CS228431B1 - Process for separating of optic isomers of phenylalaninederivates being substituted in p-position - Google Patents

Description

The invention relates to a process for resolving the optical isomers of phenylalanine derivatives substituted in the β-position. These phenylalanine derivatives are part of synthetic peptides, which can serve, for example, as selectively natriuretically acting substances and must be prepared in optically uniform form for this purpose.

It has previously been found that penicillin amide hydrolase (EC 3.5.1.11) can be used to cleave the acyl group from the L-form of the amino acid (Lucente 6, Romeo A., Rosel S .: Experientie 2i, 317 (1965); British Patent 1 369 462, Czechoslovakia 209 633 and Vojtíšek V., Slezák J .: Folia Microbiologica 20, 224 (1975), Simek P. et al .: Collect. Furthermore, it has been found that the specificity of the enzyme is not absolutely absolute and that the D-form of the amino acid is also released from the acylated derivative, but for the β-substituted phenylalanine derivatives this enzymatic cleavage method has not yet been verified.

The present invention provides a method for the enzymatic resolution of the optical isomers of phenylalanine derivatives substituted in the β-position of formula (I),

XCH ² -CH-COOH ² I

NH '(I), wherein X is C1 -C4 alkyl or halogen, wherein the D, LN-phenylacetyl phenylalanine derivative, in particular D, LN-phenylacetyl-p-ethylphenylalanine, is incubated with enzymes having penicillin amide hydrolase activity, preferably and their insoluble forms, in a batchwise or continuous manner, at a temperature range of 15 to 45 ° C, at pH values of 7.0 to 8.5, preferably 7.6 to 7.8, and the resulting mixture of L-phenylalanine derivative and DN-phenyl228431 of the phenylalanine acetylderivative, in particular Lp-ethylphenylelanine and DN-phenylacetyl-p-ethylphenylalanine, and are separated, preferably by means of an ion exchanger. Due to the optimum composition of the product, it is advantageous to stop the enzyme reaction when a conversion is achieved which still does not cleave the acyl from the D-form of the amino acid. Procedure using penioillin amide hydrolase (EC 3.5.1.11) or insoluble forms of this enzyme, eg enzy »megaterium cell-anchored enzyme (MS AO No. 208 931), bound E. coli cells (MS AO No. 203 607) having the retained foamyilinamidohydralysis activity, cross-linked and permeabilized production cells of microorganisms (No. AO No. 201 621) or aggregated cells (No. AO No. 197 101) is particularly advantageous in terms of product ease of isolation and column capability. In order to obtain an optically uniform product, it is necessary to monitor the progress of the reaction by a suitable method, for example liquid or gas chromatography, and to stop the reaction at a suitable degree of conversion, preferably 50%, otherwise the acyl is gradually cleaved from the D-form.

The method of splitting optical antipodes is explained in the following examples:

Example 1

L-p-ethylphenylalanine

To a suspension of D, L-p-ethylphenylalanine (2 g) in 214 NaOH (7 mL) was portionwise at 10 ° C over 30 min. Phenylacetyl chloride (3 mL) was added and another 214 NaOH was added to bring the pH of the mixture to 12. The suspension was then stirred at room temperature for 2 h. The reaction mixture was acidified with concentrated hydrochloric acid to pH 2 and stored in the refrigerator overnight. The product was aspirated, washed with water, dried and recrystallized from 30% ethanol. 2.65 g (82%) of m.p.

Mp 146-149 ° C.

_Rp 0.93 (S 1), 0.50 (S 2), 0.86 (S 3), 0.61 (S 4).

For C ₁₉ H ₂₁ NO ₃ (311.4) calculated: 73.29% C, 6.80% H, 4.50% N;

Found:% C, 72.98;% H, 6.67;% N, 4.32.

The resulting intermediate (phenylacetyl-DL-p-ethylphenylalanine 1.1 g) was suspended in water (40 mL) and the pH was adjusted to 7.5 by addition of 0.1 14 NaOH (3.1 mL). To the resulting solution was added 0.214 phosphate buffer (13 ml) at pH 7.5 and the bound E. coli cells obtained according to U.S. Pat. AO No. 203 607 in an amount of 1 g and the mixture was stirred at 40 ° C.

The progress of the cleavage of MeOH-0.05% trifluoroacetic acid (80:20) was monitored by liquid chromatography: k-values: phenylacetic acid - 1.19, phenylacetyl-p-ethylphenylalanine - 2.11, p-ethylphenylalanine - 2.84. After 3 h, the reaction mixture was filtered, acidified with 114 HG1 (10 mL), filtered, and loaded onto a sulfonate latex column (60 mL). After washing with water, the product was eluted with 15% pyridine and evaporated. 234 mg (68.5%) of Lp-ethylphenylalanine was obtained by thin-layer chromatography in four solvent systems and by electrophoresis at two different pHs consistent with the standard. The optical purity determined by high pressure liquid chromatography with a chiral mobile phase (0.008 mol.l ^- L-phenylalanine and 0.004 mol.l ^- CuSO4-methanol; (55:45) was > 99% and this value was confirmed by incubation of the sample. with L-amino acid oxidase The product was recrystallized from 1M HCl;

206 DEG -208 DEG C., -23.9 DEG (c 0.12, water). Literature (Zhuze A. L., Jošt K., Kasafírek E.,

Sudinger J .: Collect. Czech. Chem. Gommun. 29, 2648 (1964)), [.alpha.] D @ 23 = -23.1 DEG (c 0.12; water). ®>

Example 2

L-p-Methylphenylalanine

Phenylacetyl-D, L-p-methylphenylalanine was incubated in the same manner as in Example 1, except that an enzyme anchored on Bacillus megaterium cells (MS AO δ. 20-931) was used. After isolation, pure L-p-methylphenylalanine was obtained in a yield of 65% identical to that of the standard (Zhuze A. L. et al .: Gollect. Czech. Chem. Commun. 29, 2648 (1964)). The optical purity was verified in the same manner as in Example 1.

Example 3

L-p-chlorophenylalanine

Phenylacetyl-D, L-p-chlorophenylalanine was incubated as in Example 1 except that cross-linked and permeabilized cells were used (ref. AQ No. 201 621). Conventional work-up gave L-p-chlorophenylalanine in 52% yield, the optical purity of which was verified as in Example 1.

Claims (2)

A method of enzymatic resolution of optical isomers of phenylalanine derivatives substituted in the β-position of general formula (I), X · (I) wherein X is alkyl having 1 to 4 carbon atoms or halogen, wherein D _r l ~ N ~ fenylacetylderivát phenylalanine, in particular D, LN-phenylacetyl-p-ethylphenylalanine, is incubated with enzymes we with penicilinamidohydrolázovou activity , preferably with their insoluble forms, in a discontinuous or continuous manner in the temperature range of 15 to 45 ° C, at pH values of 7.0 to 8.5, preferably 7.6 to 7.8, and the resulting L- The phenylalanine and the DN-phenylacetylderivative of phenylalanine, in particular Lp-ethylphenylalanine and DN-phenylacetyl-p-etbylphenylalanine, are separated, preferably by means of an ion exchanger. 2. The method of claim 1, wherein the enzyme reaction is stopped when a conversion is achieved that does not yet cleave the acyl from the D-form of the amino acid.