CS209871B2 - Method of preparation of the d-ureido acids and respective d-amino acids - Google Patents

Abstract

1506067 Preparation of D-carbamoylamino acids; the corresponding D-amino acids SNAMPROGETTI SpA 11 May 1976 [12 May 1975] 19444/76 Addition to 1452591 Heading C2C The invention comprises a process for producing a D-carbamoyl amino acid of Formula I wherein X is H, halogen, OH, NO 2 , alkoxy, carboxy or a hydrocarbon radical by enzymatic hydrolysis of a racemic hydantoin of Formula II in the presence of, as enzyme, hydropyrimidine hydrolase obtained from calf liver at a pH in the range 8 to 9, and optionally hydrolysing the product by boiling in water to obtain the free amino acid of Formula III

Description

(M) A method for preparing D-ureido acids and corresponding D-amino acids

The invention relates to a process for the preparation of D-ureido acids and corresponding D-amino acids by enzymatic hydrolysis of substituted hydantoins.

Some amino acids having the D-configuration (eg, phenylglycine, p-hydroxyphenylglycine) have recently become of considerable importance as intermediates for the preparation of large-scale compounds used in industry.

The chemical processes used hitherto for the separation of optical antipodes or enantiomers have utilized sulfcamphoric acids.

Sulfocaproic acid is a costly substance, can only be obtained in low yield and is not readily available, so the costs associated with the chemical process are very high.

Another method consists in first acylating the racemic amino acid, then in the second step selectively hydrolyzing the D-form acylase enzyme and finally isolating it. However, D-acylases are relatively rare and always contaminated with L-acylase, so that during the hydrolysis process of the acylated derivative, non-negligible amounts of L-antipode or L-enantiomer are formed, resulting in a product having low optical purity.

From the description of the Czechoslovak patent no.

AOP 222 177 discloses a process for preparing L-ureido-amino acids and the corresponding amino acids by an enzymatic route starting from substituted hydantoins, which are subjected to enzymatic hydrolysis in a pH range of from 6 to 11. The reaction is developed towards the formation of final levorotation compounds non-hydrolyzed forms.

It has now been found that when a particular hydantoin is substituted with residues that contain at least one aromatic group attached to an asymmetric carbon atom and the reaction is carried out within a narrower pH range, the reaction proceeds selectively and quantitatively to form the D-form itself.

The process for the preparation of the D-ureido acids and the corresponding D-amino acids according to the invention is characterized in that the compounds of the formula

CO-NH

NH-CO

where?

X - represents a hydrogen atom, an OH group or OCH3, undergoes enzymatic hydrolysis using a hydropyrimidine hydrolase from calves of the liver, whereupon - the ureido acids obtained are optionally hydrolyzed by boiling.

The action of the hydropyrimidine hydrolase (EC 3522) from the liver of calves on the racemic form of the 5-substituted hydantoins of the general formula results in a strictly selective hydrolysis of the D-form. The hydrolysis is preferably carried out at pH 8.5, the temperature can be maintained between 10 and 70 ° C, preferably at 30 degrees Celsius.

The hydrolysis proceeds according to the scheme

COOH l (D)

CH-NH-CO-NH4 + / CO-NH

CH 1 (L) 4 NH-CO

Since the D-ureido derivative formed during the hydrolysis is acidic, it is necessary to maintain the pH at the initial value by adding alkali as the reaction proceeds. It has been found that at the above-mentioned pH values hydrolysis proceeds and at the same time the remaining L-hydantoin is racemized according to the scheme

CH-CO <

NH-CO

NH

CH-CO NH-CO) nh '(Dp

For this reason, the continuous removal of D-hydantoin produced by the enzymatic reaction results in the final yield of the reaction being only the ureido derivative in the optical - D-form. The rate of racemization of L-hydantoin is a function of both temperature and pH, and the higher the higher the temperature and the more basic the pH. However, at a pH close to 8.5, the rate is so high that it is not a rate determining reaction rate.

It has also been found that the D-ureido derivative decomposes according to the equation when dissolved in water and heated to boiling under certain conditions.

CH — COOH NH-CO-NH,

HzO

CH-COOH + NH + CO 3 *

NH ₂ thus gives rise to an optically active acid which - can be isolated - in the form of - very high chemical and optical purity. In addition, a further technical and economic improvement of the present invention can be achieved by encapsulating the enzyme into fibrous structures as set forth in Italian Patent No. 836,462. In this way, the enzyme can be reused instead of once, thereby reducing labor costs . In addition, the reaction proceeds in a purer manner since the enzyme proteins do not remain in the reaction mixture.

