JPS6156100A - Determination of dipeptidylaminopeptidase ii activity - Google Patents

Abstract

PURPOSE:To enable the specific determination of the titled substance, in high sensitivity, by contacting a substrate with the titled substance in the presence of an agent to inhibit the activity of an aminipeptidase, and determining the produced 7-amino-4-methyl-coumarin. CONSTITUTION:At least one of the peptide derivative of formula (R1 and R2 are H or amino-protecting group) and its acid addition salt is made to contact with an animal-originated specimen containing dipeptidylaminopeptidase II, in an aqueous solution, in the presence of an agent for inhibiting the enzymatic activity of amono-epetidase, e.g. N-ethylmaleimide, EDTA, o-phenanthroline, puromycin, iodoacetic acid, etc. The activity of dipeptidylaminopeptidase II can be determined by determining the isolated 7-amino-4-methylcoumarin.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an improvement in a method for measuring the activity of Noveptinolami-7 Bebutiguse.

[&Double] Dipeptidyl amide 7-beptiguse■ (hereinafter abbreviated as DAP■) is known as a powerful enzyme for early detection in the diagnostic range of cancer, for example (McDonald).

J , Ni, , Re1ly, T, J , +ZeiL+a
a+t, Fi, BandE 1lis, S: J
, B iol, Cbem, , 243.2028
2037 (1968)).

The present inventors have discovered that a peptide derivative represented by the general formula (wherein R, , R, represents a hydrogen atom or an amine/protecting group) can be used to improve the fluorescence of DAPII and/or its synthetic intermediate. It was published as a book, and he proposed to R: J. Ro that it could be used to measure his drunkenness and death.

However, when actually diagnosing cancer, etc., it is necessary to use crude enzyme materials such as serum as fermentation materials, which often results in variations in measured values, which may be caused by the presence of enzymes present in the crude enzyme materials. The peptide-induced AP A by Ami/Beptigase (hereinafter abbreviated as AP)
1% of P body (Lys Ala-MCA-+Ala-M
CA-IA MC).

Based on the above knowledge, the present inventors have conducted extensive studies, and have found that
Peptide derivative represented by the above general formula and D of animal polished rice
When a sample containing AP is brought into contact with a sample containing AP in an aqueous solution in the presence of an enzymatically active soundproofing agent, 7-7 and 7-4-
We have discovered that by quantifying methylcoumarin, it is possible to specifically and sensitively quantify and measure only DAP [activity] without being affected by AP, and have completed the present invention.

The peptide derivative represented by the above general formula used in the present invention may be in the form of an acid addition salt, and the acid used in the addition may be a mineral acid such as hydrochloric acid or sulfuric acid, or an organic acid such as vinegar or toluenesulfonic acid. 1. To obtain 4HH in its free form, the above acid addition salt can be neutralized with an alkali'7).

This peptide derivative includes those in which the amino group of the lysine constituting it is protected, and when R, R2 is a protecting group for the amine 7 group, the protecting group includes a carbobenzoxy group, a -bunaloxy carbonyl group, L-amyloxycarbonyl group (, trityl group, p-nitrocarbobenzoxy group, formyl 9, trifluoroacetyl group,
Examples include amine protecting groups commonly used in peptide synthesis such as 7-taloyl group, benzoyl group, acetyl group, tosyl group, and succinyl group. The protection method using these protecting groups or the method for removing the protecting group can be carried out using the means commonly used in the synthesis of peptides.

This peptide derivative can be prepared by combining, for example, 7-7 ranyl amine/-4-methylcoumarin with lysine or an active ester thereof in which the amine 7 group is protected, and a J11°i carrier such as dicyclohexylcarboxylate, for example, in accordance with a method commonly used for peptide synthesis. It can be easily produced by reacting in the presence of boimide and, if desired, removing the protecting group of the amine 7 group of the reaction product.

Preferred examples of the above-mentioned active dimethyl form include p-nitro heptadyl ester and N-hydroxysuccinimide ester.

When using a solvent, for example, trimethylformamyl' (
DMF) and water are preferably used. This reaction may proceed at room temperature or may be accelerated by heating if desired.

To isolate the above peptide derivative using reaction mixture J,
For example, the anti-1,15 mixture is dried to dryness, the residue is purified by column chromatography, and then! The multiplication factor is -・2.

