JPS5919532B2 - peptide derivative - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel peptide derivatives useful as substrates for specific enzymes.

In order to investigate the action and potency of various enzymes, the usual method is to prepare a substance that is specifically affected by the enzyme and compare the state before and after the action of the enzyme. In this case, the substances used may be naturally occurring substances, but it is advantageous to use synthetic substances because they can be supplied in large quantities and can be obtained in a pure state. There is an increasing demand for compounds that can serve as substrates for these enzymes. In addition, such compounds are expected to be used as medicines because they act as potent anti-substances in the metabolic system of living organisms. The present invention meets these needs and provides a synthetic substrate that exhibits specificity for enzymes such as trypsin, kallikrene, thrombin urokinase, and Captogani blood coagulation enzyme.

That is, the compound of the present invention has two types. It is a peptide derivative represented by CON□N02 (CHO)3 N00 C=NH (wherein R is a serylglycyl group, a leucylglycyl group, a valylserylglycyl group, a glycyl group, a valylprolyl group, and a prolyl group).

), these are all new compounds that have not been published in any literature. This peptide derivative may have the form of an acid addition salt,
In addition, the guanidino group of arginine constituting the molecule may be protected, such as N-guanidino protecting groups commonly used in peptide synthesis such as nitro group, tosyl group, p-methoxybenzenesulfonyl group, and other acid addition salts. Those with added protons can be used. In all of these peptide derivatives of the present invention, the amino group or imino group at the α-position has a substituent commonly used as a protecting group during peptide synthesis, such as an acyl group such as acetyl or benzoyl.
It may be protected by a carbobenzoxy group, a tertiary alkyloxycarbonyl group, a tosyl group, or a glutaryl group. Further, when serine is contained in the molecule, the hydroxyl group may be protected with a protecting group commonly used in peptide synthesis, such as an alkyl or benzyl group. The amino acids constituting the peptide derivative of the present invention can be either L-unit or D-unit.

The peptide derivative of the present invention can be produced using arginine-P-nitroanilide as a starting material according to a method commonly used for peptide synthesis.

For example, amino group-protected glylene and arginine P
- nitroanilide in the presence of a condensing agent such as synchhexylcarbodiimide, or after reacting an active ester of glycine with a protected amino group with arginine-P-nitroanilide, Glycylarginine-P-nitroanilide is obtained by removing the protecting group, and this is further reacted with an active ester of serine with protected amino and hydroxyl groups to remove the protecting groups for the amino and hydroxyl groups. For example, serylglycylarginine-P-nitroanilide is obtained. others,
The peptide derivative of the present invention can be produced, for example, by the same condensation reaction as described above in which an active ester of an amino acid is reacted, or by employing the protective group protection method and removal method commonly used in peptide synthesis.

All of these condensation reactions are preferably carried out in a suitable solvent such as dimethylformamide, dimethylsulfoxide, water, or a mixture thereof.

The carboxyl group component to be reacted with the amino group component is advantageously used in the form of an active ester, and examples of this active ester include N-hydroxysuccinimide ester,
P-nitrophenyl ester and the like are preferred. Although the reaction using this active ester proceeds satisfactorily even at room temperature,
If desired, the reaction can be accelerated by heating. After the reaction is complete, the reaction mixture is concentrated to dryness, the residue is purified by column chromatography, and then freeze-dried.

The compounds obtained in this way are usually white powders. Among these compounds, those that have a protecting group on the amino or imino group can be removed using a normal method for removing the protecting group. I can do it.

For example, a carbobenzoxy group can be reacted in HBr monoacetic acid or hydrogen fluoride for 30 minutes at room temperature, and a tert-butyloxycarbonyl group can be reacted in trifluoroacetic acid for 30 minutes at room temperature, or in acetic acid, etc. can be removed by reaction with toluenesulfonic acid in a solvent of 90 minutes. The compound of the above general formula, which is a peptide derivative of the present invention, can be converted into an acid addition salt in a free form, and an acid addition salt can be converted into a free form, depending on the manufacturing conditions.

Examples of acid addition salts include hydrochloride, sulfate, nitrate,
There are inorganic acid salts such as phosphates, and organic acid salts such as acetates, oxalates, tartrates, succinates, citrates, and toluenesulfonates. The fact that the compound produced as described above has a chemical structure corresponding to the general formula is that
This was confirmed by comparing elemental analysis, amino acid analysis, ultraviolet spectrum, and ultraviolet absorption spectrum after hydrolysis with trypsin with P-nitroaniline.

