JP3270127B2 - New peptides - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel peptide which inhibits the action of an angiotensin converting enzyme which is useful, for example, as a medicament, a health food or the like for preventing hypertension.

[0002]

2. Description of the Related Art Looking at the status of medical examinations by major disease in Japan, those with hypertension are increasing year by year. This is thought to be largely due to the influence of food, such as the change in Japanese eating patterns to Western-style eating. In addition, it is known that if hypertension continues for a long period of time, the incidence of heart disease and cerebrovascular disease increases, and early improvement is required.

At present, various methods are used to improve hypertension. Among them, renin, a protein hydrolase which is said to be closely related to diet, and angiotensin, a causative substance of hypertension, are used. Adjustment with is one of the most effective methods.

[0004] To regulate this renin-angiotensin system, angiotensin converting enzyme is used.
Enzyme (hereinafter referred to as ACE). ACE is a dipeptidyl carboxylase that releases a dipeptide from a peptide, and promotes the production of angiotensin II, a blood pressure increasing peptide that increases blood pressure from angiotensin I, and is involved in the degradation of bradykinin, a hypotensive substance that lowers blood pressure . Therefore, substances that inhibit this ACE action have a blood pressure lowering action.

On the other hand, it has recently been reported that ACE inhibitory peptides are present in various foods such as soybeans, rice bran and fish meat [Food Industry, Vol. 33, No. 2, 20-30 (1990), Vol.
4, 20-25 (1990), Vol. 34, No. 22, 18-26 (1991)
]. And to date, many A
A CE inhibitory peptide has been isolated.

[0006]

However, in order to isolate and purify the ACE inhibitory peptide from such foods, various chromatographies and the like must be used, the operation is complicated, and all of them have extremely low yield. There are many cases. The present inventors have already found that an ACE inhibitory peptide is present in royal jelly, and have filed a patent application for a peptide obtained by a method of hydrolyzing it with an enzyme (Japanese Patent Application No. 3-42022).

[0007] The present invention has been made in view of the above conventional technique, and an object of the present invention is to provide a novel peptide which has an ACE inhibitory action, is easy to produce, and can be obtained in a high yield.

[0008]

SUMMARY OF THE INVENTION The present invention provides a Phe-Val-Ty
peptide consisting of r-Thr-Pro made structure (below, SP5
). Note that a peptide having the structure of Leu-Tyr-Leu-Pro is hereinafter referred to as SP4, and is referred to as Trp-Lys-Tyr.
The peptide having the following structure is hereinafter referred to as SP3. Phe means phenylalanine, Val means valine, Tyr means tyrosine, Thr means threonine, Pro means proline, Leu means leucine, Trp means tryptophan, and Lys means lysine.

[0009] Chemical synthesis methods for peptides are almost established in the art, and the peptides of the present invention can also be synthesized by known methods. The amino acid used is usually in the L-form, and the side chain functional groups and the like are protected with known protecting groups, if necessary.

The structure of the obtained peptide can be confirmed by, for example, a fully automatic protein primary structure analyzer PSQ-1 manufactured by Shimadzu Corporation. In addition, amino acid composition analysis
It can be performed by a PICO-TAG system manufactured by rs.

Next, in order to use the thus obtained peptide as a food (especially a functional food) based on its ACE inhibitory action, it is necessary to use an enzymatic synthesis method instead of a chemical synthesis method due to safety issues. Is considered appropriate.

A number of excellent reviews have already been reported on the enzymatic synthesis of peptides ("Development of New Functions of Enzymes", pp. 75-129, publisher: Kodansha Scientific (1989)). There is difficulty in synthesis depending on the type of bond, and it is necessary to select an appropriate enzyme according to the type of peptide bond. Therefore, unlike the chemical synthesis method, the enzyme synthesis method is not a complete method and is not yet an established technique, and it is necessary to carry out a detailed study for each target peptide.

