JPH0417960B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel peptide derivative useful as a fluorescent substrate for measuring specific enzyme activity or a synthetic intermediate thereof, and uses thereof. As a result of extensive research in order to obtain enzyme substrates that can be used to easily and sensitively measure the activity of specific enzymes, the present inventors discovered the general formula We succeeded in synthesizing a peptide derivative shown in , and furthermore, this new peptide derivative was used to synthesize a specific enzyme, namely dipeptidyl aminopeptidase type (McDonald.JK, Reilly.
T.J., Zeitman.BB and Ellis.S; J.Biol.
Chem. 2432028-2037 (1968)) and found that its enzyme activity could be measured easily and with high sensitivity, leading to the completion of the present invention. The peptide derivative of the present invention may be in the form of an acid addition salt, in which case the acid may be a mineral acid such as hydrochloric acid or sulfuric acid, acetic acid,
Organic acids such as toluenesulfonic acid can be used. To obtain its free form, the acid addition salt may be neutralized with an alkali. The peptide derivative of the present invention can be obtained by reacting 7-alanylamino-4-methylcoumarin with amino-protected lysine or an active ester thereof in the presence of a condensing agent such as dicyclohexylcarbodiimide, according to a method commonly used for peptide synthesis. It can be easily produced by removing the protecting group of the amino group of the reaction product as desired. Preferred examples of the active ester include p-nitrophenyl ester and N-hydroxysuccinimide ester. When using a solvent, for example, dimethylformamide (DMF) or water may be used. This reaction proceeds at room temperature, but the reaction can be accelerated if desired by heating. To isolate the peptide derivative of the present invention from the reaction mixture, for example, the reaction mixture is concentrated to dryness, the residue is purified by column chromatography, and then lyophilized. In order to produce a target compound in which lysine and alanine constituting the molecule are either L-form or D-form, optically active lysine, alanine or a derivative thereof, which is the starting material used for the production of the target compound, is produced. Alternatively, a DL-isomer of the target compound may be produced and subjected to optical resolution. The peptide derivatives of the present invention are selectively hydrolyzed by the dipeptidyl aminopeptidase type and are therefore suitable as fluorescent substrates for this enzyme. In addition, when using the peptide derivative of the present invention as a substrate, alanine constituting the molecule is preferably L-form, and when other amino acids constitute the molecule, the amino acid may be either L-form or D-form. However, it is particularly preferable that both are L-forms. Enzyme activity measurement using the peptide derivative of the present invention is carried out as follows. That is, a sample solution containing a desired enzyme is brought into contact with 7-L-lysyl-L-alanyl-4-methylcoumarin, and then,
Enzyme activity in a sample solution can be measured by quantifying 7-amino-4-methylcoumarin. 7-L-lysyl-L-amanyl-4-methylcoumarin can be used if an enzyme, ie dipeptidyl aminopeptidase type, is present in the sample solution.
L-lysyl-L-alanyl and 7 in proportion to the amount.
- decomposes and liberates amino-4-methylcoumarin,
Enzyme activity can be measured with high sensitivity by measuring the fluorescence intensity of 7-amino-4-methylcoumarin under fluorescence of Ex 380 nm and Em 460 nm. Therefore,
The peptide derivative of the present invention allows cancer diagnosis to be performed extremely accurately and easily. The peptide derivative of the present invention is a safe substance with no risk of carcinogenicity as a substrate for measuring enzyme activity, and also meets various conditions essential as a substrate, such as ease of measurement of the amount of substrate subjected to enzyme action and stability. It has been confirmed that the method satisfies all the requirements such as linearity with respect to enzyme amount and contact reaction time, and it is possible to provide an enzyme activity measuring method that has important medical significance, particularly in cancer diagnosis. Hereinafter, the present invention will be specifically explained with reference to Examples. Example 7-(N〓-t-butyloxycarbonyl-L-
Synthesis of alanyl)amino-4-methylcoumarin t-butyloxycarbonyl-L-alanine
18.9g dried tetrahydrofuran (THF)
11.3 g of dicyclohexylcarbodiimide was dissolved in 50 ml of dry THF and added little by little under ice-cooling and stirring. Thereafter, the mixture was stirred at room temperature for 50 minutes, and insoluble matter was filtered off. Add 8.75 g of 7-amino-4-methylcoumarin to the filtrate (DMF) 50
ml solution was added and stirring continued overnight. The solvent was distilled off, the residue was dissolved in ethyl acetate, and 1
It was washed with normal hydrochloric acid, 5% sodium bicarbonate solution, and water in this order, and dried with magnesium sulfate. Ethyl acetate was distilled off, and the residue was recrystallized from ethyl acetate and n-hexane.