The peptide derivatives of the present invention are all hydrolyzed by enzymes such as trypsin, kallikrene, thrombin, urokinase, and horseshoe crab blood coagulase, and therefore are suitable as synthetic substrates for these enzymes.

When using the peptide derivative of the present invention as a substrate for 4 enzymes, it is preferable that the constituent amino acids are L-units.

Hereinafter, the present invention will be explained in detail with reference to Examples.

Example 1 3.68r (10 mmol) of L-arginine-P-nitroanilide dihydrochloride and 3.0r (11 mmol) of t-butyloxycarbonylglycine-N-hydroxysuccinimide ester were dissolved in tetrahydrofuran (T
The solution was dissolved in 100 d of HF), 1.4 d of triethylamine was added thereto, and the mixture was stirred at room temperature for 20 hours, and then the solvent was distilled off.

100 ml of ethyl acetate was added to the residue to dissolve it, and the residue was extracted three times with 100 ml of 1N aqueous sodium carbonate solution. The organic layer was extracted three times with 100 d of 0.5N hydrochloric acid. The organic layer was washed with 100 d of water and then dried over magnesium sulfate. After removing the desiccant by filtration and concentrating the filtrate to dryness, it was subjected to silica gel column chromatography (
2X20c1n) Purification using the solvent system chloroformrimethanol:acetic acid-95:5:3 to 85:20:3. The main fraction was concentrated to dryness, 100 d of ether was added, and the resulting powder was collected by filtration.
Arginine-p-nitroanilide hydrochloride 4.5r (yield: 92%) was obtained. Melting point: Gradual decomposition from 100°C Example 2t-Butyloxycarbonyl-glycyl-L-arginine-P-nitroanilide. Hydrochloride 4.88t (
Add 30 ml of trifluoroacetic acid to 10 mmol) at room temperature.
After stirring for 0 minutes, trifluoroacetic acid was distilled off.

To the residue, t-butyloxycarbonyl-0-benzyl-L-serine-N-hydroxysuccinimide ester 4
．． Add 7t (12 mmol) and dimethylformamide 3
0d and add about 4ml of triethylamine to bring the pH to 7.
After stirring at room temperature for 20 hours, the solvent was distilled off. The residue was dissolved in 100a of ethyl acetate, and extracted three times with 50d of a 1N aqueous sodium carbonate solution. The organic layer was extracted three times with 100 ml of 0.5N hydrochloric acid.
The organic layer was washed at 100 d and then dried over magnesium sulfate. After removing the desiccant by filtration and concentrating the filtrate to dryness, it was subjected to silica gel column chromatography (2×20 (177!)).
) Purification was performed using a solvent system of chloroformrimethanol:acetic acid=952523 to 85:20:3. The main fraction was concentrated to dryness, 100 ml of ether was added, and the resulting powder was collected by filtration. t-Butyloxycarbonyl-0-benzyl-L-serylglycyl-L-arginine-P-nitroanilide hydrochloride5.
6t (yield: 84%) was obtained. Melting point: 160℃ (decomposition)
. Specific optical rotation [α] = -21. C (C=1.3, D゛)
. Example 3 t-Butyloxycarbonyl-0-benzyl-L-serylglycyl-L-arginine-P-nitroanilide. 2 ml of anisole was added to 1.7 f (3 mmol) of the hydrochloride, and the mixture was reacted with liquid hydrogen fluoride at 0° C. for 45 minutes, and then the hydrogen fluoride was distilled off.

40 ml of ether was added to the residue, the soluble matter was removed, and t-butyloxycarbonyl-L-valine-N-hydroxysuccinimide ester 1.41f (
4.5 mmol) was added thereto, and this was dissolved in 20w11 of dimethylformamide. Approximately 4 d of triethylamine was added thereto to adjust the pH to 7, and the mixture was stirred at room temperature for 20 hours, and then the solvent was distilled off. Add 200 d of 0.1N monohydrochloric acid to the residue and add Diaion HP-20 (porous resin manufactured by Mitsubishi Kasei Corporation) 1
After adsorption with 00d, unadsorbed salts and the like were removed by washing with 400d of water.

Desorption was performed with 300 ml of methanol, and the methanol solution was concentrated to dryness.

The concentrated dry matter was subjected to silica gel column chromatography (2
X20cTn) Purification was performed using a solvent system of chloroformrimethanol:acetic acid=95:5:3 to 85:20:3.