A molecule? +Y; To prepare a desired compound after either lysine and alanine, each consisting of one L or ()-, the starting materials lysine and alanine used in the production of the desired compound are prepared. Alternatively, an optically active material may be employed as the transparent book, or a DL integral of the target compound may be produced and subjected to optical resolution.

This peptide derivative is progressively hydrolyzed by DAPn in the presence of an enzyme inhibitor of AP and is therefore suitable as a fluorescent substrate for this enzyme.

Examples of the enzymatic modification of AP used in the present invention include N-ethylmaleimide, l:': l)'j''
Examples include A, o7 enanthroline, buromain, perchloromercury-benzoic acid, and iodoacetic acid, but 0-7 enanthroline is more preferred from the viewpoint of selection specificity of DAPII.

In addition, when the above peptide derivative is used as a substrate, the alanine constituting the molecule is preferably ]0 or monolithic, and when other amino acids form the molecule (β1), the amino acid may be either L monolithic or D monolithic. However, it is particularly preferable that both are integrated.

DAPn activity measurement according to the present invention is carried out as follows.

In other words, DAPI for human, U/T, mouse, etc.
The sample solution containing I and 7-L-1j true L-alanyl-methylcoumarin was treated with ami/pebutigase (AP).
) in the presence of an inhibitor of 7-amino-4-methylcoumarin (AM
Enzyme activity during sample dissolution can be measured by quantifying . 7-L-lysyl-7-ranyl-4-methylcoumarin was added to ami/bebutiguse (A
Even if P ) is present, it does not change due to the presence of the inhibitor
Exclusively due to the action of DAP[I, L-lysyl-cycloalanine and 7-amino-4-methylcoumarin are quantitatively decomposed and liberated, and their 7-7-7-7 ions are released under the fluorescence of Ex380 Old^ and Em46 On+a. Enzyme activity can be measured with high sensitivity by measuring the fluorescence intensity of -4-methylcoumarin. Therefore, by the method of the present invention, cancer diagnosis can be performed extremely accurately and easily.

The peptide salt conductor used in the present invention is a safe substance with no risk of carcinogenicity as a substrate for enzyme activity measurement, and it is easy to measure under various conditions essential for a substrate, that is, a substrate subjected to enzyme action. , stability, 11゛, linearity with respect to the amount of alcohol and contact reaction time, ヰ, etc., all of which have been confirmed to be satisfied, and provide a method for measuring enzyme activity that has important medical significance in the diagnosis of cancer. be able to.

Seared 1 pressure Hereinafter, the present invention will be specifically explained with reference to Examples.

Production example t-2 tyloxycarbonyl-L-alanine 18.9
100ml of tetrahydro7ran (THF) dried from 8
Then, 11.3 g of dicyclohexylcarboimide was dissolved in dry FAT HF 50 +nl and added little by little under water-cooling and stirring. Thereafter, the mixture was stirred at room temperature for 50 minutes, and insoluble matter was filtered off. 7-amino/-4-methylcoumarin 8 in the filtrate
．． 501 nl of 75E DMF was added and stirring continued overnight.

The solvent was diluted, and the residue was dissolved in ethyl acetate, washed successively with 1N hydrochloric acid, 5% sodium bicarbonate, water, and ice water, and dried over magnesium sulfate. Dilute the ethyl acetate and mix the residue with ethyl acetate.
- Recrystallization from hexane gave 12.8 g (73.6%) of 7-(N'-t-butyloxycarbonyl-cycloalanyl)amino-4-methylcoumarin (I). This product has a melting point of 193-194℃ (decomposition) and a specific optical rotation [α]
”-38,1 (c=1.05.acetic acid) Rf=0.50(
Solvent system; chloroform:meth/-ol:acetic acid = 5
:5 :3).

L-lysine hydrochloride 91B (0.5 mol) and 215 g (1.5 mol) of 1-butyloxycarbonyl-and were dissolved in 75 ml of dioxane and 250 ml of water, and carbonate sorb 2108 was added thereto at 35-40°C. After stirring for two days, add 1 ml of water 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.After washing the ethyl acetate layer with water and drying) Add 60 g of dicyclohexylamine dissolved in 300 ml of ethyl acetate to the residue, shake the mixture, and then add a thin layer of ethyl acetate.

