CS244645B1 - A method for preparing oxytocin intermediates based on fragments of an oxytocin molecule - Google Patents

Abstract

A method for preparing oxytocin intermediates based on fragments of the oxytocin molecule, the essence of which is that derivatives of amino acids or peptides forming the initial fragments of the oxytocin molecule are treated with proteolytic enzymes to form connected fragments of the oxytocin molecule, whereby papain is used as proteolytic enzymes for the formation of peptide bonds Cys-Tyr, GLn-Asn and Asn-Cys, for the formation of the peptide bond Tyr-Ile of chymotrypsin or thermolysin, for the formation of the peptide bond Ile-Gln of elastase, for the formation of the peptide bond Pro-Leu of thermolysin and for the formation of the peptide bond Leu-Gly of chymotrypsin. These oxytocin intermediates are used for the preparation of the Oxytocin molecule and its analogues or have their own biological activities.

Description

The invention relates to a process for the preparation of oxytocin intermediates based on fragments of an oxytocin molecule.

It has previously been found that proteolytic enzymes (whose function in the living matter is the hydrolysis of peptide bonds) can also be used in the opposite reaction under certain conditions, i. for peptide bond synthesis / review, see: Brtnik F., Jošt K .: Chem. Listy 74, 951 (1980); Jakubke H. - D., Kuhl P.: Die Pharmazie 37, 89 (1982); Fruton J. S.: Adv. Enzymol.

23, 239 (1982)].

It has now been found that this method of preparing peptides is also advantageous for the synthesis of peptides constituting structural fragments of an oxytocin molecule. This procedure gives cleaner products and shortens the number of synthetic steps.

It can be assumed that this method of synthesis should be more economical. Vzoreem I

TL

PA CHT

Cys - Tyr - Ile Gin As Cys - Pro - Leu - GlyNH ₂ (1) the oxytocin molecule is schematically shown; the arrows indicate enzymatically catalysed peptide bond formation (PA = papain, CHT chymotrypsin, E1 = elastase, TL = thermolysin). The only oeptidic bond for which no suitable enzyme has been found is Cys - Pro.

Since the enzymes are stereospecific, it is also possible to choose synthetic procedures which, when using organic-chemical reagents for peptide bond formation, could lead to racemization.

Synthesis published in literature (Tominaga M., da Silva Filho L. P., Muradian J., Seidel W. F .: Peptide Chemistry 1982. Sakakibara S. (Ed), Protein Research Foundation, p. 271, Osaka 1983), which describes two peptides and one tripeptide, does not take advantage of this advantage and is therefore not suitable from a production point of view.

It is an object of the present invention to treat the amino acid derivatives or peptides forming the starting fragments of the oxytocin molecule with proteolytic enzymes to form coupled fragments of the oxytocin molecule, and used as proteolytic enzymes to form peptide bonds Cys-Tyr, Gln-Asn and Asn-Cys papain to form a peptide bond of Tyr-Ile chymotrypsin or thermolysin, to form a peptide bond to Ile-Gln elastase, to: form a peptide bond to Pro-Leu thermolysin, and to form a peptide bond to Leu-Gly chymotryosin.

The preparation of oxytocin intermediates using proteolytic enzymes is further illustrated in the Examples.

Example 1

Benzyloxycarbonyl-S-benzylcysteinyl-tyrosine ethyl ester

Ethylenediaminotetraacetic acid (6 mg) was added to a solution of benzyloxycarbonyl-S-benzylcysteine (691 mg) and ethyl tyrosine hydrochloride (492 mg) in a mixture of ethanol (9 ml) and 0.2 M acetate buffer pH 4.8 (11 ml). cysteine hydrochloride (10 mg).

After adjusting the pH to 4.8, papain (8.5 mg) was added and the mixture was incubated for 24 h at 38 ° C. The product was extracted into ethyl acetate and the ethyl acetate solution was washed with 1 M - HCl water, 0.5 M - NaHCO 3 and water, dried over sodium sulfate and evaporated.

The residue was crystallized from ethyl acetate-petroleum ether. 440 mg (41%) of the product with m.p. 104 ° C. From the mother liquors 100 mg (9%) of the product of the same m.p. = 24 ° (c = 0.6, chloroform).

