through an enzymatic cyclization reaction, which was previously unanticipated. This is what we provide.

Claims (3)

[Claims] (1) General formula (I) ▲Mathematical formulas, chemical formulas, tables, etc.▼(I) [In the formula, R^1 and R^2 are each independently different alkyl groups, aralkyl groups, aryl groups, or substituents thereof. or R^1 and R^2 form one asymmetric cyclic compound. ]N-carbamoyl-
General formula (II) that corresponds to only one configuration in the racemic form of α-amino acid ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (II) [In the formula, R^1 and R^2 are the same as the above (I) same. * represents an asymmetric carbon. ] The enzyme of a microorganism belonging to the genus Aerobacter, Agrobacterium, Bacillus, Corynebacterium or Nocardia which has the ability to convert into the hydantoin represented by the general formula (II) is reacted with the enzyme to convert the optically active hydantoin represented by the general formula (II). After converting and separating unreacted optically active N-carbamoyl-α-amino acid from the generated hydantoin, the optically active N-carbamoyl-α-amino acid is heated and cyclized under acidic conditions to produce the corresponding optically active hydantoin. 1. A method for producing optically active hydantoins, which comprises producing optically active hydantoins. (2) R^1 and R^2 are ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [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) Optically active N-carbamoyl-α-amino acid,
The manufacturing method according to claim 1 or 2, wherein the asymmetric carbon of formula (I) is in the (S) form.