JPH06220002A - Hydroxyphenylglycine derivative and enzyme activity assay using the same - Google Patents

Abstract

PURPOSE:To provide the subject new compound useful as a new substrate to be used for the assays of enzyme activities such as angiotensinase activity or aminopeptidase activity. CONSTITUTION:The objective compound of formula I [R<1> is H, t- butyloxycarbonyl, benzoyl or L-leucyl of formula II (R<3> is H, t-butyloxycarbonyl or benzyloxycarbonyl); R<2> is OH, benzyloxy or L-histidyl-L-leucyl of formula III (R<4> is H or alkyl)], e.g. N-benzoyl-L-2-(4-hydroxyphenyl)glycyl-L-histidyl-L- leucine. Angiotensinase is made to act on the above illustrated compound to form N-benzoyl-L-2-(4-hydroxyphenyl)glycine, which is then converted into 4- hydroxybenzaldehyde by an oxidase, and this product is quantified to determine angiotensinase activity. The activity of aminopeptidase can be determined using L-leucyl-L-2--(4-hydroxyphenyl)glycine as substrate.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel hydroxyphenylglycine derivative which is a useful substrate for measuring the activity of angiotensin converting enzyme, aminopeptidase and the like, and a synthetic intermediate thereof. Furthermore, the present invention relates to a method for measuring the activity of angiotensin converting enzyme and aminopeptidase utilizing a novel enzymatic reaction in which an oxidase such as laccase converts a phenol compound such as 2- (4-hydroxyphenyl) glycine into 4-hydroxybenzaldehyde. .

[0002]

2. Description of the Related Art Angiotensin-converting enzyme (ACE) is used for renin
In the angiotensin system, it acts on angiotensin I,
Its C-terminal dipeptide, namely L-histidyl-L
-An enzyme that releases leucine to produce active angiotensin II, which has a blood pressure increasing effect. In the kinin-kallikrein system, it is also an enzyme that inactivates bradykinin by hydrolyzing the C-terminal of bradykinin with a dipeptide. Since it plays such an important role in vivo, clinically, the measurement of ACE activity in blood is used to diagnose active sarcoidosis, chronic renal failure, acute hepatitis, chronic hepatitis, cirrhosis, etc. ing.

The most common method for measuring ACE activity in conventional clinical tests is the Cushuman's spectrophotometric method or its modification, but it has problems in simplicity and accuracy.

As a conventional method for measuring the enzyme activity of ACE,
Known methods include (1) a method using fluorescence, (2) a method using a radioisotope, (3) a method using high performance liquid chromatography, and (4) a method using a spectrophotometer.
However, the methods (1) to (3) are not used as general methods because they require a special analyzer.
As the method (4), there is the above-mentioned Kashman method. The Kashman method uses hippuryl-L-histidyl- as a substrate.
Hypuric acid generated by hydrolysis with ACE using L-leucine was extracted with ethyl acetate, and the ethyl acetate was distilled off under reduced pressure. By quantifying the acid
This is a method of measuring ACE activity. The disadvantage of this measuring method is that the measurement values vary greatly because the operation is complicated.

On the other hand, aminopeptidase is used in the kidney, small intestine,
Widely distributed in the pancreas, bile, urine, etc., in the kidney, in the renal tubules,
It is said that villi in the small intestine have a strong activity and are involved in peptide absorption. In the liver, the membrane on the bile secreting side is active, and is known as a biliary enzyme along with AIP and γ-GTP.

[0006] Aminopeptidase, which is measured by a normal clinical test, is a cytoplasmic aminopeptidase (EC3.4.
11.1, also known as leucine aminopeptidase), microsome-derived aminopeptidase (EC3.4.11.2, also known as aminopeptidase M), etc., and these enzymes are measured without distinction as leucine aminopeptidase (LAP). Pathologies with elevated LAP activity include cytoplasmic aminopeptidases such as acute hepatitis, malignant lymphoma, leukemia, and atypical pneumonia, and microsomal aminopeptidases include biliary tract cancer, bile duct stones, liver cancer, and pancreatic cancer. and so on.

[0007] As a conventional activity measuring method, there is a method for quantifying ammonia produced by an enzymatic reaction of L-leucine amide as a substrate for cytoplasmic aminopeptidase. However, this method has a problem in that it is difficult to completely eliminate endogenous ammonia in the serum, and thus the blank value becomes high. In addition, for microsome-derived aminopeptidases, there are a method using L-leucyl-β-naphthylamide as a substrate, a method using L-leucyl-p-nitroanilide as a substrate, and the like. However, these substrates are said not to act on cytoplasmic aminopeptidases.

[0008] Therefore, an object of the present invention is to provide a novel substrate that can be used for measuring enzyme activities such as ACE activity and aminopeptidase activity.

Further, an object of the present invention is to provide an accurate and simple assay method for ACE activity and aminopeptidase activity using the above-mentioned substrate.

[0010]

The present inventors have found that certain hydroxyphenylglycine derivatives can be substrates for ACE and aminopeptidase, and that they are enzymatic reaction products such as 2- (4-hydroxyphenyl) glycine. The phenolic compound of 4 is oxidized by oxidase such as laccase and converted into 4-hydroxybenzaldehyde,
The inventors have found that ACE activity and aminopeptidase activity can be measured by quantifying hydroxybenzaldehyde, and have completed the present invention.

The present invention relates to a hydroxyphenylglycine derivative represented by the following general formula [I].

[0012]

[Chemical 4]

(In the formula, R ¹ is a hydrogen atom, a tert-butyloxycarbonyl group, a benzoyl group or an L-leucyl group represented by the following chemical formula 5 (wherein R ³ is a hydrogen atom, te
rt-butyloxycarbonyl group or benzyloxycarbonyl group), R ² is a hydroxyl group, a benzyloxy group or an L-histidyl-L-leucyl group represented by the following Chemical formula 6 (wherein, R ⁴ is A hydrogen atom or a lower alkyl group). )

[0014]

[Chemical 5]

[0015]

[Chemical 6]

Further, the present invention is N-benzoyl-L-2-
(4-Hydroxyphenyl) glycyl-L-histidyl-L-leucine is reacted with angiotensin converting enzyme (ACE) to produce N-benzoyl-L-2- (4-hydroxyphenyl) glycine, and the produced N-benzoyl -L-2- (4-hydroxyphenyl) glycine is converted into 4-hydroxybenzaldehyde by an oxidase, and the produced 4-hydroxybenzaldehyde is quantified, and relates to a method for measuring angiotensin converting enzyme activity.

