JPH0676436B2 - Novel bioactive peptide - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel bioactive peptide.

[Conventional technology]

Since 1970, with advances in purification technology and analytical methods, peptide P, neuterotensin, vasoactive intestinal polypeptide, and morphine that act in a manner similar to conventional amine-based neurotransmitters from mammalian brain and digestive tract Since the discovery of a group of physiologically active peptides such as the endogenous peptides β-endorphin, methionine enkephalin, leucine enkephalin, α-neo-endorphin, and dynorphin, which have similar activities, Is being actively conducted.

[Problems to be solved by the invention]

However, on the other hand, it has been desired to develop a novel peptide having physiological activity.

[Means for solving problems]

In such an actual situation, the present inventor has extracted various physiologically active peptides from porcine spinal cord and investigated their primary structure and biological activity. It was found that there is a novel compound described above, and it was successfully produced by synthesis.

That is, the present invention has the general formula X-Tyr-Phe-Leu- Phe-Arg-Pro-Arg-Asn-NH 2 ( wherein, X is H or H-Phe-Lys-Val- Asp-Glu-Glu
-Phe-Gln-Gly-Pro-Ile-Val-Ser-Gln-Asn-Arg
-Arg-)) (hereinafter, X is H is referred to as peptide (1), X is H-Phe -...- Arg- is referred to as peptide (2)) and salts thereof. To do.

In the present specification, the abbreviations in the compounds of the present invention are commonly used in the art and have the following meanings.

Tyr; L-tyrosine, Phe; L-phenylalanine Leu; L-leucine, Arg; L-arginine Pro; L-proline, Asn; L-asparagine Lys; L-lysine, Val; L-valine Asp; L- Aspartic acid, Glu; L-glutamic acid Gln; L-glutamine, Gly; Glycine Ile; L-isoleucine, Sre; L-serine The novel bioactive peptide of the present invention can be extracted or isolated from porcine spinal cord or synthesized. You can get it.

To extract and isolate the peptide of the present invention from porcine spinal cord, for example, porcine spinal cord is homogenized in a suitable acidic solvent, for example, formic acid aqueous solution, acetic acid aqueous solution, etc. to obtain an extract by centrifuging insoluble matter, and then This extract is subjected to fractional precipitation with an organic solvent, solvent extraction, dialysis, ultrafiltration, gel filtration, ion exchange chromatography, adsorption chromatography, high performance liquid chromatography, and other known means used for peptide purification. It may be used to obtain a substance having a corresponding molecular weight.

However, industrially, it is more advantageous to obtain it by synthesis in terms of raw material acquisition, mass production, and price.

The peptide of the present invention can be synthesized by a solid phase method or a liquid phase method commonly used for peptide synthesis. [Nobuo Izumiya et al., "Peptide Synthesis", 1984, Maruzen Co., Ltd., Luminescence; The Chemical Society of Japan, "Biochemistry Laboratory (I) / Protein Chemistry" 4
Volume, pp. 208-495, 1977, published by Tokyo Kagaku Dojin].

For example, when the solid phase method is applied to the present invention, it is preferable to use benzhydrylamine resin (polystyrene) as the support for obtaining the peptide amide. In addition, the α-amino group of the amino acid used is protected by a tert-butyloxycarbonyl group (Boc group) in all cases, and aspartic acid, β of glutamic acid, and γ carboxylic acid are benzyl group (Bzl group) and arginine. Guazino group is a tosyl group (T
os group), tyrosine hydroxyl group is 2,6-dichlorobenzyl group (Cl ₂ -Bzl group), lysine ε-amino group is benzyloxycarbonyl group (Z group), serine hydroxyl group is benzyl group (Bzl group) It is preferable to protect each with. Furthermore, the condensation of protected amino acids is carried out by the dicyclohexylcarbodiimide (DCC) method or the DCC acid anhydride method,
For -Asn-OH and Boc-Gln-OH, it is preferable to use the active ester method.

For example, in the peptide of the present invention, first, the C-terminal amino acid asparagine is introduced into a benzhydrylamine resin by using its protected derivative, Boc-asparagine-p-nitrophenyl ester, and then the amino acid is sequentially extended to form a protected peptide resin. After the synthesis, the crude synthetic peptide can be obtained by treating it with hydrofluoric acid (HF), and further purifying the peptide if necessary.

