CS250444B1 - Method of preparation of peptides based on pressin molecule - Google Patents

Abstract

The invention relates to a method for preparing peptides based on the pressinic acid molecule of the general formula I Z—Cys—Tyr—Phe—Gin—Asn—Cys—N2H2P1] Bzl Bzl (I) where Z is a benzyloxycarbonyl protecting group, Bzl is a benzyl protecting group, N2H2P11 is a phenylhydrazide protecting group. The essence of the invention is that the protected amino acids or peptides forming fragments of the pressinic acid molecule are treated with proteolytic enzymes, papain for the synthesis of peptide bonds Tyr—Phe, Gin—Asn, chymotrypsin for the synthesis of peptide bonds Tyr—Phe, Phe—Gin and thermolysin for the synthesis of the peptide bond Tyr—Phe. This method of preparation provides purer products, is more flexible in the choice of synthetic procedure and is also more economically advantageous than the procedures used for this purpose so far.

Description

The invention relates to a process for preparing peptides based on a pressinic acid molecule and is based on the use of proteolytic enzymes for peptide bond formation.

It has been found previously that proteolytic enzymes whose function in living matter is the hydrolysis of peptide bonds can also be used in the opposite reaction under certain conditions, i. on the synthesis of peptide bonds (for review see: Brtník F., Jošt K .: Chem. listy 74, 951 (1980), Fruton JS: Adv. Enzymol. 53, 239 (1982)), Jakubke II. - D. Kuhl P. , Kěnnecke A., Angev. Chem., 24, 85 (1985).

The synthesis of peptides based on the oxytocin molecule by proteolytic enzymes is known (U.S. Patent No. 244,645). Described herein is the papain-catalyzed synthesis of peptide fragments of Cys-Tyr and Asn-Cys, which are common to both the oxytocin and pressinic acid molecule sequences.

It has now been found that this method of synthesis is advantageous 1 for the synthesis of the N-terminal protected hexapeptide of vasopressin and other structural fragments of pressinic acid. This method gives both chemically and optically pure products, carrying out the reactions and processing the resulting products is simple and economical.

The essence of the preparation of peptides based on the pressin acid molecule of formula I is that the protected amino acids or peptides forming fragments of pressinic acid are treated with proteolytic enzymes, papyrus for the synthesis of peptide bonds Tyr-Phe, Gin-Asn, chymotrypsin for synthesis of peptide bonds Tyr —Phe, Phe — Gln, and thermolysin for the synthesis of the Tyr-Phe peptide bond.

The method for preparing protected peptides of the pressinic acid sequence using proteolytic enzymes is further elucidated in the Examples.

Example 1

Tert-Butyloxycarbonylphenylalanyl-glutamine phenylhydrazide

To a solution of tert-butyloxycarbonylphenylalanine methyl ester (430 mg) and glutamine phenylhydrazide trifluoroacetate (650 mg) in a mixture of methanol (4 mL) and 0.2 mol. I- ¹ carbonate-bicarbonate buffer pH 10.5 (16 ml) was added to pH 10 by addition of α-chymotrypsin (10 mgj) and the mixture was stirred at room temperature for 3 hours, and the precipitate was filtered off and worked up in a conventional manner. 39 | 5 mg (52%] product, mp 200-202 ° C. Recrystallization from methanol-ether yielded 324 mg (43%) with mp 202-203 C ^q

[α] _D -11.4 (c 0.3, methanol).

Amino acid analysis:

Glu 1.00, Phe 1.01,

For C25H33N5O5. O. 5Ή2Ο (492.6) calculated:

% H, 6.96;% N, 14.21;

f »o I ·σρΐΎΑ ·

% C, 61.25;% H, 6.76.