In addition, the present invention provides L-leucyl-L-2-
Aminopeptidase is allowed to act on (4-hydroxyphenyl) glycine to give L-2- (4-hydroxyphenyl)
A method for measuring activity of aminopeptidase, which comprises producing glycine, converting the produced L-2- (4-hydroxyphenyl) glycine into 4-hydroxybenzaldehyde by an oxidase, and quantifying the produced 4-hydroxybenzaldehyde. Regarding

Explanation of Abbreviations Abbreviations used in the present specification are as follows. HPG: 2- (4-hydroxyphenyl) glycine or 2- (4-hydroxyphenyl) glycyl L- Leu: L-leucine or L-leucyl L- His: L-histidine or L-histidyl Boc: tert-butyloxycarbonyl Group, (CH ₃ )
₃ COCO- (protecting group for an amino group) Cbz: carbobenzoxy group or benzyloxycarbonyl _{group, C 6 H 5 CH 2 OCO-} ( carboxyl protecting group) OBzl: benzyl ester, -OCH ₂ C ₆ H ₅ Tos : p-toluenesulfonyl group (tosyl group) (protecting group for imidazolyl group of histidine)

[0019]

[Chemical 7]

TFA: trifluoroacetic acid THF: tetrahydrofuran Bz: benzoyl group Ac: acetyl group Et: ethyl group DCC: dicyclohexylcarbodiimide In the present specification, the lower alkyl group means, for example, an alkyl group having 1 to 6 carbon atoms. Therefore, specific examples thereof include methyl, ethyl, propyl and butyl.

L-leucyl-L-2- (4-hydroxy
Synthesis of Phenyl) glycine [L-Leu-L-HPG] A method for synthesizing L-Leu-L-HPG will be described according to scheme 1 of the following chemical formulas 8 and 9.

[0022]

[Chemical 8]

[0023]

[Chemical 9]

The starting material D-2- (4-hydroxyphenyl) glycine [D-HPG] (1) is a commercially available compound (for example, from Tokyo Kasei Co., Ltd.). D-HPG (1) is acetylated to give N-
Acetyl-D-HPG (2), then hydrolyzed to racemic DL-HPG (3). DL-
HPG (3) was reacted with (Boc) ₂ O to give N-Boc-DL-HPG (4) whose amino group was protected with Boc, and then with benzyl bromide to react with N-Boc which was protected with a carboxyl group. -DL-HPG-OBzl (5)
And N-Boc-DL-HPG-OBzl (5) was deprotected with TFA to give DL-HPG-OBzl (6), which was reacted with N-Cbz-L-Leu in the presence of DCC to give N- Cbz-Leu-DL-HPG-OBz
l (7) is obtained.

N-Cbz-L-Leu-DL-HPG-
OBzl (7) is L-Leu by Pd / C catalytic reduction.
-DL-HPG (8). Moreover, in the presence of DCC in the above DL-HPG-OBzl (6), N
-Boc-L-Leu is reacted to give N-Boc-LL
eu-DL-HPG-OBzl (9) was obtained, which was subjected to alkali hydrolysis to give N-Boc-L-Leu-DL-HP.
L-Leu-DL-HPG (8) can also be obtained by treating G (10) with acid hydrolysis to remove the protecting group. L-Leu-DL-HPG (8) can be isolated and purified by a known separation and purification method such as preparative HPLC.
-L-HPG and L-Leu-D-HPG can be separated. Incidentally, N-Cbz-L-Leu and N-Bo
c-L-Leu is a commercially available compound (for example, from Peptide Research Institute, Inc.).

N-benzoyl-L-2- (4-hydroxy
Cyphenyl) glycyl-L-histidyl-L-leucine
Of [N-Bz-L-HPG-L-His-L-Leu]
A method for synthesizing the synthetic N-Bz-L-HPG-L-His-L-Leu will be described according to scheme 2 of the following chemical formulas 10 and 11.

[0027]

[Chemical 10]

[0028]

[Chemical 11]

L-Boc-His (To
s) (11) and L-Leu-OEt.HCl (12)
Are all commercially available compounds (for example, from Peptide Institute). L-Boc-His
(Tos) (11) and L-Leu-OEt.HCl (1
2) is reacted with DCC in the presence of DCC to give L-Boc-Hi.
s (Tos) -L-Leu-OEt (13), followed by deprotection (de-Boc) to give L-His (Tos) -L.
-Leu-OEt (14).

This compound (14) was added to the above N-Boc-D.
L-HPG (4) was reacted in the presence of DCC to give NB
oc-DL-HPG-L-His (Tos) -L-Le
u-OEt (15) is obtained. Compound (15) was sequentially deprotected to give N-Boc-DL-HPG-L-His-L-.
Leu (16) was obtained, then DL-HPG-L-His
Obtain L-Leu (17). DL-HPG-L-Hi
s-L-Leu (17) was reacted with benzoyl chloride to give N-Bz-DL-HPG-L-His-L-Leu.
(18).

N-Bz-DL-HPG-L-His-L
-Leu (18) is a known separation and purification method, for example, preparative HP
N-Bz-L-HPG-L-His-LL by LC etc.
eu and N-Bz-D-HPG-L-His-L-Leu
Can be separated into

ACE activity measurement

[0033]

[Chemical 12]

The method for measuring ACE activity of the present invention comprises
N-benzoyl-L-2-as shown in Scheme 3 of
(4-Hydroxyphenyl) glycyl-L-histidyl-L-leucine [N-Bz-L-HPG-L-His-
L-Leu] as a substrate, N-benzoyl-L-2- (4-hydroxyphenyl) glycine [N-Bz-L-HPG] produced by hydrolysis of ACE is converted into 4-hydroxybenzaldehyde by an oxidase such as laccase. This is done by converting. 4-Hydroxybenzaldehyde can be quantified by, for example, 2,4-dinitrophenylhydrazine (2,4-D) which is a color reagent specific to aldehyde.
By using a spectroscope to change the color to red (near 500 nm) or by using 4-hydroxybenzaldehyde with an oxidase such as laccase to obtain ABTS (2,2'-azino-bis-
A method of quantifying a coloring substance produced by coupling with a chromogen such as (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) by a spectroscope can be used.
Since all of these methods are quantified on the long wavelength side, they are less likely to be affected by contaminants in serum, and the method using 2,4-D allows the use of an automatic separator.

Substrate N-Bz-L-HPG-L-H
It is preferable that the concentration of is-L-Leu is such that the reaction rate of ACE is almost maximum because it has advantages such as shortening the measurement time. The specific substrate concentration varies depending on the ACE concentration in the sample such as serum, but is, for example, 1 to 10 mM,
It is suitable to set it to 1 to 3 mM. Examples of the oxidase include polyphenol oxidase, and examples of the polyphenol oxidase include laccase, bilirubin oxidase, peroxidase, ascorbate oxidase, and ceruloplasmin. The concentration of the oxidase in the reaction system (protein concentration mg / ml) is not particularly limited as long as it is present in an excess amount with respect to the ACE activity. That is, L-HPG, which is a hydrolysis product of ACE, can be quantitatively converted to 4-hydroxybenzaldehyde, and the amount of 4-hydroxybenzaldehyde can be quantified to determine the L-HPG production rate. For example, when laccase is used as the oxidase, it can be set to, for example, 100 to 500 U / ml.
When peroxidase is used as the oxidase, the reaction is carried out in the presence of hydrogen peroxide.