Amino Acid Analysis: Tyr 1.00 Cys / Bzl / 1.12. For C ₂₉ H ₃₂ N ₂ C ₆ S 0.5 H ₂ O / 545.7 / calculated: 63.84% C, 6.09% H, 5.13% N; Found: 64, 19% C, 5.85% H, 4.95% N. The literature reports mp 103-104 ⁰ C, & _D OU = 26 ° / _c = 3.58, chloroform / / Xselin B., Feuer M ., Schwyzer R .: Helv. Chim. Acta 38, 1508 (1955).

Example 2

Benzyloxycarbonyl-S-benzylcysteinyl-tyrosyl-isoleucine methyl ester

To a solution of benzyloxycarbonyl-S-benzylcysteinyl-tyrosine ethyl ester (67 mg) and isoleucine methyl ester hydrochloride (364 mg) in a mixture of dimethylformamide (2.25 ml) and 0.25 ml) and 0.2 M carbonate-bicarbonate buffer pH 10/2 , 75 ml /, jebe ?. The pH was adjusted to 10 (2 M-NaOH), and i-chymotryosine (5 mg) was added.

The mixture was stirred vigorously at 25 ° C. After 10 min, another portion of enzyme (2.5 mg) was added. After 40 min, the mixture was acidified with 1 M-HCl (5 mL), the formed precipitate was filtered off, washed with water, 1 M-HCl, water 1 M-NaOH and again with water and dried.

40 mg (50%) of the product with m.p. Mp 162-164 ° C. The sample after crystallization from wet methanol had m.p. 162 DEG C. and [.alpha.] D = 40.7 DEG (c = 0.3, methanol). Amino Acid Analysis: Ile 1.05, Tyr 1.00, Cys (Bzl) 1.15.

For CO,3H ^NjO ^S · 0.5H HO (644.8) calculated: 63.33% C, 6.56% H, 6.52% N; found:

% C, 62.95;% H, 6.39;% N, 6.50. 135 [deg.] C. ([.alpha.] D @ 32 = 32.3 DEG (c = 2.9; Methenol) / Boissonnas R.A., Guttmann S., Jaquenoud P.A., Waller J.P .: Helv. Chim. Acta 38.

491 (1955) and m.p. 161-163 ° C / Honzl J .: Candidate Dissertation, Czechoslovak Academy of Sciences, 1956 /.

Benzyl-oxycarbonyl-S-benzylcysteinyl-tyrosyl-isoleucine phenylhydrazide was prepared in a similar manner in a yield of 50%. Mp 254 ° C FAJ _D = 33,8 ° / c = 0.2, dimethylformamide /. Amino Acid Analysis: Ile 1.09, Tyr 1.00, Cys / Bzl / 1.07.

For C ₃₉ H ₄₄ N ₅ O ₆ S.0.5 H ₂ O / 720.9 / calculated: 64.98% C, 6.29% H, 9.71% N; Found: C 65.13, H 6.18, N 9.81.

Example 3

Benzyloxycarbonylglutaminyl-asparaginyl-S-benzylcysteine phenylhydrazide

To a solution of benzyloxycarbonylglutamine (28.5 mg) and asparaginyl-s-benzylcysteine phenylhydrazide trifluoroacetate (53 mg) in a mixture of dimethylformamide (100 µl) and 0.2 M acetate buffer pH 4.8 (990/11) was added ethylenediaminetetraacetic acid. 0.3 mg (a) of cysteine hydrochloride (1 mg) and the pH of the solution was adjusted to 4.8 with 2 M-NaOH.

After the addition of papain (1.1 mg), the mixture was incubated for 24 hours at 38 ° C. The precipitate formed was filtered off and washed with 1 M-HCl, water, 0.5 M-NaHCO _3, and again with water and petroleum ether and dried on Sena. Extraction with hot methanolene gave 42 mg (62%) of the product, mp 241 ° C.

= -31 ° (c = 0.3, dimethylformamide).

Amino Acid Analysis: Asp 1.00, Glu 1.01, Cys / Bzl / 0.98. For ^C 33 ^H 39 ^N 7 ^S ° 7 ^'II 2 ^O / 695.8 / calculated: 56.96% C, 5.94% H, 14.09% N; Found:% C, 56.92;% H, 5.45;% N, 14.19

Phenylhydrazide tert. butyloxycarbonylisoleucyl-glutamine

To the methyl ester solution of tert. butyloxycarbonylisoleucine (8 mg) and glutamine trifluoroacetate phenylhydrazide (35 g) in a mixture of dimethylformamide (50 µl) and 0.2 M carbonate-bicarbonate buffer pH 10.5 (430 µl) were added a small amount of CaCl 2, the pH of the solution was adjusted to 10.2 and elastase (0.5 mg) was added to the final solution.