The enzymes used include proteases such as trypsin, chymotrypsin, papain, pancreatin, bromelain, proline endopeptidase,
Bacterial proteases such as subtilisin, thermolysin, nagase, protease A, protease B,
Protease M, protease N, protease P, protease P-6 and the like are suitable.

As a protecting group for preventing the functional group at the C-terminal of the amino acid from participating in the reaction, a short-chain alkyl group such as methyl, ethyl and propyl and an amide-type protecting group are used. An acyl group such as benzoyl and a urethane-type protecting group such as benzyloxycarbonyl and t-butoxycarbonyl are used. The reaction solvent uses a known buffer according to the optimum pH of the enzyme, and appropriately adds methanol, ethanol, propanol, butanol, glycerin, ethylene glycol,
Organic solvents such as 1,4-butanediol, dimethylformamide, dimethylsulfoxide and the like can be added.

[0015]

EXAMPLES (Example 1) A peptide having a structure of T rp-Lys-Tyr (S
P3) shows a method for synthesizing an enzyme.

[0016] 40 ml of glycerin and a carbonate buffer (pH 10.
4) 1.3 g of L-acetyltryptophan methyl ester (0.1 M final concentration) and 0.98 g of L-lysine methyl ester hydrochloride (0.1 M final concentration) were added to 7 ml of the mixed solvent to prepare a suspension. Then, while vigorously stirring at room temperature, a solution of 62.5 mg of α-chymotrypsin (final concentration: 50 μM) dissolved in 3 ml of a carbonate buffer was added dropwise over about 2 minutes, followed by stirring for further 3 minutes, and then adding acetic acid. By stopping the reaction, a kinetic control reaction in which the reaction proceeds linearly was performed.

After neutralization with aqueous ammonia, 100 ml of ethyl acetate was added, and the mixture was washed with a saturated sodium hydrogen carbonate solution and further washed several times with a saturated saline solution. Next, 50 ml of a 10% citric acid solution was added, and the aqueous layer was extracted, made basic again with sodium hydrogen carbonate, and re-extracted with ethyl acetate. The ethyl acetate layer was washed with saturated saline, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained crude product is passed through a short column of silica gel, and chloroform: methanol:
Elution was carried out with a mixed solvent of triethylamine = 51: 1: 0.1, and Ac-Trp-Lys-OMe was obtained as colorless crystals in an amount of 1.59 g (83
%).

Next, 2 ml of ethanol and the above-mentioned carbonate buffer
In 3 ml of the mixed solvent, 232 mg of Trp-Lys-Tyr-OMe (final concentration: 0.1 M), L-tyrosine ethyl ester hydrochloride 1
48 mg (final concentration 0.1 M) was added to obtain a suspension. Then, while vigorously stirring at room temperature, a solution of 3 mg of trypsin (final concentration: 20 μM) dissolved in 1 ml of a carbonate buffer was added dropwise over about 2 minutes, and the mixture was further stirred for 3 minutes, and acetic acid was added to stop the reaction. A kinetic control reaction was performed.

The yield of this reaction was determined by liquid chromatography (HPLC). That is, methanol was added to the reaction mixture to make 100 ml, and 5 μl thereof was
A chromatogram was obtained by injection into C. Then, the yield was determined from the area ratio with the chemically synthesized standard product. Ac-Trp-Lys- was obtained at a good yield of about 68%.
It was found that Tyr-OEt was obtained. In addition, HP
LC measurement conditions are as follows.

Column: ODP-50, φ4.6 × 150 mm,
Mobile phase: mixed solvent of acetonitrile / water (30/70), detection:
Ultraviolet (UV) 280 nm, temperature: room temperature, flow rate: 0.6 ml / min
The protected peptide purified according to a conventional method was subjected to deprotection of the C-terminal and N-terminal protecting groups by a known base or acid hydrolysis method to obtain SP3 as colorless crystals. The molecular weight of the obtained peptide was measured using a liquid chromatography mass spectrometer (trade name: LC-MS, QP1000EX manufactured by Shimadzu Corporation). Equipment (Wate
Structural analysis was performed using rs Co., Ltd. (trade name: PICO-TAG system), and it was found to be Trp-Lys-Tyr.