The above-mentioned enzyme reaction is preferably carried out under the optimum conditions of ACE, and the temperature is 35-40 ° C, preferably about 37 ° C, and the pH is 6-9, preferably 7-8. Is.

As a method for quantifying 4-hydroxybenzaldehyde, for example, high performance liquid chromatography (HPL) is used.
C) is used, and a method of chemically coloring and quantitatively determining with a spectroscope is available. The methods for quantifying 4-hydroxybenzaldehyde described below are all examples, and the present invention is not limited to these methods for quantifying 4-hydroxybenzaldehyde. The method using high-performance liquid chromatography (HPLC) uses a reversed-phase column using octadecylsilanized silica gel (ODS), etc., and the mobile phase can be hydrous methanol or hydrous acetonitrile with buffer added if necessary. . Detection is carried out at an ultraviolet wavelength of 260 nm to 300 nm, preferably 280 n.
m ~ 285 nm is used. At an ultraviolet wavelength of 280 nm, the molar extinction coefficient of 4-hydroxybenzaldehyde is about 14 times that of 2- (4-hydroxyphenyl) glycine, which enhances the detection sensitivity of 4-hydroxybenzaldehyde. Quantification can be performed by the absolute calibration curve method using an integrator or the internal standard method.

As a method of chemically developing the color and quantifying it with a spectroscope, for example, a color reaction of aldehyde with 2,4-dinitrophenylhydrazine can be mentioned. Generated 4-
Hydroxybenzaldehyde and 2,4-dinitrophenylhydrazine are condensed under acidic condition to form hydrazone, which is converted into red quinoid structure by applying alkaline condition, and the absorbance at around 500 nm is measured, Obtain from the calibration curve.

Method for measuring aminopeptidase activity

[0040]

[Chemical 13]

The method for measuring the aminopeptidase activity of the present invention is as follows: L-leucyl-L-2- (4-hydroxyphenyl) glycine [L
-Leu-L-HPG] as a substrate, L-2- (4-hydroxyphenyl) glycine [L-HPG] produced by hydrolysis of aminopeptidase is converted into 4-hydroxybenzaldehyde by an oxidase such as laccase. By. The 4-hydroxybenzaldehyde produced can be quantified in the same manner as in the above method. Preferably, 4-hydroxybenzaldehyde is colored red (near 500 nm) with 2,4-dinitrophenylhydrazine (2,4-D) and quantified with a spectrophotometer, or by oxidase such as laccase.
Examples of the method include quantifying a color substance produced by coupling with a chromogen such as a spectrophotometer. This quantification method is preferable because it is not affected by ammonia and exhibits a stable color on the long wavelength side, and thus is less susceptible to contaminants in serum.

As the aminopeptidase, generally, cytoplasmic aminopeptidase (EC3.4.11.1), microsomal aminopeptidase (EC3.4.11.2), cystine aminopeptidase (EC3.4.11.3), arginine aminopeptidase (EC3). .4.11.6), aspartic acid aminopeptidase (EC3.4.11.7), aminopeptidase (EC3.4.
11.11), aminopeptidase (human liver) (EC3.4.1
1.14) and so on. However, cytoplasmic aminopeptidases and microsomal aminopeptidases, which are measured as “leucine aminopeptidase (LAP) measurement” in clinical analysis due to their usefulness, can be mentioned.

It is preferable that the concentration of the substrate L-Leu-L-HPG is such that the reaction rate of aminopeptidase is almost maximized from the viewpoint that the measurement time can be shortened. The specific substrate concentration varies depending on the aminopeptidase concentration in the sample such as serum, but it is, for example, 0.5 to 3 m.
It is suitable to set M, preferably 1 to 2 mM. As the oxidase, laccase or the like can be used as described above, and the concentration can be appropriately selected depending on the activity of aminopeptidase as in the ACE activity measurement.

The above-mentioned enzyme reaction conditions are preferably carried out near the optimum conditions for aminopeptidase, and the temperature is 35-4.
0 ° C., preferably about 37 ° C., pH varies depending on the type of aminopeptidase, and for example, for cytoplasmic aminopeptidase, pH is 8 to 10, preferably 8.5 to 9.5.
Is suitable, and for microsome-derived aminopeptidases, the pH is 6 to 8, preferably 6.5 to 7.
A value of 5 is suitable.

In the measurement of ACE activity of the present invention, L-form N-Bz-L-HPG-L-His as a substrate is used.
-L-Leu is used. However, N-Bz-DL-HPG obtained in the synthesis without separating only the L-form
It is also possible to use -L-His-L-Leu as it is for ACE activity measurement. Similarly, in the aminopeptidase activity measurement, L-Leu-DL-HPG can be used as it is, instead of using L-Leu-L-HPG which is an L-form.

[0046]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a novel hydroxyphenylglycine derivative as a substrate for ACE or aminopeptidase and a synthetic intermediate thereof. Furthermore, according to the method of the present invention, it is possible to perform the measurement more easily and accurately.

[0047]

EXAMPLES Next, the present invention will be specifically described by way of examples. However, the present invention is not limited to the examples.

Example 1 L-Leucyl-DL-2- (4-hydroxyphenyl)
Synthesis of Glycine (8) (1) 5 g (30 mmol) of D-(-)-2- (4-hydroxyphenyl) glycine (D-HPG) (1) and 300 ml of acetic anhydride
Were mixed and stirred at 80 ° C. for 5 hours. After filtering the reaction solution,
Concentrate under reduced pressure to remove crude N-acetyl-D-HPG (2).
8.55 g was obtained. Add 300 ml of 1N hydrochloric acid water to the reaction mixture, and
The mixture was stirred at 00 ° C for 7 hours. The obtained reaction mixture was purified with Dowex 50W (eluting with 3N ammonia water) and further crystallized with water to obtain 1.52 g of DL-2- (4-hydroxyphenyl) glycine (DL-HPG) (3). 9.2 mmo
l) got. [Α] ²⁶ _D +1.5 ° (c2.0, 1N HC
l) ¹ H-NMR (D ₂ O, 50 ° C) δ: 4.74 (1 H, s),
6.96 (2H, d, J = 8.8), 7.33 (2H, d, J = 8.
8)

DL-HPG (3) (1.53 g, 9.2 mmol) was dissolved in water-dioxane (1: 1) (40 ml) and triethylamine was added.
2.2 ml (15.7 mmol), di-t-butyl-dicarbonate 2.74
g (13.8 mmol) was added, and the mixture was stirred at room temperature for 3 hours. After concentrating the reaction mixture, add water and ethyl acetate, and add 1N aqueous hydrochloric acid to adjust the pH of the aqueous layer.
Was adjusted to 2. After washing with water, dehydration and drying with anhydrous sodium sulfate, and concentration under reduced pressure gave crude N-Boc-D.
2.60 g of L-HPG (4) was obtained.