The following examples are intended to better illustrate the invention, but do not limit it in any way.

Example 1

176 g (1 mol) of D, L-5-enylhydantoin are stirred in 7.5 liters of a 0.1 M potassium phosphate buffer solution at pH 8.5 at 30 ° C.

To the stirred suspension was added 125 ml of a hydropyrimidine hydrolase solution from the liver (total activity 1875 micromoles per minute at 30 ° C, pH 8.5; total protein 2.75 g).

After about 6 hours, when 250 mL of 4 M NaOH was added to maintain the system at a constant pH, the reaction was discontinued and the reaction mixture was cooled to 4 ° C and the pH was adjusted to 5.5 with 6 N HCl. .

During this operation mucilaginous was obtained

With a precipitate of denatured protein that was centrifuged.

The supernatant recooled to 4 ° C was adjusted by adding 6 N. HCl to pH 2.5. During this operation, a crystalline product precipitates, which is washed with approximately one liter of cold water and dried to constant masses. The product was crystallized from a hot 70/30 v / v ethanol / water mixture.

The crystals (189 grams, 97% yield) are chromatographically homogeneous and are N-carbamoyl-phenylglycine based on IC and NMR and mass spectra and elemental analyzes.

Specific optical rotation (.alpha.) _D @ 25 = 137 DEG (C = 1% in 1N NH.sub.OH) corresponding to literature data: T. Suzuki, K. Igarashi. K. Hase, Agr. Biol. Chem. 37 (2) 411-416 (1973) for D (-). N-carbamyl-phenylglycine.

Example 2

A 10 liter 0.1 M phosphate buffer solution containing hydropyrimidine hydrolase from the liver (total activity: 7500 micromoles per minute; total protein: 11) was placed in a reactor equipped with a thermostatic jacket, nitrogen introduction, pH reading electrode and soda addition port. grams). After nitrogen bubbling for approximately 30 minutes, the solution maintained at 30 ° C was supplemented with 192 grams (1 mole) of D 6 -S-diethyloxyphenylhydantoin while maintaining a constant pH by continuous addition of 4 M NaOH. After 20 hours, when soda consumption was 250 ml, D- (-) - N -carbamoyl-p-hydroxyphenylglycine was isolated in a manner similar to that described in Example 1.

The crude product obtained was crystallized from ethanol / hexane. In this way, 152 g (72% yield) of a product having the following properties were obtained:

Melting point: 170 ° C; (alpha) D ² = -70 ° (C = 0.5 ·% in water / alcohol 50/50 v / v).

The structure of the product was confirmed by elemental analysis, IC, NMR, and mass spectra as compared to literature data in J. Eagles, W. M. Laird, E. C. Matals, Biochemical Journal 121, 425-430 (1971) Sup. Publ. No. SUP 50003, Annex 1, Annex 2

Example 3

206 grams (1 mol) of D, L-5-p-methoxyphenylhydantoin was stirred in 10 liters of 0.1 M potassium phosphate buffer solution at pH 8.5 containing hydropyrimidine hydrolase from the liver (total activity 7500 micromoles per minute; total protein 11 grams) ) at 30 ° C.

The pH was kept constant by the continuous addition of 4 M NaOH.

After 20 hours, when 250 ml of soda was consumed, the reaction was complete and D (-) - N-carbamoyl-p-methoxyphenylglycine was isolated as described in Example 1 in 90% yield. The structure of the product was verified by elemental analysis and IC, NMR and mass spectra.

Example 4

230 ml of a hydropyrimidine hydrolase solution of liver, having an activity of 10,800 micromoles per minute and having 3.45 grams of protein, was added to 2.1 kg of a solution of 150 grams of triacetylcellulose in methylene chloride while maintaining vigorous stirring. The emulsion thus obtained was spun according to the process described in Italian Patent No. 836,462.

The fiber thus obtained (300 g) was inserted in the form of a single skein into a glass column (8 centimeters diameter and 60 centimeters high) by gripping at both ends with two stainless steel stirrups. The fiber was washed by recycling 4 liters of 0.1 M potassium phosphate buffer solution at pH

Enzymatic activity in solution (4 washes, 6 hours total).