Purification of the crude synthetic peptide can be carried out by gel filtration, ion exchange chromatography, reverse phase high performance liquid chromatography (RP-HPLC), etc. according to conventional methods. Also,
The salt of the peptide of the present invention can be obtained by dissolving the peptide in an equimolar amount of acetic acid, citric acid, tartaric acid, fumaric acid, maleic acid or the like, and then freeze-drying.

Confirmation that the peptide thus extracted or synthesized has the structure of peptide (1) or peptide (2) can be confirmed by amino acid composition by an amino acid analyzer and amino acid sequence by Edman's brothein sequencer. Phenylthiohydantoin (PTH) amino acids obtained by decomposition were analyzed by reverse phase high performance liquid chromatography. The C-terminal amide was verified by digesting the purified peptide with trypsin, dansylating with dansyl chloride, and confirming the formation of dansyl asparagine amide by analysis by two-dimensional thin layer chromatography on a polyamide sheet.

The physicochemical properties of peptides (1) and (2) of the present invention are as follows.

[Action] The peptide of the present invention has an activity of contracting smooth muscle (rat uterine muscle) and an action of increasing blood pressure.

The biological properties of the peptides of the present invention were tested by the following methods. The results are shown in FIG.

(Test method) Rat [wister] Female uterine muscle was removed,
It was immersed in a Rock-Ringer solution using a 3 ml organ bath. Rock-Ringer solution in organ bath contains 95% O ₂ −
The temperature was kept at 32 ° C by passing 5% CO ₂ gas. A static pressure of 1 g was applied to the muscle sample, and the muscle sample was allowed to stand for about 1 hour. When the automatic movement of the muscle was stabilized, the peptide of the present invention was administered and the contraction of the muscle was measured for 3 to 4 minutes [isotonic transducer. : Model ME-40
12 Made by ME Commercial Co., Ltd.). After the measurement, the organ bath was washed immediately, and this was repeated every 20 to 30 minutes. A predetermined amount of the test substance was dissolved in physiological saline and administered.

(Results) As is clear from FIG. 2, the peptide (1) having 8 amino acid residues showed activity at 1.0 nM. On the other hand, the peptide (2) having 25 amino acid residues has a stronger activity than that of (1) and has a concentration of 0.35 nM.
The activity was shown at 0.7 nM, and at 0.7 nM, the duration of the activity was remarkably long. This characteristic can be effectively used for elucidation of physiological effects.

〔The invention's effect〕

Conventionally, many physiologically active peptides having an activity of contracting or relaxing smooth muscle such as digestive tract, uterus, vas deferens, gallbladder, and blood muscle are known. For example, substance P, neurotensin, which has a strong effect on contraction of guinea pig ileum,
Cholecystokinin, Neuromedin K, Neuromedin L,
Neuromedin N, catsinin and the like are known, and oxytocin, bradykinin, gastrin-releasing peptide, Neuromedin B, Neuromedin C, bombesin and the like are known to have strong effects on rat uterine muscle contraction. Further, these substances are known to have not only smooth muscle contraction and relaxation but also unique physiological actions in vivo, and it is considered that the peptide of the present invention also plays a new physiological role in vivo.

Furthermore, the amino acid sequence of a conventionally known physiologically active peptide is, for example, H-Arg-Pro-Lys-Pro in substance P.
-Gln-Gln-Phe-Phe- Gly-Leu-Met-NH 2, in the oxytocin For bradykinin, H-Arg-Pro-Pro-Gly-Phe-Ser-
Pro-Phe-Arg-OH is completely different from the amino acid sequence of the physiologically active peptide of the present invention.

Therefore, the peptide of the present invention is novel, has physiological activity, and is useful for elucidation of physiological action.

〔Example〕

 Next, the present invention will be described with reference to examples.