2.10 g of N-Boc-DL-HPG (4)
(7.9 mmol) was dissolved in 25 ml of N, N'-dimethylformamide, 3.3 ml (23.7 mmol) of triethylamine and 1.7 ml (14.3 mmol) of benzyl bromide were added, and the mixture was stirred for 14 hours. The reaction solution was transferred to ethyl acetate, washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 2.4 g of a reaction mixture. The residue was purified by silica gel column, N-Boc-
1.80 g (5.0 mmol) of DL-HPG-OBzl (5) was obtained. [Α] ²⁶ _D + 1.1 ° (c1.0, CH ₃ OH) ¹ H-NMR (CDCl ₃ ) δ: 1.43 (9H, s), 5.1
5 (2H, s), 5.28 (1H, d, J = 6.6), 5.53 (1
H, brs. ), 6.73 (2H, d, J = 8.8), 7.17 (2
H, d, J = 8.8), 7.19-7.21,7.29-7.30 (5H, m)

N-Boc-DL-HPG-OBzl
(5) Dissolve 1.80 g (5.0 mmol) in 30 ml of methylene chloride,
10 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 2 hours. After the reaction solution was concentrated under reduced pressure, 200 ml of ethyl acetate,
Saturated aqueous sodium hydrogen carbonate solution (100 ml) and 1N aqueous sodium hydroxide solution (5 ml) were added, the solvent was extracted, washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain DL-
1.20 g (4.7 mmol) of HPG-OBzl (6) was obtained. ¹ H-NMR (CDCl ₃ ) δ: 4.58 (1H, s), 5.1
0 (1H, d, J = 12.5), 5.17 (1H, d, J = 12.5)
, 6.70 (2H, d, J = 8.8), 7.17 (2H, d, J =
8.8), 7.20-7.24,7.27-7.33 (5H, m)

This compound (6) was dissolved in 100 m of ethyl acetate, and 1.34 g (5.1 mmo) of N-Cbz-L-Leu (6) was added.
l) and 1.17 g (5.1 mmol) of DCC were added, and the mixture was stirred at room temperature for 1 hour. After filtering the reaction solution, water and ethyl acetate were added to the filtrate and solvent extraction was performed. The ethyl acetate layer was washed with 0.1N hydrochloric acid and water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a reaction mixture. 2.84 g was obtained. The obtained reaction mixture is purified by silica gel column chromatography to obtain N-Cbz-L-Leu-DL-HPG-OBzl.
2.34 g (4.5 mmol) of (7) was obtained.

The obtained N-Cbz-L-Leu-DL-
2.34 g (4.5 mmol) of HPG-OBzl (7) was dissolved in 80 ml of methanol, and 240 mg of 10% palladium carbon was added,
After stirring at room temperature for 1 hour, the Cbz group and the benzyl ester group were removed by catalytic reduction. The reaction solution was concentrated to dryness under reduced pressure, and L-leucyl-DL-2- (4-hydroxyphenyl) gucillin (L-Leu-DL-HPG) (8) was added to 1.
22 g (4.12 mmol) was obtained. [Α] ²⁶ _D −67 ° (c0.1, CH
₃ OH), IR (KBr) cm ⁻¹ : 3333, 2932, 2853, 1674, 1628,
1576, 1514, 1451, 1385, 1246, 1175, 1088, 1005, 89
3,837,754,708,640,521,476,415 ¹ H-NMR (D ₂ O + NaOH, 400 MHz): 0.81
(3H, d, J = 8.8), 0.83 (3H, d, J = 8.8), 1.3
3-1.53 (3H, m), 3.39 (1H, t, J = 6.6, 7.4), 5.0
3 (1H, s), 6.55 (2H, d, J = 8.4), 7.05 (2H,
d, J = 8.8)

Example 2 L-Leucyl-DL-2- (4-hydroxyphenyl)
Synthesis of gucillin (8) (2) DL-HPG-OBzl (6) obtained in Example 1 (1.
20 g) was dissolved in 30 ml of ethyl acetate to give N-Boc-LL
Eu (1.24 g, 5.4 mmol) and DCC (1.25 g, 6.1 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was filtered, transferred to ethyl acetate, washed with 0.1 N aqueous hydrochloric acid and water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a reaction mixture of 2.
59 g were obtained. The residue was purified with a silica gel column, NB
oc-L-Leu-DL-HPG-OBzl (9) 1.
97 g (4.2 mmol) was obtained. [Α] ²⁶ _D -17.8 ° (c1.0, C
H ₃ OH) ¹ H-NMR (CDCl ₃ ) δ: 0.89-0.94 (3H ×
2, m), 1.41, 1.44 (9H, s), 1.60-1.78 (3
H, m), 4.15 (1H, m), 5.10, 5.11 (1H, d,
J = 12.5), 5.15, 5.16 (1H, d, J = 12.5), 5.42,
5.60 (1H, s), 6.57,6.62 (2H, d, J = 8.
8), 7.06, 7.08 (2H, d, J = 8.8), 7.17-7.20, 7.27
-7.30 (5H, m)

N-Boc-L-Leu-DL-HPG-
1.00 g (2.2 mmol) of OBzl (9) was dissolved in 8 ml of THF, 4 ml of 0.5 N sodium hydroxide aqueous solution was added, and the mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated, ethyl acetate and water were added, and the pH of the aqueous layer was adjusted to 2.0 with 0.5N aqueous hydrochloric acid. After washing with water, it was dehydrated and dried over anhydrous sodium sulfate and concentrated under reduced pressure to give N-Boc-L-Leu-DL-HPG (10).
0.84 g (2.2 mmol) was obtained. [Α] ²⁶ _D -18.7 ° (c1.0,
CH ₃ OH) ¹ H-NMR (CD ₃ OD) δ: 0.89-0.95 (3H ×
2, m), 1.43 (9H, s), 1.46-1.55 (2H,
m), 1.66-1.69 (1H, m), 4.08-4.16 (1H,
m), 5.29, 5.30 (1H, s), 6.76 (2H, d, J =
8.8), 7.20, 7.22 (2H, d, J = 8.8)