The mixture was incubated at 37 ° C for 8 h, the precipitate formed was filtered off and washed with water and petroleum ether. 1.2 mg (8.2%) of the product with m.p. 2 ° C. Amino Acid Analysis: Glu 1.00, Ile 0.88.

Example 5

Benzyloxycarbonylprojyl-leucyl-glycine amide

To the methyl ester solution of tert. butyloxycarbonylleucine (750mg) and glycine amide hydrobromide (1.86g) in a mixture of idimethylformamide (3ml) and carbonate-bicarbonate buffer pH 1U (27ml), whose pH was adjusted to 10 with 4M-NaOH, was added p-chymotrypsin / 33 mg /. The mixture was stirred vigorously at 25 ° C for 1 h.

The reaction was quenched by quenching and carefully acidifying 1 M-HCl to pH 3.5. The product was extracted into ethyl acetate, the ethyl acetate solution was washed with H 2 SO 4 buffer (pH 2), water and evaporated. The residue was dissolved in 50% methanol and filtered through an anion exchange column in 50% methanol, and the filtrate was evaporated. The residue was triturated with petroleum ether and filtered off; 425 mg of protected dipeptide was obtained.

This was dissolved in trifluoroacetic acid (6 ml) and after standing at room temperature for 45 min, the solution was evaporated. The residue was triturated with ether and dried. 400 mg of dipeptide-amine trifluoroacetate were obtained.

mg of this preparation was dissolved in a mixture of dimethylformamide (100 µl) and 0.2 M tris-maleic acid buffer pH 7 (900 µl). Benzyloxycarbonylproline (74 mg) was added to the solution and the pH of the solution was adjusted to 7 with 4 M-NaOH.

After addition of a small amount of CaCl 3 and thermolysin (4 mg), the mixture was incubated for 2 h at 38 ° C. The product was extracted several times with ethyl acetate from the solution and the combined ethyl acetate extracts were evaporated.

The residue was dissolved in 30% aqueous methanol and filtered through a cation exchange and anion exchange column and evaporated. The residue was triturated with ether. 18 mg (43% calculated on dipeptidamide trifluoroacetate / product of m.p. Mp 162-163 ° C = 68.15 ° (c = 0.27, ethanol). The product is identical to the substance prepared in the literature by the described procedure according to high performance liquid chromatography (HPIC) and thin layer chromatography (TLG).

The literature states m.p. 165-165.5 ° C = -70 ° (c = 2.0, ethanol) / Stewart F. H. G .: Austr. J. Chem. 19, 2361 (1966); m.p. 162-163 ° C / Zaoral M., Rudinger J .: Collect. Czech. Chem. Commun. 20, 1183 (1955) and 163-163.5 ° C (Ressler, C., du Vigneaud, V .: J. Amer. Chem.

Soc. g6. 3107 (1954).

Example 6

Benzyloxycarbonylglutaminyl-asparagine phenylhydrazide To a solution of benzyloxycarbonylglutamine (281) and asparagine phenylhydrazide trifluoroacetate (681 mg) in dimethylformamide (1 ml) and 0.2 M acetate buffer pH 4.8 (9 ml) were added ethylenediaminetetraacetic acid (3 mg) (10 mg) and the pH was adjusted to 4.8 with 4 M NaOH. After the addition of papain (21 mg), the mixture was incubated at 38 ° C for 24 h.

The precipitate was filtered off and washed well with 1 M-HCl, water, 0.5 M-NaHCO 3 and again with water, petroleum ether and dried. 350 mg (72%) of the product with m.p. 253-254 [deg.] C. The sample to be analyzed was extracted with hot methanol, m.p. 253 ° C. = - 24.3 ° (c = 0.3, dimethylsulfoxide).

For C ₂₃ H ₂₈ N ₆ O _g . 0.5 Hg O (493.5) calculated: 55.97% C _; H, 5.92; N, 17.03; Found: C 55.54, H 5.80, N 16.79.