Example 1 2 kg of spinal cord 20 kg (for 550 animals) extracted from a pig immediately after being slaughtered
The endogenous protease was inactivated by boiling for 10 minutes in 1N-acetic acid containing double amount of 20 mM hydrochloric acid. After cooling this, it was extracted by homogenizing with a Polytron mixer at 4 ° C. The obtained extract is centrifuged at 12,000 x G for 30 minutes, and the supernatant is about 40
Obtained. Further, the floating fat was removed with a GF / B glass filter (manufactured by Wattman), and the mixture was desalted and concentrated by ultrafiltration (UM-2, manufactured by Amicon) to a final liquid volume of 3.3. Acetone was added dropwise to this adjustment solution at 4 ° C to a final concentration of 75
Acetone precipitation was performed by adjusting the ratio to%, and this was then centrifuged to obtain about 13 supernatants. The obtained supernatant was concentrated to dryness under reduced pressure, and the residue was dissolved again in 1N-acetic acid and freeze-dried. This dried product was dissolved in 1N-acetic acid 2 and equilibrated with 1N-acetic acid SP-Sephadex C-25.
Treated with 400 ml, the acidic fraction (SP-
I), neutral weak base fraction (SP
-II), a basic fraction (SP-III) eluted with 2M-pyridine-acetic acid (pH 5.0) was obtained.

This SP-III fraction was lyophilized to obtain a dried product 10.
5 g was obtained, and this was dissolved in 1N acetic acid to obtain Sephadex G-
Gel filtration (4.5 × 140 cm; flow rate 40 ml / hour; fraction size 50 ml / tube) was divided into 5 parts by 50. Here, the rat uterine muscle was assayed, and the fraction having contractile activity was freeze-dried, dissolved in 1N-acetic acid, and further subjected to gel filtration (7.5 × 135 cm; flow rate 6) with Sephadex G-25.
0 ml / hour; fraction size 100 ml / tube). Again, rat uterine muscle was assayed to obtain fractions A to F as shown in FIG.

The peptide (1) can be obtained by purifying the F fraction.

The F fraction was further subjected to cation exchange HPLC. Conditions [Column: TSK gel CM-2sw, 4.0 x 250 mm (made by Toyo Soda);
Flow rate 1.0 ml / min; solvent system (A) 10 mM HCOONH ₄ (pH 6.6): acetonitrile = 90:10, (B) 1.0 M HCOONH ₄ (pH 6.6): acetonitrile: 90:10, (A): ( From (B) = 100: 0 to (A): (B) = 8
Straight line gradient up to 5:15 for 48 minutes (A): (B) = 85: 1
A 48 minute linear gradient was applied from 5 to (A) :( B) = 50: 50. By this HPLC, a fraction having uterine muscle activity was obtained at a retention time of 103 to 105 minutes. This fraction was further subjected to reverse phase HPLC. Conditions [Chem ChemSorp (Chem
cosorb) 5 ODS-H, 4.6 x 250 mm (Chemco); flow rate 1 m
l / min, solvent system (A) water: acetonitrile: 10% trifluoroacetic acid (TFA) = 90: 10: 1, (B) water: acetonitrile: 10%
A linear gradient of 80 minutes from TFA = 40: 60: 1, (A) :( B) = 100: 0 to (A) :( B) = 0: 100 was applied. ] The major peak of retention time 39 minutes was collected to obtain a substantially pure peptide.
About 1 μg of (1) was obtained.

The peptide of (2) is obtained from the B fraction of Sephadex G-25. 2.6 g of the B fraction was dissolved in 10 ml of 1N acetic acid and subjected to reverse phase HPLC in seven portions. Conditions [Column: TSK gel OD
S-120A, 20 × 250 mm (Toyo Soda); Flow rate 5 ml / min;
Solvent system (A) water: acetonitrile: 10% TFA = 90: 10: 1, (B)
Water: acetonitrile: 10% TFA = 40: 60: 1, (A) :( B) = 90: 1
A 250 minute linear gradient from 0 to (A) :( B) = 30: 70 was applied. By this HPLC, a fraction having uterine muscle activity was obtained at a retention time of 128 to 136 minutes. This fraction was then subjected to cation exchange HPLC. Conditions [Column; TSK gel CM-
2sw, 4.6 × 250 mm (manufactured by Toyo Soda); flow rate 1 ml / min; solvent system (A) 10 mM HCOONH ₄ : acetonitrile = 90:10, (B) 1.
0M HCOONH ₄ : acetonitrile = 90:10, (A): (B) = 100:
A 100 minute linear gradient from 0 to (A) :( B) = 50: 50 was applied. ] A fraction having uterine muscle activity was obtained at a retention time of 68 to 71 minutes. Further, this fraction was subjected to reverse phase HPLC. Conditions [column; Chemcosolve 7-diphenyl,
4.6 × 250 mm (made by Chemco); flow rate 1.5 ml / min, solvent (A) water:
Acetonitrile: 10% TFA = 90: 10: 1 (B) Water: Acetonitrile: 10% TFA = 40: 60: 1, (A): (B) = 80:20 to (A): (B) =
A 128 minute linear gradient to 0: 100 was applied. A fraction having uterine muscle activity was obtained at a retention time of 27 to 8 minutes. The active fraction was subjected to reverse phase HPLC for final purification. Conditions [Column; ChemcoSorp 3 ODS-H, 4.6 x 75 mm
(Chemco); flow rate 1 ml / min; solvent system (A) water: acetonitrile: 10% TFA = 90: 10: 1 (B) water: acetonitrile: 10% T
FA = 40: 60: 1, (A): (B) = 80:20 to (A): (B) = 0: 100 19
A 2-minute linear gradient was applied. ] Retention Time 3
The major peak at 4 minutes was collected to obtain about 3.5 μg of substantially pure peptide (2).