Obtained N-Boc-L-Leu-DL-
HPG (10) (840 mg) was dissolved in 15 ml of methylene chloride, 5 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 2 hours. After concentration, it was dissolved in 20 ml of water, and Diaion WA-30 was added to adjust the pH to 4.6. This was filtered off and the filtrate was concentrated to dryness under reduced pressure to give L-Leu-DL-HPG (8) 0.43.
g (1.5 mmol) was obtained. [Α] ²⁶ _D -14.5 ° (c1.0, 1N
HCl) ¹ H-NMR (D ₂ O + NaOH) δ: 0.85-0.94 (3
H × 2, m), 1.36-1.58 (2H, m), 1.63-1.71
(1H, m), 3.45 (2H, dd, J = 5.8, 13.2), 5.0
6, 5.08 (1H, s), 6.59, 6.60 (2H, d, J = 8.
1), 7.09, 7.11 (2H, d, J = 8.1)

Example 3 L-leucyl-L-2- (4-hydroxyphenyl) g
Separation and purification of lysine L-Leu-L-HPG and L-Leu-D-HPG were distinguished from each other by using L-Leu-D-HPG synthesized from D-HPG by the same method as in Example 1. That is, HP
LC analysis (column: YMC-Pack ODS A-302 (4.6 x 150 mm),
Mobile phase: Methanol / 20 mM phosphate buffer (pH 6.8) (3/7)
, Flow rate: 0.5 ml / min, detection: UV280 nm) L-
The retention times of Leu-L-HPG and L-Leu-D-HPG were 4.8 minutes and 5.9 minutes, respectively, and two diastereomers could be distinguished.

L-leucyl-DL-synthesized in Example 1
2- (4-hydroxyphenyl) glycine (L-Leu
-DL-HPG) (8) to L-leucyl-L-2-
(4-hydroxyphenyl) glycine (L-Leu-L
-HPG) for separation and purification, L-Leu-DL-
HPG 1.2 g methanol: 20 mM phosphate buffer (pH 6.8)
(2: 8) Dissolve in 50 ml, and divide 5 ml each 10 times in succession to perform preparative HPLC (column: YMC-Pack ODS S-343-15).
(2 x 25cm), mobile phase: methanol / 20mM phosphate buffer (pH6.8) (2/8), flow rate: 7.0ml / min, detection: UV280nm
), And the peak with a retention time of 15.5 minutes was collected. still,
The L-Leu-DL-HPG fraction was desalted under the same conditions as in the preparative high performance liquid chromatography described above except that the mobile phase was water, and the desalted L-Leu-L- was eluted.
The HPG peak was collected, freeze-dried and L-Leu-L.
-560 mg of HPG as sodium salt was obtained. ¹ H-NMR (D ₂ O + NaOH): 0.86 (3H, d,
J = 7.4), 0.88 (3H, d, J = 7.4), 1.36-1.58 (2
H, m), 1.63-1.71 (1H, m), 3.45 (2H, dd, J =
5.8, 13.2), 5.06 (1H, s), 6.59 (2H, d, J =
8.1), 7.09 (1H, d, J = 8.1)

Example 4 N-Boc-DL-HPG-L-His (Tos) -L
-Synthesis of LeuOEt (14) 20 ml of dichloromethane was added to 0.98 g (5.0 mmol) of L-LeuOEt.HCl (12) to dissolve it, 0.77 ml (5.5 mmol) of triethylamine was added and mixed, and further dissolved in 5 ml of dichloromethane. L-Boc-His (Tos)
(11) 2.05 g (5.0 mmol) was added. 18 ml of dichloromethane containing 1.13 g (5.5 mmol) of DCC while stirring under ice cooling
The solution was added dropwise. After reacting for 5 hours under ice cooling with stirring, the reaction was further continued at room temperature for 11 hours. After completion of the reaction, insoluble materials were removed by filtration and the solvent was distilled off under reduced pressure. The obtained residue was dissolved in ethyl acetate, washed with 0.1N HCl, water and saturated brine, dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure to give 2.98 g of a reaction mixture. This was subjected to silica gel column chromatography (toluene-ethyl acetate, 2:
1 Elution) L-Boc-His (Tos) -LL
Obtained 2.36 g of euOEt (13) (yield, 85.6%).

L-Boc-His (Tos) -L-Le
uOEt (13) 2.0 g (3.64 mmol) was added to dichloromethane 20
10 ml of TFA while stirring under ice-cooling,
After reacting for 1 hour with stirring under ice cooling, further at room temperature 1.
Allowed to react for 5 hours. After completion of the reaction, the solvent was distilled off under reduced pressure, the obtained residue was dissolved in ethyl acetate, ice-cooled saturated aqueous potassium carbonate solution was added for liquid separation, and the ethyl acetate layer was washed with saturated brine, followed by sulfuric anhydride. Dried over sodium and concentrated to dryness under reduced pressure to give L-His (Tos) -L-LeuO.
1.31 g (yield, 79.9%) of Et (14) was obtained.

L-His (Tos) -L-LeuOEt
(14) N-Boc prepared by dissolving 0.93 g (2.06 mmol) in 9 ml of dichloromethane and synthesizing it in the same manner as in Example 1.
A solution of 0.5 g (1.87 mmol) of -DL-HPG (4) and 6 ml of dichloromethane was added, and DCC 0.43 was added while stirring under ice cooling.
A solution of g (2.06 mmol) and 5 ml of dichloromethane was added, and the mixture was stirred under ice cooling for 13 hours and further at room temperature for 2 hours. After completion of the reaction, insoluble materials were removed by filtration and the solvent was distilled off under reduced pressure. The obtained residue was dissolved in ethyl acetate, 5% aqueous citric acid solution,
The extract was washed with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to give 1.36 g of a reaction mixture. This is subjected to silica gel column chromatography (chloroform:
Methanol = 50: 1 elution) and N-Boc-DL-
HPG-L-His (Tos) -L-LeuOEt (1
5) 0.59 g (yield, 40.8%) was obtained.