Example 7

Benzyloxycarbonylprolyl-leucine phenylhydrazide

To a solution of benzyloxycarbonylproline (25 mg) and leucine phenylhydrazide trifluoroacetate (67 mg) in a mixture of dimethylformamide (100/11) and Tris-maleic acid buffer pH 7/900 (µl), adjusted to pH 7, a small amount of CaClg and thermolysin were added. mg /.

The mixture was incubated at 38 ° C for 24 h. The precipitate formed was filtered off, washed with 1 M-HCl, water, 0.5 M-NaHCO 3, water and petroleum ether. 3 mg (68%) was obtained, which was homogeneous by HPLC and TLC and had an m.p. Mp 159-161 ° C.

D ^{= 90} ° ^(c = ²⁵ ° methanol). For ^C 25 ^H 32 ^N 4 ° 4 / 452.5 / calculated: 66.35% C,

7.13% Ά, 12.38% N; Found: 68.87 95 C, 7.13 95 H, 12.20% N.

Example 8

Phenyl hydrazide target. butyloxycarbonyl-isoleucyl-glutaminyl-asparaginyl-S-benzylcysteine

To the solution of the target. butyloxycarbonyl-isoleucyl-glutamine (36 mg) and asparaginyl-S-benzyl-cysteine phenylhydrazide trifluoroacetate (59 mg) in dimethylformamide (150/41) and 0.2 mmol.l acetate buffer pH 5.0 (850/41) were ethylenediaminetetraacetic acid (0.3 mg) and cysteine hydrochloride (1 mg) were added.

After adjusting the pH to 4.8, papain (4.2 mg) was added to the solution. The mixture was incubated at 37 ° C for 20 h. The precipitate was filtered off, washed with K 2 SO 4 buffer pH 2, water, 0.5 M NaHCO 3 water and dried. After washing with petroleum ether, 65 mg (86%) of the product were obtained with mp 235-237 ° C. Amino Acid Analysis: Asp 1.00, Glu 0.94, Ile 0.92, Cys (Bzl) 10 _D = -40.1 ° (c = 93 = dimethylformamide). For C ^ HH ^ gOgS. 0.5 H., 0 / 756.9 / calculated:

% C, 56.45;% H, 6.97;% N, 14.63; Found: C, 56.09; H, 6.73; N, 14.32.

Example 9

Benzyloxycarbonyl-S-benzylcysteinyl-tyrosyl-isoleucyl-glutaminyl-asparaginyl-S-benzylcysteine phenylhydrazide

To a solution of benzyloxycarbonyl-S-benzylcysteinyl-tyrosine (51 mg) and isoleucyl-glutaminyl-asparaginyl-S-benzylcysteine phenylhydrazide trifluoroacetate (78 mg) in a mixture of dimethylformamide (1.5 ml) and TRIS-maleic acid buffer pH 7 / 3.5 ml A small amount of GaCl ₂ was added and the pH was adjusted to 7 with 2 M-NaOH.

After addition of thermolysin (2.5 mg), the mixture was incubated at 38 ° C for 24 h. The precipitate formed was washed with water, 1 M-HCl, water, 0.5 M-NaHCO 3, water and dried. 100 mg (86.9%) of the crude product was obtained, which was further boiled with methanol, washed with methanol and ether. 83 mg (72%) of m.p. 263-264 [deg.] C.

Amino Acid Analysis: Asp 1.14, Glu 1.09, Ile 1.00, Tyr 0.97, Cys / Bzl / 1.90. [α] D = -37.9 ° (o = 0.3, dimethylformamide). For ^C 5a ^H 70 ^N 10 ° 11 ^N 2 ' ^{2 K} 2 ° 7'^'83, m / z: 58,87 «C, 6,30% H, 11,84% N. found: % H, 5.98;% N, 11.84.

Claims (1)

OBJECT OF THE INVENTION A process for the preparation of oxytocin intermediates based on fragments of an oxytocin molecule, characterized in that the amino acid or peptide derivatives constituting the starting fragments of the oxytocin molecule are treated with proteolytic enzymes to form coupled fragments of the oxytocin molecule using Cys-Tyr as the proteolytic enzymes. , Gln-Asn and Asn-Cys papain, for peptide formation of Tyr-ile chymotrypsin or thermolysin, for peptide formation of Ile-Gln elastase, for peptide formation of Pro-Leu thermolysin, and for peptide formation of Leu-Gly chymotrypsin.