Example 2 1 g (0.4meq NH ₂ / g) of benzhydrylamine resin was swelled well in 5 ml of dimethylformamide (DMF), and Boc was added to it.
-Asparagine-p-nitrophenyl ester 0.71 g
5 ml of a DMF solution (2.0 mmol) was added, and the mixture was gently stirred at room temperature and reacted for 6 hours. The mother liquor is removed and the resin is washed 3 times with DMF. The same reaction was repeated again. The progress and completion of the reaction were monitored by Kaiser test with ninhydrin.
Then, the resin was washed with DMF, methylene chloride, isopropyl alcohol, and methylene chloride in this order and dried.

In the synthesis of a protected peptide resin, all the α-amino groups of each constituent amino acid are protected by a Boc group, and among the active side chains, the hydroxyl group of tyrosine is a dichlorobenzyl group (Cl ₂ -Bz
l), the guadino group of arginine is a tosyl group (Tos),
The ε-amino group of lysine is a benzyloxycarbonyl group
The aspartic acid β-carboxylic acid was protected with a benzyl group (Bzl), the glutamic acid γ-carboxylic acid was protected with a Bzl group, and the serine hydroxyl group was protected with a Bzl group.

For the condensation of protected amino acids, the Boc group, which is the terminal amino group protecting group of the protected peptide bound to the resin, is treated with 50% trifluoroacetic acid in methylene chloride at room temperature for 20 minutes, and repeated twice. Completely removed. Then, the amino group released by de-Bocization was condensed with the carboxyl group of the Boc-protected derivative of the amino acid located next in the amino acid sequence of the target peptide. In the condensation of this protected anomic acid
Boc-Asn and Boc-Gln were used as p-nitrophenyl ester in an amount of 5 equivalents, and the reaction of reacting for 10 hours was performed twice. Other Boc-amino acids were condensed in 5 equivalents using dicyclocarbodiimide as a condensing agent. When the reaction was not completed by this operation, the same operation was repeated.
The progress and completion of the reaction were monitored by Kaiser test with ninhydrin.

In this way, 1 g of benzhydrylamine gave Boc-Tyr
_{(Cl 2 -Bzl) -Phe-Leu} -Phe-Arg (Tos) -Pro-Arg
A part (500 mg) of (Tos) -Asn-NH-benzhydryl resin [hereinafter referred to as the protected peptide resin (A)] was taken out at the stage of synthesis. The rest is further subjected to an N-terminal extension reaction and Boc-Phe-Lys (Z) -Val-Asp (OBzl) -Glu.
(OBzl) -Glu (OBz) -Phe-Gln-Gly-Pro-Ile-Val
-Ser (Bzl) -Gln-Asn-Arg (Tos) -Arg (Tos)-)
_{Tyr (Cl 2 -Bzl) -Phe-} Leu-Phe-Arg (Tos) -Pro-A
1460 mg of rg (Tos) -Asn-NH-benzhydryl resin [hereinafter referred to as the protected peptide resin (B)] was obtained.

