DE2621076A1 - PROCESS FOR THE PREPARATION OF D-CARBAMYLAMINO ACIDS AND THE CORRESPONDING D-AMINO ACIDS - Google Patents

Description

Dr. F. Zumstein Sr. - Dr. E. Assmann - Dr. R. Koenigsberger Dipl.-Phys. R. Holzbauer - Dipl.-Ing. F. Klingseisen - Dr. F. Zumstein jun.

PATENT LAWYERS

• 8 MUNICH 2,

BRÄUHAUSSTRASSE 4

TELEPHONE: COLLECTIVE NO. 225341 TELEGIRAMS: ZUMPAT TELEX 529979

Case 857
12/10 / bs

SJTAMPROGETTI S.ρ.Α., Milan / Italy

Addendum to the patent (Deutsche Patentanmeld. P 24 22 737.0-42)

The invention relates to the splitting or separation of optical Antipodes or enantiomers of amino acids by an enzymatic route to obtain the amino acid with the D configuration.

Some amino acids with the D configuration (such as phenylglycine, p-Hydroxyphenylglyein) have so far been of great importance as Obtained intermediates for the manufacture 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 expensive compound that can only be recovered with low yield and is difficult to access, so that the cost of the chemical process is very high.

Another method involves the steps of acylating the racemic amino acid, the selective hydrolysis of the D-form with the enzyme acylase and its isolation. However, the D-Acy "

609848/1023

read relatively rarely and always contaminated by L-acylase, so that during the hydrolysis process of the acylated derivative not negligible amounts of the L-antipode or L-enantiomer are present, whereby a product with poor optical purity is obtained.

From the German patent specification (German patent application P 24 22 737.0-42 or DOS 2 422 737) by the same applicant is a process for the preparation of 1-carbamylamino acids and the corresponding amino acids known by enzymatic means that start from substituted hydantoins, the latter being subjected to enzymatic hydrolysis in a pH range from ~ 6 to 11. the The reaction proceeds in the direction of the formation of laevoroti er end products, with the non-hydrolyzed form being recycled can be.

As the subject of the present invention has now been found that, if the hydantoin in question is substituted by radicals, the at least one aromatic group bonded to the asymmetric carbon atom, and the reaction is carried out within a narrower pH range, the reaction is both selectively and quantitatively with formation the D-shape runs alone.

Surprisingly, it was found that this was extracted from the liver of calves and classified as hydropyrimidine hydrolase Enzyme (E.C. 3.5.2.2.) On the racemic form of compounds the general formula

NH- CO

wherein X has the meaning of -OH, a hydrocarbon residue, a halogen, an alkoxyl group, -UO ₂ , a carboxyl group, acts with strictly selective hydrolysis of the D-form,

60 9 848/1023

The process "consists essentially in subjecting compounds of the above formula known as 5-substituted hydantoins" to enzymatic hydrolysis. The hydrolysis should be carried out under pH conditions in the range of 8 and 9, preferably at 8.5 , be performed. The temperature can be preferably maintained between 1O ⁰ C and 7O ⁰ C, at 30 ⁰ C. The hydrolysis proceeds according to the following scheme:

COOH
^ - CH-NH-CO-NH (D)

If the D-carbamyl derivative of If the pH is acidic, the pH should be kept at the original value by adding alkali as the reaction progresses will. It has been found that at the above pH values during the course of the hydrolysis reaction, one is the same early racemization of the remaining L-hydantoin occurs according to the following scheme:

Because of the continuous removal of the D-hydantoins by enzymatic ^ hydrolysis of the entire carbamyl derivative in the D-Porm. The rate of racemization of L-hydantoin is a puncture of both temperature and the pH value and is greater the higher the temperature and the more basic the pH value. But if you work for one pH close to 8.5, the speed is so great that it does not have a rate-limiting puncture

609648/1023

exercising the reaction.

It has also been found that the D-carbamyl derivatives dissolved in Water and heated to boiling under "certain conditions, can be decomposed according to the following equation:

COOH 2 "£> -CH COOH + NH, + CO.

NH-CO-NH ₀

thereby obtaining the optically active amino acids which are easily isolated with great chemical and optical purity can. Furthermore, an additional technical and economic improvement of the present invention can finally be made can be achieved if you turn the enzymes into fibrous Structures as described in Italian patent specification 836 462 (or German patent specification 1 932 426), includes. In this way, the enzyme can be used repeatedly instead of a single use, thereby reducing the Operation cost can be reduced. In addition, the reaction proceeds in a cleaner way since it is not enzymatic Proteins remain in the reaction mixture.