Example 2

Tert-butyloxycarbonylphenylalanyl-glutamylnylasparaginyl-S-benzylcysteine phenylhydrazide

To a solution of t-butyloxycarbonylphenylalanyl-glutamine (200 mg) and asparaginyl-S-benzylcysteine phenylhydrazide trifluoroacetate (320 mg) in a mixture of ethanol (1.5 ml) and 0.2 mol.l ^-1 acetate buffer pH 5.8 (3, Ethylendiaminotetraacetic acid (3 mg) and cysteine hydrochloride (5 mg) were added. The pH of the solution was 4.8. After the addition of papain (20 mg), the mixture was incubated at 38 ° C for 20 hours. The precipitate formed was filtered off and treated in a conventional manner. 345 mg (87%) of the product with mp 228-229 ° C were obtained. Crystallization from methanol-ether gave 315 mg (80%) of mp 229-230 ° C.

[Α] _D -35.9 (c 0.2, dimethylformamide).

Amino acid analysis:

Asp 1.02, Glu 1.00, Phe 1.00, Cys (Bzl) 0.99, For C 39 H 50 N 8 O 8 S1. O. 5H2O (799.9) calculated:

% H, 6.43;% H, 14.01;

nflloyonn '

H, 6.21; N, 13.87.

Example 3

Tert-Butyloxycarbonyltyrosyl-phenylalanine phenylhydrazide

To the solution of the target. butyloxycarbonyltyrosine (565 mg) and phenylalanine phenylhydrazide trifluoroacetate (815 mg) in a mixture of ethanol (4 mL) and 0.2 mol. Ethylendiaminotetraacetic acid (6 mg) and cysteine hydrochloride (20 mg) were added to I- ¹ acetate buffer pH 4.8 (16 mL). After adjusting the pH to 4.8, papaline (85 mg) was added and the mixture was incubated at 38 ° C for 25 hours. The product was extracted many times into ethyl acetate and the solution was washed HSOá ^- buffer pH 2 and with water, 0.5 mol. I ¹ NaHCO3, water, dried over sodium sulfate and evaporated. The residue was crystallized from a mixture of ethyl acetate and petroleum ether. 370 mg (36%) of the product with mp 203-205 ° C were obtained. A sample for analysis was recrystallized from ethyl acetate, mp 207-208 ° C.

5 β 4 4 4 [a] _D -3.1,9 (c 0.3, dimethylformamide).

Amino acid analysis:

Tyr 0.93, Phe 1.07,

For C29H34N4O5 (518.6) calculated:

H, 6.61; N, 10.80. found

H, 6.55; H, 10.56.

Example 4

Benzyloxycarbonyl-S-benzylcysteinyl-tyrosyl-phenylalanine methyl ester

To a solution of benzyloxycarbonyl-S-benzylcysteinyl-tyrosine (51 mg) and phenylalanine methyl ester hydrochloride (25 mg) in a mixture of dimethylformamide (0.5 mL) and 0.2 mol. I ^"1 Tris-maleate buffer pH 7 (2 ml) was added calcium chloride (0.5 mg) and pH was adjusted to 7.0. After addition of thermolysin (2 mg), the mixture was incubated for 24 hours at 38 ° C. The mixture was acidified with 1 mol. . I ^"1 HC1 and the product extracted into ethyl acetate. The ethyl acetate solution was washed with 1 mol. ^{1 -1} HCl, water, 0.5 mol. 1 ^x water, dried over sodium sulfate and evaporated. The residue was triturated with ether. 40 mg (60%) of the product with mp 162 ° C were obtained.

(α) _D -36.5 (c 0.3, methanol).

Amino acid analysis:

Tyr 0.99, Phe 1.03, Cys (Bzl) 0.98.

The literature reports mp 154 ° C, (α) _D -33.7 (c 1.5, methanol) (Boissonnas RA, Guttmann St., Jaquenoud P. -A., Waller J. -P .: Helv, Chim. Acta 39, 1421 (1956)).