-(N〓-t-butyloxycarbonyl-L-alanyl)amino-4-methylcoumarin () 12.8g
(73.6%). This substance has a melting point of 193-194℃
(decomposition), specific rotation [α] ²⁰ _D = -38.1 (c = 1.05, acetic acid) Rf = 0.50 (solvent system; chloroform: methanol: acetic acid = 95:5:3). N〓,N〓-di-butyloxycarbonyl-L-
Synthesis of lysine-p-nitrophenyl ester 91 g (0.5 mol) of L-lysine hydrochloride and 215 g (1.5 mol) of t-butyloxycarbonyl-azide were dissolved in 750 ml of dioxane and 250 ml of water, and dissolved in sodium carbonate.
After adding 210g and stirring at 35-40℃ for two days, add water.
Add 750 ml and remove the resulting oil by extracting with ether. Add 1N hydrochloric acid to the aqueous layer to adjust the pH to 2 and extract with ethyl acetate. The ethyl acetate layer was washed with water, dried and concentrated, and 60 g of dicyclohexylamine dissolved in 300 ml of ethyl acetate was added to the residue, which was then shaken.
Ethyl acetate is distilled off. The residue was crystallized by adding n-hexane, and recrystallized from ethyl acetate and n-hexane to obtain N with a melting point of 141-141.5°C and a specific optical rotation [α] ²² _D = 8.1 (c = 4.06, methanol). 〓、
N-di-t-butyloxycarbonyl-L-lysine dicyclohexylamine salt () 149g
(56.4%). 79.2 g (0.15 mol) of the solution was dissolved in ethyl acetate, mixed with 1N sulfuric acid, dicyclohexylamine sulfate was removed from the aqueous layer, and the ethyl acetate layer was washed with water and dried. Subsequently, P-nitrophenol 21
g (0.15 mol) and stir while cooling on ice.
After adding 32 g (0.17 mol) of dicyclohexylcarbodiimide and stirring at room temperature overnight, insoluble matter was filtered off, the mother liquor was concentrated, and the residue was diluted with ethyl acetate.
100 ml and 300 ml of n-hexane were added to remove the soluble materials, and the residue was dissolved in 300 ml of ethyl alcohol and recrystallized. As a result ^, the _N
，
N-di-t-butyloxycarbonyl-L-lysine-p-nitrophenyl ester () was obtained. 7-(N〓,N〓-di-t-butyloxycarbonyl-L-lysyl-L-alanyl)amino-4-
Synthesis of methylcoumarin () 3.46 g (10 mmol) and trifluoroacetic acid
After adding 15 ml and stirring at room temperature for 15 minutes, ethyl ether was added to the residue that was concentrated under reduced pressure to obtain a white powder, which was filtered and dried to obtain 7-(N〓-L-alanyl)amino-4-methylcoumarin.・Trifluoroacetate () was obtained. This product showed a single spot at Rf=0.05 (solvent system: chloroform:methanol:acetic acid=95:5:3) in silica gel thin layer chromatography. () Add 30 ml of DMF to the total amount, dissolve, and neutralize by adding 1.5 ml of triethylamine. ()
5.6 g was added and stirred at room temperature for two days. Distill the solvent, dissolve in ethyl acetate, add 10% sodium carbonate water, 0.5
After thoroughly washing with normal hydrochloric acid and water and drying with magnesium sulfate, add 100% ethyl ether to the concentrated residue.
ml was added and dissolved, cooled and allowed to stand to obtain crystals. The yield was 2.2 g of 7-(N〓,N〓-di-t-butyloxycarbonyl-L-lysyl-L-alanyl)amino-4- with a melting point of 179°C (decomposition).
Methylcoumarin () was obtained. This material was measured by silica gel thin layer chromatography with Rf = 0.55 (solvent system; chloroform: methanol: acetic acid = 95:5:
3) shows a single spot. Further, the specific optical rotation [α] ²⁰ _D = -59.1 (c = 0.77, acetic acid). 7(L-lysyl-L-alanyl)amino-4-
Synthesis of methylcoumarin 2-toluenesulfonate (2) Add 10ml of acetic acid and 1.0g of toluenesulfonic acid monohydrate to 1.0g, stir at room temperature for 90 minutes, then add ethyl ether to obtain a white powdery precipitate. . This product was filtered and washed with ethyl acetate to obtain 1.29 g of 7-(L-lysyl-L-alanyl)amino-4-methylcoumarin 2-toluenesulfonate (). No impurities of 1% or more were found in this product by high pressure liquid chromatography (HPLC), and the specific optical rotation [α] ²⁰ _D -28.6 (c =
1.3450% acetic acid). After hydrolysis in a sealed tube at 110° C. for 24 hours in 6N hydrochloric acid, the actual content composition determined by an amino acid analyzer was 1.00 alanine and 0.99 lysine, which was in good agreement with the theoretical value of 1:1. Elemental analysis Measured values C = 53.09%, H = 6.11%, N = 7.17% Logical values C = 53.00%, H = 6.06%, N = 7.17% (Logical values are C ₃₃ H ₄₂ N ₄ O ₁₀ S ₂・H ₂ O・AcOH) 7-(L-lysyl-L-alanyl)amino-4
- Fluorescent substrate test of methylcoumarin 2-toluenesulfonate (Lys-Ala-MCA Tosylate) Lys-Ala-MCA Tosylate was dissolved in water to a concentration of 2 mmol to prepare a substrate solution. The dipeptide diaminopeptidase (DPPII) homogeneously purified from rat kidney was used as an enzyme solution.
The operations shown in 1 were performed sequentially (the enzyme used in the present invention was prepared using F. Fukasawaetal. Toluol; Biochimica et Biophysica Acta
(1983) according to the method described in BBA31603. ). Next, similar operations were performed while changing the incubation time. Fluorescence analysis was used to determine the relationship between (1) the PH activity curve and (2) the reaction time and the amount of 7-amino-4-methylcoumarin (MCA) produced.