The peptide of (1) was released from the resin and purified by the following method. Protected peptide resin (A) 300 mg anisole
Put it in a HF reactor together with 1.5 ml and introduce 8 ml of HF at 0 ° C.
And reacted for 60 minutes. Then, excess HF was distilled off, and ether was used.
Anisole is removed by washing with 25 ml, and 14.2 ml of 1N-acetic acid is added to extract the product. The resin and insoluble matter were separated, further diluted 10 times with water, and then adsorbed on a column (2.0 × 40 cm) packed with 90 ml of ODS resin [LC-sorb (manufactured by Chemco)],
After washing well with N-acetic acid, elute with 200 ml of 60% acetonitrile containing 0.1% trifluoroacetic acid (TFA). The eluate was distilled off under reduced pressure and then freeze-dried to obtain about 90 mg of crude peptide (1). This was dissolved in 6 ml of 0.1% trifluoroacetic acid and divided into 6 times and subjected to reverse phase HPLC. Condition [Column: T
SK gel ODS120A, 2.0 × 250 cm; flow rate 5 ml / min; solvent system (A)
Water: Acetonitrile: 10% TFA = 90: 10: 1, (B) Water: Acetonitrile: 10% TFA = 40: 60: 1, (A): (B) = 75:25 to (A):
(B) A 100-minute linear gradient of 25:75 was applied. ]
This operation was repeated 6 times to collect the main peak. Acetonitrile was distilled off from this fraction under reduced pressure and freeze-dried to obtain the desired peptide (1) H-Tyr-Phe-Leu-Phe-Arg-Pr.
58.2 mg of o-Arg-Asn-NH ₂ was obtained.

The peptide (2) was released from the resin and purified by the following method. Anisole 2 with 600 mg of protected peptide resin (B)
It is put in a reactor of HF together with 10 ml of HF, and 10 ml of HF is introduced, and it is 60 at 0 °
It was made to react for minutes. Then, after distilling off excess HF, 30 ml of ether
After washing with water to remove anisole, 24.2 ml of 1N-acetic acid was added to extract the product. Separate the resin and insoluble matter, and further with water.
90 ml ODS resin after 10-fold dilution [LC-sorb (Chemco)]
Adsorbed on a column (2.0 × 40 cm) packed with EDTA, washed well with 0.1N-acetic acid, and washed with 60% acetonitrile containing 0.1% TFA 200 ml
Elute with. As the eluent, acetonitrile was distilled off under reduced pressure and freeze-dried to obtain about 228 mg of crude peptide (2). This crude peptide (62.2 mg) was dissolved in 7 ml of TFA and divided into 5 times and subjected to reverse phase HPLC. Conditions (column; TSK gel, ODS120A, 2.0 x 25 cm;
Flow rate 5 ml / min, solvent system (A) water: acetonitrile: 10% TFA
= 90: 10: 1, (B) water: acetonitrile: 10% TFA = 40: 60: 1,
A 100-minute linear gradient from (A) :( B) = 75: 25 to (A) :( B) = 25: 75 was applied. This operation was repeated 5 times to collect the main peak. Acetonitrile was distilled off from this fraction under reduced pressure and freeze-dried to obtain the desired peptide (2) H-Phe-Ly.
s-Val-Asp-Glu-Glu-Phe-Gln-Gly-Pro-Ile-Va
l-Ser-Gln-Asn-Arg-Arg-Tyr-Phe-Leu-Phe-Ar
the g-Pro-Arg-Asn- NH 2 was obtained 11.5mg.

[Brief description of drawings]

FIG. 1 is a drawing showing the results of gel filtration of the basic fraction (SP-III) with Sephadex G-25 in Example 1, showing the absorption curve at 280 nm and the relative activity. FIG. 2 is a drawing showing changes over time in contraction length when the peptide of the present invention was administered to a rat uterine muscle specimen.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tetsuji Sudo 3-13-5 Nihonbashi, Chuo-ku, Tokyo Inside Daiichi Pure Chemicals Co., Ltd. (56) References [Peptides (Fayetter file, NY) 6 ( Suppl. 3) 245-248 (1985) [Pept. Struct. Func t. , Proc. Am. Pept. Sym p. , 9th 643-646 (1985) [Biochem. Biophys. Res. Commun] 130 (3), 1078-1085 (1985)

Claims (1)

[Claims] 1. A general formula X-Tyr-Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH ₂ (wherein X is H or H-Phe-Lys-Val-Asp-Glu-Glu.
-Phe-Gln-Gly-Pro-Ile-Val-Ser-Gln-Asn-Arg
-Arg-) and a salt thereof.