Example 5

Benzyloxycarbonyl-S-benzylcysteinyl-tyrosyl-phenylalanine phenylhydrazide (preparation by thermolysin)

To a solution of benzyloxycarbonyl-S-benzylcysteinyl-tyrosine (.51 mg) and phenylalanine phenylhydrazide trifluoroacetate (47 mg) in a mixture of dimethylformamide (0.5 ml) and 0.2 mol. .1 ^-1 Tris-maleate buffer pH 7 (2 ml) was added calcium chloride (0.5 mg) and the pH was adjusted to 7.0. After addition of thermolysin (2 mg), the mixture was incubated for 6 hours at 38 ° C. The precipitate formed was filtered off and treated in a conventional manner. 70 mg (94%) of the product with mp 243-245 ° C was obtained. Crystallization from methanol gave 63 mg (84%) of the product, mp 244-245 ° C.

[Α] D -41.2 (c 0.3, dimethylformamide).

Amino acid analysis:

Tyr 0.95, Phe 1.11, Cys (Bzl) 0.94.

For C42H4N35O6S1. O. 5H2O (754.9) calculated:

% C, 66.82;% H, 5.87;% 9.27; found:

% C, 66.45;% H, 5.73;% 9.36.

Example 6

Benzyloxycarbonyl-S-benzylcysteinyl-tyrosyl-phenylalanine phenylhydrazide (prepared by chymotrypsin)

To a solution of benzyloxycarbonyl-S-benzylcysteinyl-tyrosine ethyl ester (67 mg) and phenylalanine phenylhydrazide trifluoroacetate (185 mg) in a mixture of dimethylformamide (2.25 ml) and 0.2 mol. l ~ ^x carbonate-bicarbonate buffer pH 10.5 (2.75 mL) after adjusting pH to 10 was added a-chymotrypsin (5 mg) and the mixture was stirred for 1 hour at room temperature. The mixture was diluted with 1 mol. I ^-1 HC1, and the precipitate was filtered off and worked up in conventional manner. 62 mg of crude product were obtained which contained the starting dipeptide residue. Crystallization from methanol gave 50 mg (54%) of a homogeneous product, mp 243-244 ° C.

Amino acid analysis:

Tyr 0.97, Phe 1.11, Cys (Bzl) 0.92.

Example 7

Benzyloxycarbonyl-S-benzylcysteinyl-tyrosyl-phenylalanyl-glutamine phenylhydrazide

To a solution of benzyloxycarbonyl-S-benzylcysteinyl-tyrosine (102 mg) and phenylalanyl-glutamine phenylhydrazide trifluoroacetate (110 mg) in a mixture of dimethylformamide (3 ml) and 0.2 mol. Even Trismaleinanového ^-1 buffer pH 7.3 (7 ml) was added calcium chloride (5 mg) (solution pH 7.0) and thermolysin (5 mg). The mixture was incubated at 38 ° C for 20 hours. The precipitate formed was filtered off and treated in a conventional manner. 138 mg (79%) of the product with mp 228-231 ° C were obtained. Crystallization from methanol gave 124 mg (71%) of the product, mp 231-233 ° C.

(a] _at -27.6 (c 0.3, dimethylformamide).

Amino acid analysis:

Glu 1.0, Tyr 0.9, Phe 1.0, Cys (Bzl) 0.9.

For C47H51N7O8S. IH2O (892.1)

S3 4 4 4 calculated:

% C, 63.27;% H, 5.99;% N, 10.99;

found:

% C, 63.36;% H, 5.87;% N, 11.23.

