JPS6342637B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel peptide derivatives and acid addition salts represented by formula ().

Compound 1 of the present invention is a compound useful as a substrate for enzymes.

Although various methods have been known for measuring enzyme activity, there is a need for a more sensitive and convenient method for measuring the activity of a specific enzyme.

The substance of the present invention acts as a substrate that is hydrolyzed by various enzymes, and is useful for highly sensitive measurement of enzyme titer, purity, etc.

The peptide derivative of the present invention can be produced as follows. By converting N〓-protected-alanine into an active ester by a conventional method and reacting it with 7-arginyl-amino-4-methylcoumarin, 7-(N〓-protected-alanyl-arginyl)-4-methyl-coumarin is obtained. is obtained. After removing the protecting group, 7-
(N〓-protected-leucyl-alanyl-arginyl)
-4-methyl-coumarin is obtained. Similarly, when the protecting group is removed, 7-(leucyl-alanyl-arginyl)-4-methyl-coumarin is obtained.
The reaction for producing the peptide derivative of the present invention may be carried out by a commonly used method. For example, the solvent may preferably be dimethylformamide (DMF), dimethylsulfoxide (DMSO), water, or a mixture thereof.

Further, the carboxyl group component to be reacted with the amino group component is advantageously used as an active ester, and N-hydroxysuccinylimide ester is preferred. The reaction temperature may be around room temperature. After the reaction is completed, the reaction mixture is concentrated to dryness, and the residue is purified by column chromatography and freeze-dried.
The protective group of the compound obtained here can be removed by a conventional method, if desired. For example, a tertiary butyloxycarbonyl group can be removed using commonly used agents such as trifluoroacetic acid or hydrochloric acid-dioxane.

Next, the present invention will be illustrated.

Example 7-L-Arginylamino-4-methylcoumarin hydrochloride 1/2 hydrate 754 mg in DMF 25 ml and water 25 ml
N-hydroxysuccinylimide ester of carbobenzoxy-L-alanine is dissolved in
After adding 833 mg and stirring at room temperature overnight, the mixture was concentrated to dryness. Using this residue as a solvent, chloroform/methyl alcohol/acetic acid (95/5/3 to 90/20/5)
Purification was _performed by silica gel column chromatography using a mixed solvent of
-(N〓-carbobenzoxy-L-alanyl-L
-Arginyl)amino-4-methylcoumarin hydrochloride (900 mg) was obtained.

Melting point: approximately 182℃ (decomposition point).

IR, 3200, 1715, 1690, 1660 cm ^-1 .

7-(N〓-carbobenzoxy-L-alanyl-L-arginyl)amino-4- obtained here
650 mg of methylcoumarin hydrochloride was suspended in 80 ml of methanol, 150 mg of 10% palladium on carbon was added, and hydrogen gas was passed through the mixture for 3 hours. After the reaction, palladium on carbon was removed by filtration, the mother liquor was concentrated under reduced pressure, and anhydrous ether was added to the residue to obtain 440 mg of 7-(L-alanyl-L-arginyl)amino-4-methylcoumarin hydrochloride. Melting point: approximately 98℃ (decomposition point).

IR 3300, 1670, 1610cm ^-1 .

400 mg of 7-(L-alanyl-L-arginyl)amino-4-methylcoumarin hydrochloride obtained here was added to
Dissolve in 20 ml of DMF, add 400 mg of N-hydroxysuccinylimide ester of t-butyloxycarbonyl-L-leucine, and stir overnight at room temperature.
The reaction solution was concentrated to dryness, and the residue was purified by silica gel column chromatography (30 g of SiO ₂ ) using a mixed solvent of chloroform/methyl alcohol/acetic acid (80/20/4) as a solvent. -t-
270 mg of butyloxycarbonyl-L-leucyl-L-alanyl-L-arginyl)amino-4-methylcoumarin hydrochloride was obtained.

Melting point: approximately 70℃ (decomposition point).

IR 3300, 1650, 1610cm ^-1 .

7-(N〓-t-butyloxycarbonyl-L
200 mg of -leucyl-L-alanyl-L-arginyl)amino-4-methylcoumarin hydrochloride is suspended in 3 ml of dioxane, 5.0 g of 9.8% hydrochloric acid-dioxane solution is added, and the mixture is stirred at room temperature for 3 hours and 30 minutes. After adding 5 ml of methyl alcohol to the reaction solution and stirring for another hour, the solvent was distilled off at a low temperature, and anhydrous ether was added to the residue to precipitate a white powder, 7-(L-
Leucyl-L-alanyl-L-arginyl)amino-4-methylcoumarin hydrochloride is collected. 140mg.

Melting point: approximately 116℃ (decomposition point).

IR 3350, 1650, 1615cm ^-1 .

7-(t-butyloxycarbonyl-L-leucyl-L-alanyl-L-arginyl)amino-
Trypsin activity measurement method using 4-methyl-coumarin as a substrate 7-(t-butyloxycarbonyl-L-leucyl-L-alanyl-L-arginyl ) Amino-4-methyl-coumarin
Use the solution dissolved to a concentration of 0.1mM as the substrate solution.
2 ml of this substrate solution and 0.1 ml of trypsin solution of various concentrations.
After adding and incubating at 37℃ for 30 minutes, 17%
Add 2 ml of acetic acid solution. The 7-amino-4-methyl-coumarin liberated by hydrolysis by the enzyme is measured as the fluorescence intensity at an excitation wavelength of 380 nm and a fluorescence wavelength of 460 nm. This released 4-amino-4-methyl-coumarin (AMC) corresponds to the activity of the enzyme.

The results at each concentration level of trypsin measured by this method are shown in the figure. In the figure, the vertical axis represents nmole of AMC released by trypsin in 30 minutes.
The horizontal axis shows trypsin in ng.

[Brief explanation of the drawing]

The figure shows the results of measuring enzyme activity using the compound of the present invention.

Claims (1)

[Claims] 1 Formula () A peptide derivative represented by