The following examples serve to illustrate the invention without restricting it.

example 1

176 g (1 mole) of D, L-5-phenylhydantoin were dissolved in 7.5 1 of a 0.1 M potassium phosphate buffer solution of pH 8.5 at 30 ⁰ C

dissolved.

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

Were iTach about 6 hours to maintain a constant to adjust the pH value of the system, 250 ml of 4 m-UaOH added, the reaction was stopped and the reaction mixture at 4 ⁰ C cooled and the pH

609848/1023

- 5 was through. Addition of 6 n-HCl adjusted to 5.5.

A slimy precipitate was obtained during this operation of proteins removed by centrifugation.

The supernatant liquid, cooled again to 4 ^° C., was brought to pH 2.5 with 6N HCl. A crystalline product precipitated out and was washed with about 1 liter of cold water and dried to constant weight. The product was crystallized from ethanol-water, 70/30 (vol / yol) with heating.

The crystals (189 g, yield 97 $) were chromatographic homogeneous and proved to be IT-carbamylphenylglycine on the basis of the IR, NMR and mass spectrum as well as the elemental analysis.

The specific optical rotation is [a] ^ ⁵ = 137 ° (C = 1 $ in ΝΗ, ΟΗ, Ιη), as stated in the literature: T. Suzuki, K. Igarashi, E. Hase, Agr. Biol. Chem. 37 (2) 411-416 (1973) for D (-) F-carbamyl-phenylglycine.

Example 2

A reactor, equipped with a thermostatic jacket, with an inlet for nitrogen, an electrode for reading the pH value and an inlet for adding soda, was charged with 10 1 0.1 M phosphate buffer solution, the hydropyrimidine hydrolase from liver (Total activity: 7500 micromoles per minute; total proteins: 11 g). After bubbling nitrogen for about 30 minutes, held at 30 ⁰ C solution of 192 g (1 mol) was D, L-p-hydroxyphenyl hydantoin added 5-!, Wherein the pH value by continuous addition of 4 N NaOH was kept constant. After 20 hours, when the consumption of soda was 250 ml, D (-) - U-carbamyl-hydroxyphenylglycine was isolated in the same manner as in Example 1.

A crude product was obtained which crystallizes from ethanol / n-hexane became. In this way, 152 g (yield $ 72) of a product with the following properties were obtained:

609848/1023

Melting point: 17O ⁰ C; [α] 5 ° = -170 ° (C = 0.5 $ in water / alcohol,

■ "50/50 vol / vol).

The structure of the product was confirmed by the results the elemental analysis, the IR, ITME and mass spectrum im Comparison with data from the literature J. Eagles, W.M. Laird, E.C. Matais, Biochemical Journal, 121, 425-4-30 (1971) Sup. Publ. Fo. STJP 50003, Annex 1, arm ex 2.

Example 3

206 g (1 mole) of D, L-5-p-methoxyphenyl-hydantoin were suspended in 10 1 0.1 M-potassium phosphate buffer of pH 8.5 at 30 ⁰ C, the Hydropyrimidin hydrolase from liver (total activity: 7500 Micromoles per min .; total proteins: 11 g).

The pH was determined by the continuous addition of 4 m-KaOH kept constant.

After 20 hours, when 250 ml of soda has been consumed, the reaction was interrupted and D (-) - IT-carbamyl-p-methoxyph'fenylglycine was obtained as described in Example 1 with a yield of $ 90. Confirmed the structure of the product through elemental analysis, IR, NMR and mass spectrum.

Example 4

230 ml of a solution of hydropyrimidine hydrolase from liver with an activity of 10800 micromoles per minute and 3.45 g Proteins, were added to 2.1 kg of a solution of 150 g of cellulose triacetate in methylene chloride, which was kept under vigorous stirring was joined. The suspension thus obtained was made according to that described in Italian patent 836,462 Process spun.

The thus obtained paser (300 g) was in the form of a single Strands placed in a jacketed glass column (8 cm diameter, 60 cm height), which at both ends with 2 brackets made of stainless Steel was secured. The fiber underwent two washes by recycling 4 liters of 0.1 M aluminum phosphate buffer

609848/1023

washed from pH 8.5 until the enzymatic activity in the solution disappeared. (4 washes, 6 hours in total).