Example 8

Benzyloxycarbonyl-S-benzylcysteinyl-tyrosyl-phenylalanyl-glutaminyl-asparaginyl-S-benzylcysteine phenylhydrazide (preparation by thermolysin 2 + 4)

To a solution of benzyloxycarbonyl-S-benzylcysteinyl-tyrosine (102 mg) and phenylalanyl-glutaminyl-asparaginyl-S-benzylcysteine phenylhydrazide trifluoroacetate (180 mg) in a mixture of dimethylformamide (3.5 mL) and 0.2 mol. 1 ^_1 Trysmaleinanového pH 7 buffer (6.5 ml) was added calcium chloride (5 mg) (solution pH 7.0) and thermolysin (5 mg). The mixture was incubated for 20 hours at 38 ^and C. The precipitate formed was filtered off and worked up in a conventional manner. 202 mg (86%) of the product was obtained, mp 260-261 ° C.

[α] -35.3 (0.2, diinethylformamide).

Amino acid analysis:

Asp 1.07, Glu 1.10, Tyr 0.92, Phe 1.06,

Cys (Bzl) 1.71.

For C 6 H 16 N 10 O 1 S 2. 31120 (1 235) calculated:

% H, 6.04;% H, 11.34; found *

59.38 ° C, 5.58% H, 11.61% N

Example 9

Benzyloxycarbonyl-S-benzylcysteinyl-tyrosyl-phenylalanyl-glutaminyl-asparaginyl-S-benzylcysteine phenylhydrazide (preparation by chymotrypsin 3 + 3)

To a solution of benzyloxycarbonyl-S-benzylcysteinyl-tyrosyl-phenylalanine (13.2 mg) and glutaminyl-asparaginyl-S-benzylcysteine phenylhydrazide (12 mg) in a mixture of dimethylformamide (0.3 ml) and 0.2 mol.l ^-1 Trys- maleic buffer pH 7.0 (0.7 ml) was added α-chymotrypsin (5 mg) and incubated for 20 hours at 38 DEG C. The product was treated in the same manner as above to give 20 mg (85%) of the product. mp 248-250 ° C. Crystallization from methanol afforded 15 mg (64%) of the product mp 258-260 ° C.

[a] _at -34.4 (c 0.2, diinethylformamid).

Amino acid analysis:

Asp 1.14, Glu 1.13, Tyr 0.92, Phe 0.95, Cys (Bzl) 1.71.

Example 10

Benzyloxycarbonyl-S-beuzylcysteinyl-tyrosyl-phenylalanyl-giutaminyl-asparaginyl-S-benzylcysteine phenylhydrazide (preparation by papain 4 + 2)

To a solution of benzyloxycarbonyl-S-benzylcysteinyl-tyrosyl-phenylalanyl-glutamine (16 mg) and asparaginyl-S-benzylcysteine phenylhydrazide trifluoroacetate (13 mg) in a mixture of dimethylformamide (0.3 mL) and 0.2 mol. Ethylenediaminotetraacetic acid (0.3 mg) and cysteine hydrochloride (1 mg) were added to I- ¹ acetate buffer pH 4.8 (0.7 mL). After adjusting the pH to 4.8, papain (2.1 mg) was added and the mixture was incubated for 20 hours at 38 ° C. The product was processed in the same manner as above. 15 mg (64%) of the product were obtained, mp 248-251 ° C. Crystallization from methanol gave 8 mg (34%) of the product, mp 257-259 degrees Celsius.

Amino acid analysis;

Asp 0.97, Glu 1.08, Tyr 0.96, Phe 0.97, Cys (Bzl) 2.04.

Claims (1)

Process for the preparation of peptides based on the pressin acid molecule of the formula (I) - Cys - Tyr - Phe - Gln - Asn - Cys - N 2 H 2 PI1 Bzl Bzl (I) wherein Z is a benzyloxycarbonyl protecting group, Bzl is a benzyl protecting group pina, N2H2PI1 is a phenylhydrazide protecting group characterized in that protected amino acids or peptides forming fragments of a pressin acid molecule are treated with proteolytic enzymes, a papain for peptide synthesis —Phe, Gln — Asn, chymotrypsin for the synthesis of Tyr-Phe, Phe-Gin peptide bonds, and thermolysin for the Tyr-Phe peptide bond synthesis.