DE2621076B2 - Process for the production of D-amino acids - Google Patents

Description

a) the racemate of a 5-substituted hydantoin of the general formula

CH

NH C == O

• N-FI

hydrolyzed on the hydropyrimidine hydrolast,

b) separating the selectively obtained D-form of the corresponding carbamoylamino acid from the hydrolysis product and

c) hydrolyzed in boiling water at a pH of 4 to 5 to D - (-) - phenylglycine, D - (- H-hydroxyphenylglycine or D- (-) -4-methoxyphenylglycine.

Some D-configuration amino acids (such as phenylglycine, p-hydroxyphenylglycine) have a large one Obtained importance as intermediates for the preparation of compounds widely used in industry.

The chemical methods that have hitherto been used to separate the optical antipodes or enantiomers are based on the use of Camphor sulfonic acid. This is an extremely costly connection that only uses low yield can be recovered and in poorly accessible, so that the cost of chemical process are very high.

Another method involves the steps of acylation of the racemic amino acid, the selective Hydrolysis of the D-form with the enzyme acylase and its isolation. However, the D-acylases are relative seldom and always contaminated by L-acylase, so that during the hydrolysis process of the acylated Derivative not to be neglected amounts of the L-antipode or L-enantiomer are present, whereby a product with poor optical purity is obtained.

DE-OS 24 22 737 discloses a process for the production of L-carbamoylamino acids and the corresponding amino acids by enzymatic means 2> known that starts from substituted hydantoins, the latter being an enzymatic hydrolysis in be subjected to a pH range of 6 to 11. The reaction proceeds in the direction of formation of laevorotierend end products, whereby the not »ο hydrolyzed form is returned to the process can be.

Surprisingly, it has now been found that, if the hydantoin in question has certain residues is substituted and the reaction is carried out within a certain pH range, the Reaction proceeds both selectively and quantitatively with formation of the D-form alone.

The invention therefore relates to the subject matter described in the claim.

The hydropyrimidine hydrolase extracted from calf liver is called an enzyme (E.C. 33.2.2.).

In the process according to the invention, the hydantoin hydrolysis is preferably carried out at pH 8.5. The hydrolysis temperature is preferably maintained at 3O ⁰ C. The hydrolysis proceeds according to the following scheme:

(D)

CO -NH

NH-CO

CH-NU —CO —NH,

CO -NH

NH-CO

(L)

If the D-carbamoyl derivative formed during hydrolysis is acidic in nature, the pH should be kept at the original value by adding alkali as the reaction progresses. It has it has been shown that at the above pH values during the course of the hydrolysis cactite a simultaneous racemization of the remaining L-hydantoin occurs.

Because of the continuous removal of the D-hydantoin by enzymatic hydrolysis, the entire carbamoyl derivative is therefore obtained in the D-form. the Racemization speed of L-hydantoin is

a function of both temperature and pH and is greater the higher the temperature and the more basic the pH is. However, if you work at a pH value in the vicinity of 8.5, the Speed so great that it does not

H ₂ O CH-COOH

NH-CO-NH ₂

thereby obtaining the optically active amino acid that can be easily isolated with great chemical and optical purity. It is technically and economically particularly favorable, the enzyme enclosed in fibrous structures, as described in DE-PS 19 32 426, to use. This way the enzyme can be repeated instead of a single use can be used, thereby reducing the cost of the operation. In addition, the React in a clean manner as no enzymatic proteins remain in the reaction mixture.

The following examples serve to illustrate the invention.

A) Preparation of the D-carbainoyl amino acids example 1

176 g (1 mol) of DL-5-phenylhydantoin were dissolved in 7.5 l of a 0.1 m-Kalhnnphosphat buffer solution of pH 8.5 at 30 ⁰ C

The stirred suspension was treated with 125 ml of a solution of Hydropyrimidin-HydroU e calf liver added (total activity 1875 micromoles per minute at 30 ⁰ C, pH 8.5; total proteins 2.75 g).

After about 6 hours, in order to keep the pH of the system constant, 250 ml of 4 M NaOH were added, the reaction was terminated and the reaction mixture was cooled to 4 ° C. and the pH was raised adjusted to 5.5 by adding 6 n-HCl.