[Table] Potassium phosphate, boric acid, barbital
The fluorescence intensity at 380nm/460nm was measured (Ex/Em), and the MCA produced by the enzyme was measured.
The amount was determined using the following formula. (E-C)/(S-B)×0.3 n mol The results are shown in Table-2 and Table-3.

【table】

[Table] From the above results, it can be seen that it is optimal for this substrate to have a pH of 5 to 6, and that MCA is produced in proportion to the reaction time (linear relationship). Next, the above experiment was carried out by varying the amount of enzyme solution, and after incubation at 37°C for 30 minutes, the fluorescence intensity was similarly measured to determine the amount of MCA produced by the enzyme. The results are shown in Table-4.

【table】

[Table] As is clear from the above results, it was confirmed that this substrate gave MCA in proportion to the amount of enzyme used. Therefore, by applying this substrate to a sample solution according to the method described above, measuring the amount of MCA produced, and comparing it with the standard relationship, the desired amount of enzyme in the sample solution can be accurately measured. It turns out that it is useful for diagnosing diseases. Next, we investigated the Lys levels in the serum of normal people and cancer patients.
The hydrolysis activity of -Ala-MCA was determined. The results are shown in Table-5.

【table】

[Table] As is clear from the above results, there is a significant difference in enzyme activity between normal people and patients with oral squamous skin cancer at a 1% risk rate. By using 1~3μ
It can be seen that ultra-trace measurements are possible with the following serums.

Claims (1)

[Claims] 1. General formula Peptide derivatives and acid addition salts thereof. 2. The peptide derivative and acid addition salt thereof according to claim 1, wherein the alanine constituting the molecule is L-form. 3. The peptide derivative and acid addition salt thereof according to claim 1, wherein the lysine constituting the molecule is L-form. 4 General formula 7-, which is freely produced by an enzymatic reaction, is brought into contact with a dipeptidyl aminopeptidase type-containing sample, which is characterized by containing at least one of the peptide derivative represented by the formula and its acid addition salt.
A fluorescent substrate for measuring the above enzyme activity by measuring amino-4-methylcoumarin by a fluorescence method. 5. The substrate according to claim 4, wherein the enzyme reaction solution is an aqueous solution having a pH value in the range of 4 to 8. 6. The substrate according to claim 4, wherein the sample is human serum.