As described above, SP3 can be obtained relatively easily and in good yield by the enzyme synthesis method. And AC
This peptide having E-inhibiting action is used as a pharmaceutical composition or a health food for the purpose of preventing hypertension, alleviating the tendency of hypertension or adjusting blood pressure, as a composition having an appropriate form and form together with a suitable non-toxic carrier for oral administration. Can be used. (Example 2) Next, the Phe-Val-Tyr-Thr of the present invention was used.
A method for producing a peptide (SP5) having a structure of -Pro by a chemical synthesis method will be described. The abbreviations are as follows.

Boc: tert-Butoxycarbonyl CBZ: Benzyloxycarbonyl AAn: Amino acid having a substituent at the n-th atom from the C-terminal First, proline methyl ester, which is the C-terminal amino acid, is dissolved in dichloromethane and CBZ- or Boc-AA
₂ Add (threonine) (1 equivalent), N-hydroxysuccinimide (1.2 equivalent), dicyclohexylcarbodiimide (1.2 equivalent), and conduct a condensation reaction by stirring in a refrigerator for 20 to 40 hours. Was. After the completion of the reaction, insolubles (dicyclohexylurea) were distilled off, and the organic layer was separated by 5 parts.
Wash with 10% sodium carbonate, water, 10% citric acid, water,
After drying over anhydrous sodium sulfate, the mixture was concentrated under reduced pressure to obtain the dipeptide Boc-Thr-Pro methyl ester. In addition, you may refine | purify by silica gel column chromatography as needed.

Next, one equivalent of each of tyrosine methyl ester and the above dipeptide was condensed under the above-mentioned condensation conditions to obtain the tripeptide Tyr-Thr-Pro. Further, the produced tripeptide and Boc-Val were similarly condensed to obtain a tetrapeptide Val-Tyr-Thr-Pro.
Finally, the tetrapeptide and Boc-Phe were similarly condensed. The N-terminal protecting group was removed by catalytic reduction in the case of a CBZ group and by trifluoroacetic acid in the case of a Boc group. Thereafter, the same operation was repeated, and finally, the side chain protecting group and the methyl ester at the C-terminus were removed by a conventional method, whereby the target peptide SP5 was obtained. The yield of this peptide was good at 39%.

Thus, SP5 can be obtained easily and in good yield by the chemical synthesis method. The structure of the obtained peptide was confirmed using a fully automatic protein primary structure analyzer PSQ-1 manufactured by Shimadzu Corporation. Also, Waters' PICO-T
Amino acid composition analysis was performed by the AG system. The results are shown below. (Primary structure of SP5) Phe-Val-Tyr-Thr-Pro (amino acid composition of SP5) Phe19.9%, Val20.4%, Tyr20.7%, Thr19.8%, Pro19.4% (Example 3) Next, in the same manner as in example 2, the process for producing be described by chemical synthesis of peptides (SP4) with L eu-Tyr-Leu-Pro made structure.

In Example 2, CBZ- or Boc-
AA ₂ and (threonine) (1 eq) in place of CBZ- or Boc-AA ₂ (leucine) (1 eq) to give the dipeptide (Boc-Leu -Pro) by a condensation reaction. Next, a tyrosine methyl ester was added thereto and a condensation reaction was carried out to obtain a tripeptide (Tyr-Leu-Pro). Finally, Boc-Leu was added to this and a condensation reaction was carried out to obtain a target tetrapeptide SP4.