The residue was crystallized by adding n-hexane, and recrystallized from ethyl acetate and n-hexane to give a melting point of 141 to 1.
41.5° C1 specific optical rotation [αHz=3.1 (c=4.0
6. 149 g (56.4%) of N,N-butyloxycarbonyl-L-rinone dicyclohexylamine salt (If) of methanol) was obtained.

79.2 g (0.15 mol) of (It) 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, 21 g of l]-nitrophenol (0,1
5 mol), stirred while cooling with water, added 32 g (0° 17 mol) of dicyclohexyl carbonimide,
After stirring overnight at room temperature, insoluble materials were removed by filtration, and the mother liquor was diluted with 1
After shrinkage, 300+nl of ethyl acetate 1001111,11-hexane was added to the residue, soluble materials were removed, and the residue was dissolved in 30(')+nl of ethylfurfur and recrystallized. As a result, the melting point was 119~]2]°C1 specific optical rotation [αH
”=-24,5 (c=1.1.ethyl alcohol) N
, N - No 1. -Butyloxycarbonyl-L-
1jnon-1]-nitrophenylnister (II).

(1) To 3.46 g (10 mmol), add 15+ nl of truffle-free acetic acid, stir at room temperature for 15 minutes, then reduce the pressure; add ethyl ether to the concentrated residue to make a white powder, )kk Separate φν operation, 7-(N″-L-alanyl)
Ami/-4-methylcoumarin trifluoroacetate (IV
) was obtained. This product was analyzed by silica sol thin layer chromatography with R[=0.05 (Soluble!i!4 system; Chloroform:
A single spot was shown in 95:5:3).

(IV) Add 30 + + l of DMF to the whole amount, dissolve it, add 1.5 + ol of triethylamine to neutralize it, and then ([
[[)5.6E was added and stirred at room temperature for two days. Dilute the solvent, dilute with ethyl acetate, add 10% sodium carbonate water, 0°5
After thoroughly washing with normal hydrochloric acid and water and drying with magnesium sulfate, add ethyl ether 1001111 to the concentrated residue.
By adding and sharpening it, cooling it and putting it into a device, we obtained Sukosho. 'J'l'W(is 2.2r, te, r, It,
+ Point i 7 indicates 'C (decomposition)? -(N
,N-)"-butyloxycarbonyl-alanyl)amino/-4-methylcoumarin (
V) was obtained. This product showed a single spot at Rf=0.55 (solution WM; chloroform:methanol:vinegar p=95:5:3) by silica sol thin layer chromatography. Also, specific optical rotation [α]”=−59,1(c=(
L77. Qja) and ivy.

To 1.0 g of (V), 10+ nl of acetic acid and 1.0 g of toluene sulfone dihydrate were added, and after stirring at room temperature for 90 minutes, ethyl ether was added to obtain a white powdery precipitate.

By filtering this and washing with ethyl acetate,
7-(L-linol-mono-alanyl)ami/-4-methylcoumarin 2-toluenesulfonate (Vl) 1.29g
I got it. These are high pressure liquid chromatography (HP)
No impurities of 1% or more were observed by LC), and the luminosity [
α]2o""28,6 (c=1.34.50% acetic acid).

After hydrolysis in a 6N hydrochloric acid tube at 110°C for 24 hours, the actual content of the composition based on the amino acid resolution was 1.
．． The ratio was 0.99 to lysine, which was in good agreement with the theoretical ratio of 1 to 1.

Actual elemental analysis values C = 53.09%, 11%, N = 7,1
7%JIiiQ I1α C=53.00%, 11
= 6.06%, N = 7.17% (theoretically correct is C3:
+1I (2N401°S2・lI20・8cOH) Example Lys Ala-MCA TosylaLe in water to a concentration of 2 mmol or Q, 5 mmol B
M and used as a substrate solution. DA homogeneously purified from human serum
P■ was used as an enzyme (skin), and o-7 enanthroline was added to it as an inhibitor of 7mi/beptigose (AP).
The procedures shown in Table 1 were performed sequentially with addition and depletion so that the concentration was 0 mmol (used in the present invention).
Preparation of 1F cords was performed by K, ['uknsawa et al.
al, :Biochimica et Bio
physica AcLa (1983) B
By the method described in BA31603. ).