The main fraction was concentrated to dryness, 100 d of ether was added, and the resulting powder was collected by filtration. %) was obtained. Melting point: Gradually decomposes from 110°C.

Specific optical rotation [α] 3δ = -30.8° (C = 0.95,8
0%DMF) Example 4t-butyloxycarbonyl-
15 d of trifluoroacetic acid was added to 2.44 f (5 mmol) of glycyl-L-arginine-P-nitroanilide hydrochloride, stirred at room temperature for 30 minutes, and then the trifluoroacetic acid was distilled off.

The residue contains t-butyloxycarbonyl-L-leucine-
N-hydroxysuccinimide ester 1.80V (5
．． 5 mmol) and 20 ml of dimethylformamide.
2d of triethylamine was added to the solution to make the solution 7. After stirring at room temperature for 20 hours, the solvent was distilled off. The residue was dissolved in 10011LI of ethyl acetate, and extracted three times with 100 d of 1N aqueous sodium carbonate solution. The organic layer is 0.
Extraction was performed three times with 100 d of 5N hydrochloric acid. The organic layer was washed with 100 ml of water and then dried over magnesium sulfate. After removing the desiccant by filtration and concentrating the filtrate to dryness, silica gel column chromatography (2 x 20 cTn) solvent system chloroformrimethanol:acetic acid = 95:5:3 to 85:2
Purified at a ratio of 0:3. Concentrate the main fraction to dryness and ether 100%
d and the resulting powder was collected by filtration to obtain t-butyloxycarbonyl-L-leucylglycyl-L-arginine-P-
900η of nitroanilide hydrochloride (yield: 3096) was obtained. Melting point: Gradually decomposes from 80°C. Specific optical rotation [α] Tomoichi 49. r (C=0.85, DMF'). Example 5 30 d of trifluoroacetic acid was added to 4.88 f (10 mmol) of t-butyloxycarbonyl-glycyl-L-arginine-P-nitroanilide hydrochloride, and the mixture was stirred at room temperature for 30 minutes, and then the trifluoroacetic acid was distilled off.

To the residue was added 1.7 f (15 mmol) of glutaric anhydride, dissolved in 50 d of dimethylformamide, and 4 d of triethylamine was added to make the solution 7. After stirring at room temperature for 20 hours, the solvent was distilled off. Add 200 d of water to the residue, dissolve it, adsorb it on Diaion HP-2060d, and then add 200 d of water.
Unadsorbed salts and the like were removed by washing with d. Next, desorption was performed with 2001 Lt of methyl alcohol, and the methanol solution was concentrated to dryness. 300 d of ethyl acetate was added to the residue to remove soluble materials, and the residue was solidified in 300 d of ethyl acetate and collected by filtration. After drying, it was dissolved in 100 d of acetic acid to remove insoluble matter, and then lyophilized to give glutarylglycyl-L-arginine-P-nitroanilide 3.6P (yield: 729
6) was obtained. Melting point: Gradually decomposes from 80°C. Specific rotation [
α] Swamp-10.5° (C-1.81, DMP) Example
6 L-arginine-p-nitroanilide dihydrochloride 3.6
8f (10 mmol) and t-butyloxycarbonyl-L-proline-N-hydroxysuccinimide ester 3.43f (11 mmol) were dissolved in THF5Od,
Add 1.4 d of triethylamine to make 7 and then 2 at room temperature.
After stirring for 0 hours, the solvent was distilled off.

Add 10011t1 of ethyl acetate to the residue and dissolve it.
Extraction was performed three times using 100 d of a specified aqueous sodium carbonate solution. The organic layer was washed with 100 d of 0.5N hydrochloric acid and then dried over magnesium sulfate. After removing the desiccant by filtration and concentrating the filtrate to dryness, a silica gel column graphite (2 x 20 CI
! L) Solvent system Chloroformrimethanol: Acetic acid = 95:
Purification was performed at a ratio of 5:3 to 85:20:3. The main fraction was concentrated to dryness, 100 d of ether was added, and the resulting powder was collected by filtration.
Butyloxycarbonyl-L-prolyl-L-arginine-p-nitroanilide hydrochloride 2.1f (yield: 40%
) was obtained. Decomposition from melting point 153°C Specific optical rotation [α] Hekiichi-70.36 (C-0.65, DMF) Example 7 t-Butyloxycarbonyl-L-prolyl-L-arginine-p-nitroanilide hydrochloride 2 .64f (15i of trifluoroacetic acid was added to 5 mmol), stirred at room temperature for 30 minutes, and then the trifluoroacetic acid was distilled off.