The yield was as good as 47%. This peptide was analyzed for primary structure and amino acid composition. The results are shown below. (Primary structure of SP4) Leu-Tyr-Leu-Pro (Amino acid composition of SP4) Leu49.8%, Tyr25.8%, Pro24.4% (Example 4) Next, as in Example 2 , T A method for producing a peptide (SP3) having a structure of rp-Lys-Tyr by a chemical synthesis method will be described.

In Example 2, tyrosine methyl ester was used in place of proline methyl ester, and CBZ- or Boc-AA ₂ (threonine) (1 equivalent) was replaced with C
BZ- or Boc-AA ₃ (lysine) (1 equivalent)
A dipeptide (Boc-Lys-Tyr) was obtained by the condensation reaction. Next, Boc-Trp was added thereto to cause a condensation reaction,
The target tripeptide SP3 was obtained.

The yield of this tripeptide was as good as 53%. This peptide was analyzed for primary structure and amino acid composition. The results are shown below. (Primary structure of SP3) Trp-Lys-Tyr (Amino acid composition of SP3) Trp was decomposed and could not be measured by the PICO-TAG system. Therefore, the remaining Lys was 51.1% and Tyr was 48.9%. The measurement result of the three components by the PSQ-1 was Trp 3
2.1%, Lys 34.8% and Tyr 33.1%. (Example 5) Next, measurement of the ACE inhibitory activity of the peptide will be described. (Measurement of ACE inhibitory activity) 1 g of rabbit lungs were precipitated in acetone, dried powder (Sigma) was dissolved in 5 ml of phosphate buffer (pH 8.3), and centrifuged at 10,000 G for 30 minutes. After the treatment, the supernatant is diluted 3-fold with the above buffer, and ACE is diluted.
As an enzyme solution, enzyme inhibition was measured. The measurement method is Bio
chem.Pharm., 20, p1637-1648 (1971) and Anal.Bioch
em., 84, p361-369 (1978).

That is, a 100 mM potassium phosphate buffer solution (300
pH 8.3) containing 1 mM mM sodium chloride
100 μl of mM tripeptide (Hip-His-Leu, manufactured by Peptide Research Laboratories), 100 μl of ACE enzyme solution and 100 μl of sample solution
Add 30 μl, react at 37 ° C for 30 minutes, and add
This is a method in which the reaction is terminated by heating for a minute, the hippuric acid of the reaction product is derivatized with a trichlorotriazine reagent, and the absorbance at a measurement wavelength of 382 nm is colorimetrically determined.

The inhibition rate was calculated by the following equation. Inhibition rate = (E _O −E _S ) / E _O × 100 (%) E _O : Absorbance at 382 nm when no inhibitor is contained E _S : Absorbance at 382 nm when an inhibitor is contained And an inhibition rate of 50% The sample concentration at the time is IC ₅₀ (μg / m
l) The lower the IC ₅₀ value, the higher the ACE inhibitory activity. As a comparative example, Japanese Patent Application No. Hei 3-4
No. 2022, namely Tyr-Asn-Glu
_{-Val -Pro (RJP 5), Ser} -Leu -Pro -Lys -
_{Leu -His -Glu -Trp (RJP 8)} , Ser -Leu -Pr
was measured in the same manner as in IC ₅₀ also o -Ile -His -Glu -Trp -Lys (RJP 9). As a result, the results shown in the following Table 1 were obtained.

[0031]

[Table 1]

As shown in Table 1, each of Examples 5-1 to 5-3
The value of the IC ₅₀ of approximately equally low as the control example 5-4～ 5-6, it is found to have sufficient ACE inhibitory action.

[0033]

Peptides of the present invention as described in detail above, according to the present invention is a novel peptide, which has a angiotensin enzyme inhibitory activity, easy high yield obtained preparation,
It has an excellent effect that it is useful as a pharmaceutical or food for the purpose of preventing or treating hypertension.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C07K 7/06 CA (STN)

Claims (1)

(57) [Claims] From [claim 1] Phe-Val-Tyr-Thr- Pro made structure
Made peptide.