The amounts of 7-amino-4-methylcoumarin (AMC) produced were then measured and compared by (1) a fluorescence method and (2) a high performance liquid chromatography (HPLC)/fluorescence method. The amount of AMC produced is 38 On +o
The fluorescence C intensity at /460 n +n is measured (Ex/Efn) and determined by the following formula.

(EC)/(S-B)Xo, 3n+nol
However, E: experimental, C: control, S: standard, and B are the respective fluorescence intensities when blank.

In-1 Moxibustion 1! (3-Thursday (pl=5, :l)
40μ22+IIN(or8+nM) Lys Ala 54CA 25μ! ! (
0.5or2.0+JI) human serum
10 At 237°C 130 min ink elation 1,
-N-1 Sea 2==nee AlaMCノ＼→ l, y6
Ala + ノ＼ MC Kukaraban Houn <)I
PCL/fluorescence method>E x380n+n/F Q4
60 old n E x380+u++/F 1 4
80n+nAMC peak column: M aHnuspl+ereC-18250
rv+
(0.2M boric acid, 0.05M citric acid adjusted to pH 5.3 with sodium phosphate) As a result of the above, the normal drinking and drinking method and IT p l, C / Firefly - Shibai Tote are the same as those of AMC. Hotaruzu C strong j branch Nawamo A\IC (::, ・The opposite amount is different, 0-7 enanthroline 1.0 mmol, 7 degrees is ami/bebnadase (AP) tko, Lys-Ala
-It is not possible to completely inhibit the decomposition of MCA, and H
It can be seen that approximately 8% of Lys Ala MCA, which is the fluorescent substrate of DAP■, and its decomposition products, Alu MCA and AMC, can be quantified by combining PLC and SIL of the fluorescence method.

Figures 1 and 2 show the 7-ami/-
4-Methylcoumarin (AMC) layer formation and fireflies
6>) Shows the calculation with the support, and the fluorescence intensity is proportional to the concentration of AMC; a linear relationship is obtained. Therefore, 11. If the line is prepared in advance, D A in the sample solution
The enzymatic activity of Pu can be quantified. Table 2 shows the constant conditions for the second case.

Table-2 Measurement conditions Column; Magouspherc C-18
(7μIll) 250m+n x 4.6mm I,
D buffer: 1ffl 10mM potassium phosphate (p
H3,0) Flow rate: 0,7J/+oin Open when held; A M C19,5m1nA la
MCA 6.0+n1nL ys A l
a-M CA 4,2+nin th;3 and l)(P
0-7 engineered nanthroline concentration by LC/fluorescence likelihood method and heM
The relationship with C beak (%) is shown, and song 1 glands A and B are respectively 7-7 minnow 11-methylcoumarin (AMC), 7
-(L,-alanyl)amino-4-methylcoumarin (A
lu −M (′, A) is shown. Repetition conditions are 0
-47 Same as Table-1 except that the concentration of 7-functional nanthroline was changed.
It is.

It is clear from the figure that -) is added with o-7 enanthroline.
As I increases, the A M CA generator decreases (Curve B), and along with this the A M C peak (
%) decreases and becomes constant (curve A), which means that near the zero point of curve A, i.e. in the absence or at low concentrations of 07 enanthroline as an enzyme inhibitor of ami/bebutiguse (AP). (As shown in the following equation (+), (2), AP n Lys - Δla - MCA ]7
y s- to lashi AMC (1) P Lys- to 1a-HC8 -111---→ 1. vz
+Δl a −? I C,'+ノ＼P-) 1. ys to 18 upper + r\i (C(2)Lys
This shows that Ala-MCA is simultaneously degraded by DAPIf and AP. On the other hand, when the concentration of 0-7 nanthroline exceeds a certain level, the enzymatic activity of AP is lost due to its inhibition;
(This shows that only a reaction occurs.

Figures 4 (a) and (b) show the HP of DA II-containing samples homogeneously purified from the rat sword and hypothalamus without and with the addition of 0-7 enanthroline 1. No. 0 4) i Show the results. In the figure, A is Ala
-N4cA, Blj:1. ,ys Ala MCA
, C indicates AMC. Other measurement conditions and results are shown in Table 3.

