JPS6026793B2 - 7-Leucylamino-4-methylcoumarin and its production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a new compound useful as a poppy plant and a photosensitive substrate for measuring oxygen activity, and a method for producing the same.

More specifically, the present invention relates to 7-leucylamino-4-methylcoumarin, which is useful as a substrate for the photoactive synthesis of poppies, and a method for producing the same, for aminopeptidases such as leucine aminobebutidase. It is known to perform microquantification of oxygen activity using chromogenic and fluorescent synthetic substrates, and in particular, methods using opium poppy and photosensitive synthetic substrates are widely used because of their good sensitivity.

Until now, leucyl 3-naphthylamide or leucyl p-nitroanilide has been used to measure the activity of leucine aminopeptidase, but sufficient sensitivity has not yet been obtained for trace quantitative determination, and there are no satisfactory results. I couldn't say that. In addition, the use of 8-naphthylamine, a component of leucyl-8-naphthylamide, which is a poppy photosubstrate, is being banned as a carcinogenic substance, so the development of an alternative poppy photosubstrate is awaited. . As a result of intensive research to obtain a new photosensitive substrate for aminopeptidases that can be measured with even higher sensitivity, the present inventors discovered that 7-leucylamino-4-methylcoumarin, shown by the structural formula, The synthesis was successful, and the inventors discovered that the above object could be achieved through the synthesis, leading to the present invention.

This 7-L-leucylamino-4-methylcoumarin is
Colorless acicular crystals containing 1/3 molecule of water in the form of hydrochloride, melting point 248-251°C (decomposition) and light intensity [Q] color o + 4
2.5o (C=1.6 in ethyl alcohol). 7-Leucylamino-4-methylcoumarin of the present invention is produced by reacting leucine with a protected amino group with chlorocarbonic acid in the presence of an organic base, and then reacting the reaction product with 7-amino-4-methylcoumarin. It can then be obtained by removing the protecting group of the amino group according to a conventional method.

At this time, the protecting groups for the amino group of leucine used as the starting material include carbobenzyloxy group, tert-butoxycarbonyl group, formyl group, trifluoroacetyl group,
Examples include amine/group protecting groups that are commonly used in peptide synthesis, such as phthaloyl groups.

The reaction between leucine having a protected amino group and the chlorocarbonate is carried out in the presence of an organic base,
As this organic base, tertiary amines such as trimethylamine, triethylamine, tripropylamine, and pyridine are suitable.

These are used in approximately equimolar proportions to leucine. Further, as the chlorocarbonic acid ester, alkyl esters of chlorocarbonic acid such as ethyl ester, propyl ester, butyl ester, and isobutyl ester, and cycloalkyl esters such as cyclobentyl ester and cyclohexyl ester are suitable.

These are used in equimolar amounts or in slight excess relative to leucine. The condensation reaction of oisine with a protected amino group and 7-amino-4-methylcoumarin according to the invention is advantageously carried out in an inert solvent.

As this inert solvent, cyclic ethers such as dioxane and tetrahydrofuran are preferred. This integrated reaction is usually carried out by stirring at room temperature or under cooling. The reaction time varies depending on the reaction conditions, but is generally in the range of 10 to 30 hours. In this way, a condensate of leucine and 7-amino-4-methylcoumarin, which has a protected amino group, is obtained, but the removal of the protecting group for the amino group from this condensate is the process of peptide synthesis. This can be easily carried out according to the means commonly used to remove the protecting group of an amino group.

For example, when the protecting group is a carpobenzyloxy group, catalytic hydrogenation may be performed, and when the protecting group is a tert-butoxycarbonyl group, it may be treated with hydrogen halide in an organic solvent.
In the case of a formyl group, it may be treated with alcoholic hydrochloric acid. When this protective group elimination reaction is carried out under acidic conditions, the target compound is often obtained in the form of an acid salt, but this can be easily converted into a free form by neutralization with an alkali.

When the activity of leucine aminopeptidase was measured using the compound of the present invention, it was found that 7-amino-4-methylcoumarin, a hydrolyzate thereof, was detected under light from Ex38m and Em44m poppies. Since it shows a relative light intensity of 30M ago, measurement sensitivity can be significantly improved.

In addition, it goes without saying that this product is superior to the previously used chromogenic substrate leucyl P nitroanilide.
Even compared to 8-naphthylamine, which is a hydrolyzate of the poppy photosubstrate leucyl 3-naphthylamide, it exhibits a higher relative poppy and light intensity of more than 1 note, and enables measurements with higher sensitivity. It is. Next, the present invention will be explained in more detail with reference to Examples.