The column containing the fiber was then placed in a circuit which had a peristaltic pump for recycling the reaction mixture, a jacketed beaker with an electrode for continuous control of the pH and an inlet for the addition of 4 m-HaOH, operated by a pH- Staten, exhibited. 5 liters of 0.02 m potassium phosphate buffer with a pH of 8.5 and 176 g (1 mol) of D, L-5-phenylhydantoin were introduced into the system, while the pump was used to recycle the reaction mixture and the pH stat for continuous addition were set in motion by soda. After 6 hours, when 250 ml of 4 m-HaOH had been consumed, the reaction was interrupted. The reaction mixture was taken out. The fibers were washed with 4 liters of water. The reaction mixture and washings of Paser were concentrated in vacuo to about 3 1, cooled to ⁰ C and 2 to 6-n HCl to pH 2.5 was added. After digesting for 30 minutes, the precipitate was collected on a filter and dried to constant weight.

The product thus obtained (180 g, 93 $ yield) turned out to be D - (-) - K-carbamylgienylglycine Vom P. = 200 ⁰ C; [a] | ° = -135 ° (C = 1 $ in 1 Xi-HH ₄ OH).

Example 5

The same system as in Example 4 was used to obtain D - (-) - N-carbamyl-p-hydroxyphenylglycine from D, L-5-p-hydroxyphenylhydantoin, except that the jacketed beaker was closed and a nitrogen inlet was added. From 192 g (1 mol) of D, L-5-p-hydroxyphenylhydantoin, after 60 hours of reaction, as described in the preceding examples, 160 g (yield 76 $) of D - (-) - IT-carbamyl-para hydroxyphenylglycine from P. = 200 ⁰ C; [oc] | ° = -175 ° (C = 0.5 $ in alcohol / water 50/50 vol / vol.)

Example 6

Using the same system as in Example 4, 206 g (1 mole) of D, L-5-p-methoxyphenylhydantoin gave 200 g

609848/1023

_ 8 ($ 89 yield) D - ^ - IT-carbamyl-para-methoxyphenylglycine.

Example 7

194 g (1 mole) of D-t-J-li-carbamylphenylglycine, as in Example 4

obtained were slurried in 5 liters of water. The suspension was adjusted to pH 4 with saturated NapCO ^ solution.

The suspension was kept boiling for 8 hours, the pH, which rose, was occasionally adjusted to 5 to 4 ^{by adding Amberlite IR 120 (H +).} (Total addition: 400 ml resin). The hot solution was then stripped from the resin, concentrated to 1 liter in vacuo, cooled and brought to pH 2 with 6N HCl. The obtained crystals were collected on a filter, washed with water and dried in vacuo to constant weight.

88 g (0.045 mol) of D - (-) - 1-carbamylphenylglycine} [a] 2 ° = -136.9 ° were obtained in this way.

From the crystallization liquor of this product, by adjusting the pH to 6 and concentrating in vacuo, 70 g (0.46 mol) of D - (-) - phenylglycine; [cc] ^ ⁰ = -154 °; (0 = 1 $ in 1 n-HCl.)

The structure of the product was confirmed by 4 ¹¹¹ C ¹¹ elemental analysis, IR, HMR and mass spectrum.

Example 8

In a similar manner as in Example 7, but £ ääte.r carrying out the reaction under a Stickstoffatmosphä ^ e _s there was obtained from 210 g (1 mole) of D - (-) - lT-Garbamyl-para-hydru ^ yphtenylglycin after 9 hours Boiling 67 g (40% yield) D - (-) - p-Hydroxyphenylglycine [a] ^ ° = -154 ° (C = 0.5 $ in 1N HCl).

The non-hydrolysed D - (-) - H "-carbamyl-p-hydroxyphenylglycine found to be 100 g ($ 47 yield); [a] £ ° = -175 ° (C = 0.5 $

6 0 9 8 4 8/1023

in ethanol / water, 50/50 vol / vol).

Example 9

Tor was carried out as in Example 7, starting from 224 g (1 mol) D - (-) - N-carbamyl - p-methoxyplienylglycine, as obtained in Example 3, and obtained 80 g (yield 44%) of D - (-) - p-methoxyphenylglyoin.

609848/1023

Claims (3)

Claims Process for the preparation of D-carbamylamino acids and the corresponding D-amino acids, characterized in that compounds of the formula CO where X is the meaning of -OH, hydrocarbon radicals, halogen, Has alkoxy, -HOp, cartoxy residues at a pH of 8 to subjected to enzymatic hydrolysis and then hydrolyzed the resulting carbamylamino acid with boiling. 2. The method according to claim 1, characterized in that the enzymatic hydrolysis of the reaction at a temperature of 10 to 70 ⁰ C is carried out. 3. The method according to any one of the preceding claims, characterized in that the enzyme hydropyrimidine hydrolase starts from the liver of calves. 609848/1023