Table-3 Experimental animals; Wistar rat (9 weeks old), male,
11=5 o-7 nanthroline concentration; 1+nM hypothalamus
; I, C3±0.26 (n+nol/l6in/m
H7 white) 1: Knee 1.15±0.25 (n+nol
+nin/m□-” white) As is clear from the figure (
In either case (a) or (b), when ○-7 enanthroline is added, the decomposition product by AP is A la -
The peak of MCA (A) completely disappeared, and the peak of MCA (A) disappeared completely, and the peak of MCA (A) disappeared, and the peak of AMC (
Only the peak C) appears.

As is clear from the above results, aminopeptigase (
By adding an inhibitor of D A P ), the degradation of Lys-Ala-MCA, a fluorescent substrate, by AP is completely inhibited.
It was found that the amount of AMC・2 was increased in proportion to the amount of rXc.

Therefore, adding this substrate to the sample solution - 11:1 to my method
The amount of AMC produced by the action of the seal is al:I'i
Comparing this with the relationship (:λ!(1), it can be seen that the amount of the target enzyme in the sample solution can be accurately measured, which is useful for the diagnosis of handlebars as mentioned earlier.

In humans, normal people and cancer patients, serum j-yS-
Hydrolysis of Ala-MCA! , T activity/go I:, Q ;l
<Nuta 5 results are shown in Table 4.

Table-4 Normal person (serum); Lys-Δla-MC8 Enzyme activity end 0.5n+M
O,22±0.02 2, On+8 1.35 ±0.108, On+8
2. (53±0.19 cancer loyalist; DAP-11* DAr'-IV
Wood DAP II /DAP IVLys -Al
a t4c Δ (+Iy l'ro −
ICΔ2, Oh+MO,5+ total control serum (n=5) 1.35±0.10 42.73±1.52 0
．． 032 valve I serum (n = 7) As is clear from the above results, there is a significant difference in [ ) AP II activity between normal people and cancer patients at a 1% risk rate, and DA in cancer patients compared to normal people
Pn/DAPIV value is increasing. Therefore, according to the method of the present invention, it is possible to perform ultra-trace measurements with high sensitivity using serum of several micrometers or more.

As explained above, the present invention is characterized in that the presence of an enzyme inhibitor completely inhibits the decomposition of fluorophore C base by ami/bebutigase (Ar'). It is possible to specifically and highly sensitively quantify only cancer, making it a powerful method for diagnosis and early detection of cancer.

[Brief explanation of the drawing]

The drawings show examples of the present invention, and FIGS. 1 and 2 are graphs showing the relationship between 7-7 my/-4-methylcoumarin concentration and fluorescence intensity, respectively, and FIG. - It is a graph showing the relationship between the concentration of heptonanthroline and the AMC peak (%), where curve A is AMC and curve B is Al
aMCA is shown. Figures 4(a) and 4(b) show the IF, G, and DAPl-containing samples uniformly prepared from the striated muscle and hypothalamus. This is a graph showing the results of (PLO analysis) (in the case of no addition of 7 enanthroline and the addition of 1 enanthroline), where A is A
B indicates Lys-Ala-MCA, and C indicates AMC. Mochi+i'l Applicant: Ajino-Cumulative Co., Ltd. Figure 1 7-7mi/-4-methylcoumarin, a61 [-1-nc
1] 1st] 7-amino-4-methylcoumarin, degree [1m01] Figure 3-109 [0-phenanthroline] Figure 4 (a) (b)

Claims (2)

[Claims] (1) At least one of the peptide derivatives and acid addition salts thereof represented by the general formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, R_1 and R_2 represent hydrogen atoms or amino protecting groups.) A dipeptidyl aminopeptidase II-containing sample from the source is contacted in an aqueous solution in the presence of an enzyme activity inhibitor of aminopeptidase, and then free produced 7-amino-4-methylcoumarin is quantified. Method for measuring peptidyl aminopeptidase II activity. (2) A method for measuring dipeptidyl aminopeptidase II activity, wherein the inhibitor according to claim 1 is o-phenanthroline.