N-Boc-DL-HPG-L-His
(Tos) -L-LeuOEt NMR (CDCl ₃ ) δ: 0.76, 0.84, 0.87, 0.94 (3H
× 4, d, J = 6.6 Hz), 1.28, 1.29 (3H × 2,
t, J = 7.3 Hz), 1.47 (9H × 2, brs), 1.40
-1.52 (2H × 2, m), 1.62 (1H × 2, m), 2.48
(3H × 2, s), 2.75-2.90 (1H × 2, m), 2.92
-3.02,3.10-3.20 (1H × 2), 3.79 (1H × 2, br
s), 4.17, 4.18 (2H × 2, q, J = 7.0 Hz), 4.
39-4.49 (1H × 2, m), 4.74 (1H × 2, m), 5.
03,5.09 (1H × 2, brs), 5.64,5.74 (1H ×
2, d, J = 5.9 Hz), 6.69 (2H × 2, d, J = 8.
4 Hz), 6.77 (1H × 2, d, J = 8.4 Hz), 7.0
5,7.13 (1H × 2, s), 7.14,7.20 (2H × 2,
d, J = 8.4 Hz, 7.40 (2H × 2, d, J = 8.1 H
z), 7.84, 7.85 (2H × 2, d, J = 8.4 Hz), 7.
90,7.92 (1H × 2, s) IR cm ⁻¹ (KBr): 3410, 3326 (br), 2965,2
872,1665 (br), 1615,1597,1514 (br), 1379,122
1,1192,1175,1080,1051,1026,893,839,704,677,590,542 UVnm (MeOH): 2670 (E ^1% _1cm = 32), 230.4 (26
6) FAB-MS (pos, matrix; NBA) m / z 700 (M
+ H) ⁺ [α] ²⁵ _D +6.7 ° (c0.3 CHCl ₃ )

Example 5 N-Boc-DL-HPG-L-His (Tos) -L
Synthesis of -Leu (16) N-Boc-DL-HPG-L-His obtained in Example 4
(Tos) -L-LeuOEt (15) 0.50g (0.72mm
(3) THF (3 ml) to dissolve, and water with stirring under ice cooling.
After adding 5 ml and 15 ml of 1N NaOH, the mixture was stirred under ice cooling for 2 hours, and further stirred at room temperature for 20 hours. After completion of the reaction, water was added to dilute, THF was distilled off under reduced pressure, n-butanol was added, and the pH of the aqueous layer was adjusted to 3 with 1N HCl under ice cooling to n-.
Butanol extraction was performed. The obtained n-butanol layer was concentrated to dryness under reduced pressure to obtain 0.67 g of a reaction mixture. The resulting reaction mixture was purified by Sephadex LH-20 column chromatography to give N-Boc-DL-HPG-
0.37 g of L-His (Tos) -L-Leu (16) was obtained.

N-Boc-DL-HPG-L-His-
L-LeuOH (16) NMR (CD ₃ OD) δ: 0.84, 0.91, 0.92, 0.97 (3H
× 4, d, J = 6.2 Hz), 1.42, 1.44 (9H × 2,
s), 1.50-1.67 (1H × 2, m), 1.71 (2H × 2,
m), 3.08 (1H, dd, J = 15.0, 7.0Hz), 3.17
(1H, dd, J = 15.0, 5.9Hz), 3.22-3.34 (1H
X2, m), 4.32, 4.39 (1H x 2m), 4.72 (1H x
2, q, like, J = 5.9 Hz), 4.96,5.00 (1H
× 2, s), 6.74,6.76 (2H × 2, d, J = 8.4 H
z), 6.82, 7.28 (1H × 2, s), 7.17, 7.18 (2H
X2, d, J = 8.4 Hz), 8.53, 8.63 (1H x 2,
s) IR cm ^-1 (KBr): 3310, (br), 2961,1659,
1514,1454,1393,1370,1248,1167,1053,895,841,685,627 UVnm (MeOH): 276.8 (E ^1% _1cm = 29), 221.8
(239) FAB-MS (pos, matrix; NBA) m / z 518 (M
+ H) ⁺ [α] ²⁵ _D -15.5 ° (c1.01, CH ₃ OH)

Example 6 Synthesis of DL-HPG-L-His-L-Leu (17) N-Boc-DL-HPG-L-His obtained in Example 5
To 0.36 g (0.69 mmol) of -L-Leu (16), 3 ml of dichloromethane was added to suspend, and 1 ml of TFA was added with stirring under ice-cooling, followed by stirring at room temperature for 2 hours. After completion of the reaction, the solvent was distilled off from the reaction solution under reduced pressure, and TFA was azeotropically removed using toluene and water, respectively. The obtained residue was dissolved in water, and Diaion WA-30 was added little by little while stirring at room temperature to adjust the pH to 4.6. This was filtered off, and the filtrate was concentrated to dryness under reduced pressure to obtain DL-HPG-L-His-LL.
0.32 g of eu (17) was obtained.

FAB-MS (pos, matrix; glycerin) m / z: 418 (M + H) ⁺ , ¹ H-NMR (CD ₃ OD): 0.84 (3H, d, J = 6.2)
Hz), 1.50-1.80 (3H × 2, m), 3.02-3.28 (2H
× 2, m), 4.27 (1H, dd, J = 10.6, 3.7Hz),
4.33 (1H, dd, J = 10.6, 3.7Hz), 4.72 (1
H, t, J = 5.9 Hz), 4.75 (1 H, t, J = 5.9 H)
z), 4.96 (1H × 2, s), 6.58 (1H, brs), 7.2
3 (1H, brs), 6.81 (2H, d, J = 8.4H
z), 6.86 (2H, d, J = 8.4 Hz), 7.29 (2H,
d, J = 8.4 Hz), 7.30 (2H, d, J = 8.4 H)
z), 8.30 (1H, brs), 8.37 (1H, brs) [α] ²⁶ _D −1.6 ° (c1.1, CH ₃ OH)

Example 7 N-benzoyl-L-2- (4-hydroxyphenyl)
Synthesis of Glycyl-L-Hisudil-L-Leucine DL-HPG-L-His-L-Leu obtained in Example 5
(17) 0.26 g (0.62 mmol) was dissolved in 15 ml of water, and 86 μl (0.74 mmol) of benzoyl chloride and aqueous sodium carbonate (1 mmol) were added in 4 portions over 2 hours while stirring under ice-cooling. The reaction was carried out for 2 hours. After completion of the reaction, the reaction mixture was washed with ether, and the aqueous layer was continuously divided into 5 ml portions in three times and preparative HPLC (column: YMC-Pack ODS A-302 S-343-1).
5 (2 x 25 cm): Mobile phase: methanol / 20 mM phosphate buffer (pH6.8) (45/55), flow rate: 7.0 ml / min, detection: UV28
(0 nm) and the peak having a retention time of 18.4 minutes was collected. still,
Bz-D-HPG-L-His-L-Leu is retention time
It eluted at 20.9 minutes.

N-Bz-L-HPG-L-His-L-
Leu and N-Bz-D-HPG-L-His-L-Le
u is distinguished from N-Bz-D-HPG-L-H synthesized from D-HPG by the same method as in Examples 3, 4, 5, and 6.
It was performed using is-L-Leu. That is, HPLC analysis (column: YMC-Pack ODS A-302 (4.6 × 150 mm), mobile phase: methanol / 20 mM phosphate buffer (pH 6.8) (45/55), flow rate:
0.5 ml / min, detection: UV280 nm) N-Bz-L-H
PG-L-His-L-Leu and N-Bz-D-HPG
The retention time of -L-His-L-Leu is 7.4, respectively.
Min, 8.3 min, and the two diastereomers could be distinguished. Fractionated Bz-L-HPG-L-His
Desalination of the -L-Leu fraction is carried out under the same conditions as in the preparative high performance liquid chromatography described above except that water is used as the mobile phase,
Bz-L-HPG-L-His desalted and eluted
-L-Leu peak was collected, freeze-dried and Bz-L-
HPG-L-His-L-Leu as sodium salt
103 mg (0.2 mmol) was obtained.