Example N - Carbobenzyloxy L-0 Isine 1.4 (5
mmol) and triethylamine 0.5 mmol (5 mmol)
and dissolved in 10 pieces of tetrahydrofuran [0 to -5
Isobutyl chlorocarbonate 685 9 (5
Add Milmol) and stir for 10 minutes.

Next, while maintaining this temperature, 60 ml of a tetrahydrofuran solution containing 9 (5 mmol) of 7-amino-4-methylcoumarin 876 was added, and after stirring for 1 hour, the temperature was raised to room temperature and further stirred overnight. After removing the solvent from the reaction mixture thus obtained under reduced pressure, ethyl acetate is added to the residue to dissolve it, and the solution is washed in the order of 10% hydrochloric acid, water, saturated aqueous sodium bicarbonate solution, and water. . The ethyl acetate layer was separated and dried over anhydrous sodium sulfate, and the ethyl acetate was distilled off under reduced pressure. 1.4 units of 7-(N-carbobenzyloxy-L-leucyl)-4-methylcoumarin (yield 83%) are obtained. 7-(N-carbobenzyloxyleucyl)-4-methylcoumarin 846 (2 mmol) obtained as above was dissolved in 30' of ethyl alcohol containing an equivalent amount of hydrochloric acid, and 10% Add palladium on carbon and catalytically reduce for 4 hours. After removing the catalyst by filtration, the solvent was distilled off under reduced pressure and the remaining oil was recrystallized from acetic acid to give 7-L-leucylamino-4-methylcoumarin hydrochloride 507 (yield 78%) with a melting point of 248- 25r0(
Decomposition) as colorless needle-like crystals. The optical rotation [Q] arc/D of this product is 142.5o (in ethyl alcohol,
C=1.6). Further, the results of elemental analysis are as follows. Elemental analysis value (C, 6 days 2,03N2CI・
(as 1 group 20) Calculated value: C58.09, day 6.60
, N8.47, CIIO. 72 actual value; C58.37,
Sun 6.54, N9.42, CIIO. 83 Also, PH8
．． The ultraviolet absorption spectra and optical spectral values of 0.0 and 50 mmol in CI buffer are as follows.

Ultraviolet absorption spectrum: Exmax 324nm (G = 2 total 00) Fluorescence spectrum Exmax 33m, E
mmax 386 nm Reference example 7-leucylamino-4-methylcoumarin hydrochloride was added to a solution containing 0.5% dimethyl sulfoxide in 50 mmol Tris-HCI buffer as a substrate solution from 0.03 to 0.
．． Using a solution dissolved to a concentration of 5 mmol, Part 3 [
Leucine aminopeptidase (Sigma type W) 0.03
~0.3 mc/50 mc of a similar solution was added, and while reacting at 25°C, the changes in poppy light at Ex 38 m and Em 44 m were continuously recorded.

As a result, we found that the tendency of increase in light intensity for poppies was linearly related to the reaction time over 10 minutes, and that the light intensity for poppies was linearly related to the reaction time in the range of 0.11 to 1.1 mm/desk. It turns out that there is.

Also, from the Lineweaver-Park plot, Km is 0.
．． It was confirmed that the specific activity was 16 mmol and the specific activity was 4.0 mmol/female yeast acid/min, which are comparable to those of leucyl-8 naphthylamide, a known poppy photosubstrate. be.

Claims (1)

[Claims] 1. 7-Leucylamino-4-methylcoumarin represented by the structural formula ▲ Numerical formula, chemical formula, table, etc. ▼. 2. The compound according to claim 1 in the form of an acid salt. 3 Leucine with a protected amino group is reacted with chlorocarbonate in the presence of an organic base, and then the reaction product is reacted with 7-amino-4-methylcoumarin, and the protecting group of the amino group is removed. A method for producing 7-leucylamino-4-methylcoumarin, which comprises separating the 7-leucylamino-4-methylcoumarin. 4. The method according to claim 3, wherein the protecting group for the amino group is a carbobenzyloxy group. 5. The method according to claim 3, wherein the organic base is a tertiary amine. 6. The method according to claim 3, wherein the reaction is carried out in an inert solvent. 7. The method according to claim 6, wherein the inert solvent is a cyclic ether.