The residue contains t-butyloxycarbonyl-L-valine-N
-Hydroxysuccinimide ester (5.5 mmol) was added, dissolved in dimethylformamide 20d, triethylamine 2i was added, and the mixture was adjusted to 7. After stirring at room temperature for 20 hours, the solvent was distilled off. 100 d of acetic ester in the residue
was added and dissolved, washed with 100 d of sodium chloride water and dried over magnesium sulfate. After removing the desiccant by filtration and concentrating the filtrate to dryness, it was subjected to silica gel column chromatography (
2X20C1! L) Solvent system chloroformrimethanol:
Purification was performed using acetic acid = 95:5:3N85:20:3. The main fraction was concentrated to dryness, 100 TILt of ether was added, and the resulting powder was collected by filtration. %) was obtained. Melting point: 12
Decomposes gradually from 0℃. Specific optical rotation [α] Tomoichi 87.5° (C-1.3, DMF) L-arginine-p-nitroanilide dihydrochloride 3.68y (10 mmol) and carbobenzoxy L-proline-N -Hydroxysuccinimide ester 3.81f (11 mmol) in THF5O
1.4 d of triethylamine was added to the mixture, the mixture was stirred at room temperature for 20 hours, and the solvent was distilled off.

The residue was dissolved in 100a of ethyl acetate and extracted three times with 100ml of 1N aqueous sodium carbonate solution. The organic layer was extracted three times with 100 d of 0.5N hydrochloric acid. The organic layer was washed with 100 d of water and then dried over magnesium sulfate. After removing the desiccant by filtration and concentrating the filtrate to dryness, it was subjected to silica gel column chromatography (2 x 20 c!n) solvent system chloroformrimethanol:acetic acid = 95:5:3~
Purification was performed at a ratio of 85:20:3. The main fraction was concentrated to dryness, 100 d of ether was added, and the resulting powder was collected by filtration to obtain 3.7 t of carbobenzoxy-L-prolyl-L-arginine-p-nitocanilide hydrochloride (yield: 66%). Ta. Melting point:
154℃ o Specific optical rotation [α period = -69.2 (C = 3.87
, 80 Kenpa σ) Elemental analysis: Actual value C52.98%, H
5.84%, Nl7. l8%C25H32N7O6Cl
Calculated values as C53.42%, H5.74%, Nl
A portion of the 7.45% peptide derivative of the present invention was subjected to a trypsin hydrolysis reaction.

The purity of the sample used was determined by silica gel thin layer chromatography, solvent system CHCl3:MeOH:AcOH=85:1.
5:5 (glutarylglycyl-L-arginine-p-nitroanilide hydrochloride only n-BuOH:AcOH:H2
0=4:1:1), one spot is given.

Ultraviolet absorption spectrum (ε3
The intensity ratio to the ultraviolet absorption spectrum (ε40,) after hydrolyzing 1,) with trypsin was determined, and the results are shown below.

The ε,1, of the substrate is approximately 14,500, and the ε6 when this substrate is completely hydrolyzed by trypsin is approximately 10,600.
It is known that in this case ε31, A40,
= 14,500/10,600 ki 1.37.

This value indicates that the peptide derivative of the present invention was completely hydrolyzed by trypsin in the above experiment.
Next, using the peptide derivative of the present invention, the hydrolytic activity of Captocrani blood coagulase (ClOttingenzyme) was investigated.

From Japan (Tachypleus tridentatus
) and American (LimuluspOlyphemus
) using the hemocyte extract of endotoxin (Salm Onellam).
After adding innesOtaR595), the hydrolysis activity of various synthetic substrates was examined. 0.1mM Tris-HCl buffer (
PH8. Synthetic substrate solution (0.1mM) dissolved in O)
0.8T! Ll, 50 μl of 0.5MMgC12 and 0.1% EndOxinsOlutlOn (Salm
Onellamines OtaR595) 20 μl of the mixture was reacted at 37° C. for 3 minutes.

Claims (1)

[Claims] 1. A peptide derivative represented by the general formula ▲ Numerical formula, chemical formula, table, etc. ▼. In the formula, R is a serylglycyl group, a valylserylglycyl group, a leucylglycyl group, a glycyl group, a valylprolyl group, and a prolyl group.