N-Bz-L-HPG-L-His-L-
Leu FAB-MS (pos, matrix; glycerin) m /
z: 522 (M + H) ⁺ , 544 (M + Na) ⁺ , ¹ H-NMR (CD ₃ OD): 0.90 (3H, d, J = 4.0).
Hz), 0.91 (3H, d, J = 4.0 Hz), 1.60-1.70
(3H, m), 3.08 (2H, d, J = 5.5 Hz), 4.3
2 (1H, dd, J = 9.9, 4.0 Hz), 4.70 (1H,
t, J = 5.5 Hz), 5.60 (1H, s), 6.47 (1H,
brs), 6.78 (2H, dd, J = 6.6, 1.8Hz), 7.25
(2H, dd, J = 6.6, 1.8 Hz), 7.43-7.55 (4
H, m), 7.85 (2H, dd, J = 8.6, 1.3 Hz)

Reference Example 2- (4-hydroxyphenyl) glycine was used as a substrate using laccase (protein concentration: 2.3 mg / ml) derived from Coriolus versicolor IFO 9791 which is a wood-rotting fungus of the genus Agaricale. A comparison was made between the method for measuring the activity of laccase and the conventional method for measuring the activity of laccase using syringaldazine as a substrate.

The former method for measuring the activity of laccase was carried out by using 200 μl of an aqueous solution prepared by dissolving 2- (4-hydroxyphenyl) glycine in 50 mM phosphate buffer (pH 6.0) to a concentration of 50 mM.
Put l into an Eppendorf tube and add 2μ of enzyme solution to it.
l was added, and the mixture was stirred and reacted at 30 ° C. Generate 4-
The quantification of hydroxybenzaldehyde was performed using high performance liquid chromatography. That is, the column: YMC-Pack A
-302 ODS (4.6 x 150 mm), mobile phase: 30% (v / v) methanol / 0.02M phosphate buffer (pH 6.8), flow rate: 0.5 ml / mi
n, detection: UV280 nm, analytical sample volume: 20 μl, and 4-hydroxybenzaldehyde eluting at a retention time of 9.6 minutes was automatically quantified using an integrator by the absolute calibration curve method using a standard sample. After the start of the reaction, the measurement was performed twice for 5 minutes and 20 minutes when the laccase concentration was 0.023 μg / ml or more, and 10 minutes and 40 minutes when the concentration was lower than that, and the production rate of 4-hydroxybenzaldehyde (nm
ol / min / ml) was calculated and used as the laccase activity.

A method for measuring laccase activity using a syringaldazine substrate is described by Leonowicz et al.
z. Microb. Technol. 3, 55-58, 1981). That is, 0.2 ml of 0.2 mM syringaldazine in ethanol was added to 2.7 ml of 50 mM phosphate buffer (pH 6.0) immediately before the reaction.
Then, add 3 μl of the enzyme solution and stir to obtain an optical path length of 1 cm.
The reaction was started in the cell. The increase in absorbance at 525 nm was measured with time in a constant temperature cell holder at 30 ° C., and the increase in absorbance per unit time (ΔE / min) was determined and used as the laccase activity. Table 1 shows the results of 6 measurements by each activity measuring method.

[0073]

[Table 1]

As can be seen from the results in Table 1, the method using 2- (4-hydroxyphenyl) glycine as the substrate has higher detection sensitivity and a smaller coefficient of variation (CV) than the method using syringaldazine as the substrate. ) Indicates that the reproducibility is excellent.

Example 8 (Method for measuring activity of angiotensin converting enzyme) N-benzoyl-L-2- (4-hydroxyphenyl) glycyl-L-histidyl-L-leucine was added to 0.2 M borate buffer (pH 7.5). Was dissolved in 2 mM and sodium chloride was dissolved to 0.1 M to obtain a substrate solution. 100 μl of this substrate solution was placed in an Eppendorf tube, and 4 μl of laccase (derived from Coriolus versicolor IFO9791, specific activity 4.8 × 10 ³ U / mg, protein concentration 2.3 mg / ml) was added, and preheated at 37 ° C for 3 minutes or more. After that, the standard serum (made by Sigma, ACE CONTROL-N,
No. A6040) (100 μl) was added, and the mixture was kept warm at 37 ° C. for enzyme reaction for 1, 2 and 3 hours. The reaction was stopped by adding 10 μl of a saturated solution of 2,4-dinitrophenylhydrazine in 1N hydrochloric acid, and after leaving it at room temperature for 6 to 8 minutes,
Color was added by adding 10 μl of 5N sodium hydroxide aqueous solution, the absorbance at 500 nm was measured, and 4-hydroxybenzene in the measurement sample was analyzed from the calibration curve obtained by similarly developing color from known concentration of 4-hydroxybenzaldehyde. The concentration of benzaldehyde was determined. The measurement results are shown in Table 2.

[0076]

[Table 2]

The production rate of 4-hydroxybenzaldehyde was calculated from the obtained measured values and found to be 0.0338 μM / min.
(Correlation coefficient = 0.9989). Therefore, the production rate of 4-hydroxybenzaldehyde in standard serum was 0.0338.
× 2.24 = 0.0757 to 0.0757 μM / min. Then, in the same manner as above, N-benzoyl-L-2- (4-hydroxyphenyl) glycyl-L was added to 0.2 M borate buffer (pH 7.5).
-Histidyl-L-leucine was dissolved in 2 mM and sodium chloride to be 0.1 M, and the substrate solution was preheated at 37 ° C for 3 minutes or more, and then standard serum (Sigma ACE CONTRO).
LN, No. A6040) 100 μl was added, and the mixture was kept warm at 37 ° C. and the reaction solution was sampled over time to decompose the substrate by ACE to produce N-benzoyl-L-2- (4-
Hydroxyphenyl) glycine was quantified using HPLC. HPLC analysis conditions are as follows: Column: YMC-Pack A-302 S-5 12
0A ODS (6 x 150 mm), mobile phase: 40% (v / v) methanol / 0.
02M phosphate buffer (pH 6.8), flow rate: 1.0 ml / min, detection:
UV-230nm, analytical sample volume: 5 μl, N-benzoyl-L-2- eluting at retention time 5.8 minutes
(4-Hydroxyphenyl) glycine was automatically quantified using an integrator by the absolute calibration curve method using a standard sample. The results are shown in Table 3.