During this operation a slimy precipitate of proteins was obtained by centrifugation was removed. The supernatant liquid, cooled again to 4 ° C., was added to the with 6N HCl brought pH 2.5. A crystalline product precipitated out, which was washed with about 11% cold water and found constant weight was dried. The product was made from ethanol-water, 70/30 (v / v) under Heating crystallizes.

The crystals (189 g, 97% yield) were chromatographic homogeneous and proved on the basis of the IR, NMR and mass spectrum as well as elemental analysis as N-carbamoylphenylglycine.

The specific optical rotation is [often -137 ' (C- 1% in NH ₄ OH, ln>, as stated in the literature: T. Suzuki. K. Igarashi, K. Hase, Agr. Biol. Chem. 37 (2 \ 411-416 (1973) for D (-) N -carbamoylphenylglycine.

Example 2

A reactor equipped with a thermostat see controlled jacket, with an inlet for nitrogen, an electrode for pH reading and an inlet for adding soda was added to 101 0.1 M phosphate buffer solution charged, the hydropyrimidine HydroJatc from calf liver (total activity: 7500 micromoles per minute; total proteins: 11 g) Exercises a determinant function on the reaction. The D-carbamoyl derivatives are under the im Claim for process stage c) decomposed conditions according to the following equation:

COOH + NH ₃ + CO ₃

After nitrogen had been blown in for about 30 minutes, the ^{solution kept at 30 °} C. was in? ' 192 g (1 mol) of D, L-5-p-hydroxyphenylhydantoin were added, the pH being kept constant by continuously adding 4N NaOH. After 20 hours, when the consumption of soda was 250 ml, D (-) -N-carbamoyl-p-hydroxyphenylglycine was isolated in the same manner as in Example 1

μ A crude product was obtained which was made from ethanol / η-hexane was crystallized. In this way, 152 g (72% yield) of a product containing the following properties:

Melting point: 170 ⁰ C; [α]? -170 ° (C = 0.5% in

Water / alcohol, 50/50 vol / vol) l

The structure of the product was confirmed by the Results of the elemental analysis, the IR, NMR and mass spectrum in comparison with the data from Literature J. Eagles, W. M. Laird, EC Matais, Bioche-

w mical Journal, 121, 425-430 (1971) Sup. Publ. No. SUP 50 003. Annex I. Annex 2.

Example 3

206 g (1 mole) of D, L-5-p-methoxyphenyl-hydantoin r> were slurried in 101 0.1 M-potassium phosphate buffer of pH 83 at 30 ⁰ C, the Hydropyrimidin hydrolase from liver (total activity: 7500 micromoles per min .; total proteins: 11 g) contains.

The pH was determined by continuous addition of 4 m w NaOH kept constant.

After 20 hours, when 250 ml of soda had been consumed, the reaction was interrupted and D (- J-N-carbamoyl-p-methoxy-phenylglycine was as described in Example 1, obtained with a yield of 41 90%. Confirmed the structure of the product through elemental analysis, IR, NMR and mass spectrum.

Example 4

ν * 230 ml of a solution of hydropyrimidine hydrolase from liver with an activity of 10800 micromoles per Minute and 3.45 g of proteins were added to 2.1 kg of a solution of 150 g of cellulose triacetate in methylene chloride, which was kept with vigorous stirring, added. the

The suspension obtained in this way was spun according to the process described in Italian patent 8 36 462.

The fiber thus obtained (300 g) was in the form of a single strand in a jacketed glass column (H rm

Wi diameter, 60cm height). du ·; tti bulge ends with 2 brackets made of rose-free Suhl ycMihrn. The fiber was / white Wiisi-h \ i>i>'iti |.! S "luivli Recyclisiercn washed 4 1 0.1 iii K.iliiiniphnspli.iipiilfa \ ow pH 8.5 until the s / vniiiiisvlu · Akn" iut me:

H'. We waste the solution (4 W.iv lnni> :: in>: r - >>: .- together 6 hours).