The fiber-loaded column was then connected to a pump circuit to recycle the reaction mixture, with the jacket beaker having an electrode for continuous measurement of pH and a pH-stat controlled 4 M NaOH addition port. 5 liters of 0.02 M potassium phosphate buffer solution d pH 8.5 and 176 grams [1 mol] of D, L-phenylhydantDin were added to the apparatus while the circulation pump was started. . After 6 hours at 4 ml NaOH 250 ml, the reaction was discontinued. The fiber was washed with 4 liters of water. The reaction mixture and the washings from the fiber were concentrated in vacuo to approximately 3 L, cooled to 2 ° C and treated with 6 N HCl to pH 2.5. After maturation for 30 minutes, the precipitate was filtered off and dried to constant weight.

The product thus obtained was 180 grams [93% yield] and was found to be d - (-) - N-carbamyl-phenyl-glycine (mp 200 ° C; (alpha) D ²⁰ = -135 (C)). = 1% in 1 N NH 4 OH).

Example 1 a d · 5

To obtain D - (-) - N-carbamoyl-p-hydroxy-phenylglycine from D, L-5-para-hydroxy-phenylphenylhydantoin, the same apparatus as in Example 4 was used except that the beaker was The jacket was sealed and additionally provided with nitrogen inlet. 160 g (76% yield) of D- (-) - N -carbamoyl-p- was obtained from a reaction of 192 g (1 mole) of D, L-p-hydroxyphenylhydanidine after 60 hours of reaction as described in the previous examples. DrLydropyloxyphenylglycine [mp 2 <00 ° C; [.alpha.] D @ ²⁰ = 175 DEG (C.dbd. = 0.5% in alcohol / water 50/50 Dbj.)].

Ί

Example о

Using the apparatus described in Example 4, 200 grams (89% yield) of D - (-) - N-carbamoyl-p-methoxyphenylglycine were obtained from 208 grams (1 mol) of D, L-5-p-methoxyphenylhydantoin.

Example 7

194 grams (1 mol) of D - (-) - N-carbamoylphenylglycine obtained as described in Example 4 were slurried in 5 liters of water. The suspension was adjusted to pH 4 by adding saturated solution of Magnesium.

The suspension was heated to boiling for 8 hours while adjusting the pH, which occasionally rose from 5 to 4 by the addition of Amberlite IR 120 (H ⁺ ). A total of 400 ml resin was added.

The resin was separated from the hot solution, evaporated under reduced pressure to a volume of 1 liter, then cooled and 6N HCl was added to pH 2. The obtained crystals were filtered, washed with water and dried under reduced pressure to constant weight.

88 g (0.045 mol) of D- (-) - N-carbamoylphenylglycine are thus obtained.

[.alpha.] D @ ²⁰ = -136.9 DEG. 70 g (0.46 mol) of D - (-) - phenylglycine o (alpha ²⁰ = -154 ° C = 1% v) was obtained from the mother liquors of this product after adjusting the pH to 6 and evaporating under reduced pressure. 1 N HCl).

The structure of the compound was confirmed by elemental analysis, IC, NMR and mass spectra.

Example 8

In a similar manner to that described in Example 7, however, by carrying out the reaction under a nitrogen atmosphere, 210 g (1 mol) of D - (-) - N-carbamoyl-p-hydroxyphenylglycine were obtained after boiling for 9 hours 67% (40% yield). ? - (-) - p-hydroxyphenylglycine? (Α D ²⁰ = -154 ° (C = 0.5% in 1 N HCl)).

The unhydrolyzed D- (-) - N -carbamoyl-p-hydroxyphenylglycine was 100 grams (47% yield) .alpha. (.Alpha.) _D @ ² = -175 DEG (C = 0.5% in ethanol / water 50/50 vol.) .).

Example 9

As described in Example 7, out of 224 grams (1 mol) of D - (-) - N-carbamoyl-p-methoxyphenylglycine prepared according to the procedure outlined in Example 8 yielded 80 graim (44% yield) of D - (-) - p -methoxyphenylglycine.

Claims (2)

SUBJECT OF THE INVENTION A process for the preparation of D-ureido acids and corresponding D-amino acids, characterized in that the compounds are of the general formula CO-NH NH-CO wherein X represents a hydrogen atom, an OH group or an OS, undergoes enzymatic hydrolysis at pH 8-9 using hydropyrimidine hydrolase from calves, whereupon the ureido acids obtained are optionally hydrolyzed by boiling. 2. The method according to claim 1, wherein the enzymatic hydrolysis is carried out at a temperature of 10 to 70 [deg.] C.