[0078]

[Table 3]

From the obtained measured values, N-benzoyl-L-
When the production rate of 2- (4-hydroxyphenyl) glycine is calculated, it becomes 0.0368 μM / min (correlation coefficient = 0.9978). Therefore, the substrate N-benzoyl-L- in standard serum
The decomposition rate of 2- (4-hydroxyphenyl) glycyl-L-histidyl-L-leucine, that is, ACE activity was 0.03.
From 68 × 2 = 0.0 to 0.0736 μM / min. This result is almost the same value as the production rate of 4-hydroxybenzaldehyde obtained previously. From the above results, it is understood that the ACE activity can be measured by determining the production rate of 4-hydroxybenzaldehyde. When 1 unit (U) of ACE activity is defined as the amount of enzyme that produces 1 μmol of benzoyl-L-2- (4-hydroxyphenyl) glycine in 1 minute, the value obtained by the method of the present invention is 0.0757 μM / min.
The ACE activity in this standard serum can be expressed as 75.7 mU / L.

Example 9 Benzoyl-L-2-in 0.2 M borate buffer (pH 7.5)
(4-Hydroxyphenyl) glycyl-L-histidyl-L-leucine was dissolved in 2 mM and sodium chloride was dissolved in 0.1 M to obtain a substrate solution. 100 μl of this substrate solution
In an Eppendorf tube and laccase (derived from Coriolas versicolor IFO9791 with a specific activity of 4.8 × 10 ³ U /
mg, protein concentration 2.3 mg / ml) 4 μl and add 3 at 37 ℃.
After preliminarily heating for more than 10 minutes, 100 μl of human serum was added and then kept at 37 ° C. for enzyme reaction for 50 minutes and 90 minutes, respectively. The reaction was stopped by adding 10 μl of a saturated solution of 1N hydrochloric acid in 2,4-dinitrophenylhydrazine.
After leaving at room temperature for ~ 8 minutes, 5N sodium hydroxide solution 10
μl was added for color development, and the absorbance at 500 nm was measured. As a result, the enzyme reaction time was 500 nm at 50 minutes and 90 minutes.
The respective absorbances were 0.4465 and 0.4952. From these values, the ACE activity in serum was calculated by the following formula. In addition,
The molecular extinction coefficient of the quinoid structure derived from 4-hydroxybenzaldehyde is 28200. ACE activity = (0.4952-0.4465) /40/28200×2.24=9.67×10 ^-8 M / min = 96.7m U / L

Example 10 The rates of degradation of synthetic substrate L-leucyl-L-2- (4-hydroxyphenyl) glycine by cytoplasmic aminopeptidase and microsomal aminopeptidase at various pHs were determined. The enzyme is a leucine aminopeptidase (cytoplasm) (type 3-C) manufactured by SIGMA as cytoplasm-derived aminopeptidase (abbreviated as cytoplasmic LAP).
P, pig liver-derived, protein concentration: 1.9 mg / ml), and microsome-derived aminopeptidase (microsome-derived LAP)
Abbreviated as "), leucine aminopeptidase (microsome) (type 4-S, derived from pig liver microsome, protein concentration: 2.3 mg / ml) manufactured by SIGMA was used after being diluted 5-fold.

2- (4-Hydroxyphenyl) glycine produced by enzymatic hydrolysis of the substrate was quantified by high performance liquid chromatography. The analysis conditions are shown below. Column: YMC-Pack A-302 S-5 120A ODS (60 × 150 mm) Mobile phase: 10% (v / v) MeOH / 20 mM phosphate buffer (pH 6.8) Flow rate: 1.0 ml / min Detection: UV280nm used The pH of the buffers used was pH 6.0, 6.8, 7.5, phosphate buffer, pH 8.0, 8.5, Tris monohydrochloride buffer, pH 9.0, 9.
5 used a borate buffer.

The experiment was conducted as follows. That is, L-
Leucyl-L-2- (4-hydroxyphenyl) glycine was dissolved in water to make a 3.3 mM aqueous solution, of which 50 μl
To an Eppendorf tube of 1.5 ml volume, add 50 μl of each buffer solution (final concentration, 1.65 mM) to 37 ° C.
After incubation for 10 minutes, 1 μl of each enzyme was added to start the enzyme reaction. The production rate (μmol / ml / min) was determined by quantifying 2- (4-hydroxyphenyl) glycine produced by high performance liquid chromatography at regular time intervals, and used as the enzyme activity of each enzyme. The results are shown in Table 4 below.

[0084]

[Table 4]

The above results show that the synthetic substrate L-leucyl-2
-(4-Hydroxyphenyl) glycine is derived from cytoplasmic LA
It is shown that both P 2 and LAP derived from microsomes are hydrolyzed to produce 2- (4-hydroxyphenyl) glycine. It was also found that both enzyme activities could be measured in the reaction solution at pH 6.8.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Takeo Yoshioka 1959, Kamitotsutan, Ayase-shi, Kanagawa 3-3102 Green Heights (72) Inventor Kazuhiko Okamura 2502-1 Fujisawa, Fujisawa, Kanagawa Prefecture (72) Rokuro Okamoto, Kanagawa 2-18 Hananoki, Fujisawa-shi (72) Inventor Takashi Shin, 4-5-2 Ikeda, Kumamoto-shi, Kumamoto No. 505 Yoshinaga Building (72) Sawao Murao, 2-8-12 Horikami-midori-cho, Sakai-shi, Osaka

Claims (3)

[Claims] 1. A hydroxyphenylglycine derivative represented by the following general formula [I]. [Chemical 1] (In the formula, R ¹ is a hydrogen atom, a tert-butyloxycarbonyl group, a benzoyl group or an L-leucyl group represented by the following chemical formula 2 (wherein, R ³ is a hydrogen atom, a tert-butyloxycarbonyl group or Benzyloxycarbonyl group), R ² is a hydroxyl group, a benzyloxy group or an L-histidyl-L-leucyl group represented by the following chemical formula 3 (provided that in the chemical formula 3, R ⁴ is a hydrogen atom or a lower alkyl group). It is shown).) [Chemical 3] 2. N-benzoyl-L-2- (4-hydroxyphenyl) glycyl-L-histidyl-L-leucine is reacted with angiotensin converting enzyme to produce N-benzoyl-L-2- (4-hydroxyphenyl). A method for measuring the activity of an angiotensin converting enzyme, which comprises producing glycine, converting the product into 4-hydroxybenzaldehyde by an oxidase, and quantifying the produced 4-hydroxybenzaldehyde. 3. L-Leucyl-L-2- (4-hydroxyphenyl) glycine is reacted with aminopeptidase to produce L-2- (4-hydroxyphenyl) glycine, and the product is treated with oxidase to give 4- A method for measuring the activity of aminopeptidase, which comprises quantifying 4-hydroxybenzaldehyde produced by converting it to hydroxybenzaldehyde.