The Siiiilr wniilr .im «., Lili .-. I, - ··, ■ revealing the fiber pebriiihi. »Irr ι hu- | ί · ι m.iIi n. '·. ■

Pump to recycle the reaction mixture, a jacketed beaker with an electrode for continuous PH control and an inlet for the addition of 4M NaOH operated by a pH states. In the system were 51 0.02 M potassium phosphate buffers pH 8.5 and 176 g (1 mol) of D, L-5-phenylhydantoin are introduced while the pump is used to recycle the reaction mixture and the pH stat for continuous addition were set in motion by soda. After 6 hours with consumption of 250 ml of 4 M NaOH, the Reaction interrupted. The reaction mixture was taken out. The paint was washed with 41% water. The reaction mixture and the fiber washes were concentrated in vacuo to about 3 L, cooled to 2 ° C and mixed with 6 N HCl up to pH 2.5. After digesting for 30 minutes, the Precipitate collected on a filter and dried to constant weight

The product thus obtained (180 g, 93% yield)> o turned out to be D- (-) -N-carbamoyl-phepylglycine with a melting point of 200 ° C; [a] i! - 135 ° (C = 1% in 1 n-NH ₄ ÜH).

Example5

The same system as in Example 4 was used to obtain D - (-) - N-CarbamoyI-p-hydroxyphenylglycir, from DL-5-p-hydroxyphenylhydantoin, except that the jacketed beaker was closed and a nitrogen inlet was added. From 192 g (1 mol) of DL-5-p-hydroxyphenylhydantoin, after 60 hours of reaction, as described in the preceding examples, 160 g (yield 76%) of D- (- JN-carbamoyl-para-hydroxy) are obtained -phenylglycine with a temperature of 200 ° C: [*]? -175 ° (C = 0.5% _in alcohol / water 50/50 V0I / V0L).

Example 6

Using the same system as in Example 4, 206 g (1 mole) of D, L-5-p-methoxypiienylhydantoin were obtained 200 g (89% yield) D- (- J-N-Carbamoyi-para-methoxyphenylglycine.

B) Preparation of the D-amino acid
Example 7

194 g (1 mole) of D- (-) -N-carbamoylphenylglycine, such as obtained in Example 4 were slurried in 51 water. The suspension was saturated with; Na2COr solution adjusted to pH 4.

The suspension was kept boiling for 8 hours, the pH, which rose, occasionally being ^{adjusted to 5 to 4 by adding ion exchange resin (H +} ) (total addition: 400 ml of resin). The hot solution was then stripped from the resin, concentrated to 11 in vacuo, cooled and brought to pH 2 with 6N HCl. The crystals obtained were collected on a filter, washed with water and dried to constant weight in vacuo.

88 g (0.45 mol) of D - (-) - N-carbamoylphenylgycin were obtained in this way; [a] if -1363 °.

From the crystallization liquor of this product, by adjusting the pH to 6 and concentrating in vacuo, 70 g (0.46 mol) of D - (-) - phenylglycin; [α]? -154 °; (C = 1% in 1N HCl).

The structure of the product was determined by elemental analysis each, IR, NMR and mass spectrum confirmed.

Example 8

In a manner similar to Example 7, but carrying out the reaction under a nitrogen atmosphere, from 210 g (1 mol) of D- (-) - N-carbamoyl-p-hydroxyphenylglycine, after boiling for 9 hours, 67 g (40% yield ) D - (-) - p-Hydroxyphenylglycine [*] i "-154 ° (C = 03% in 1N HCl).

The unhydrolyzed D - (-) - N-carbamoyl-p-hydroxyphenylglycine found to be 100 g (47% yield); [«]? - 175 ° (C = 0.5% in ethanol / water, 50/50 Vol / vol).

Example 9

The procedure was as in Example 7, starting from 224 g (1 mol) of D- (- J-N-carbamoyl-p-methoxyphenylglycine, as in Example 3 obtained and obtained 80 g (yield 44%) of D- (-) -p-MethoxyphenyIglycia

Claims (1)

Claim: Process for the preparation of the D-amino acids of the general formula CH- COOH NH, in which X is hydrogen, hydroxy or methoxy, using the enzymatic hydantoin hydrolysis by means of hydropyrimidine hydrolase extracted from calf liver in an aqueous medium at a pH of 8 to 9 and a temperature of 10 to 70 ⁰ C, characterized in that