JPS61268700A - Novel physiologically active peptide - Google Patents

Abstract

NEW MATERIAL:The compound of formula I (X is H or group of formula II) or its salt. USE:Smooth muscle contracting agent and hypertensive agent. PREPARATION:For example, the spinal marrow extirpated from swine is boiled in an aqueous solution of acetic acid to deactivate protease contained therein, and extracted by homogenizing with a mixer, etc. The extracted liquid is centrifuged to remove the insoluble components and purified to obtain the peptide of formula by the peptide-purification processes such as fractional precipitation with organic solvent, extraction with solvent, dialysis, high-performance liquid chromatography, etc. As an alternative method, the peptide can be produced on an industrial scale by peptide synthesis process.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to novel physiologically active peptides.

[Conventional technology]

Since 1970, with advances in purification technology and analytical methods, peptides such as Saftance P, Neuroti/Syn, and Panactive Intestai, which have effects similar to conventional amine neurotransmitters, have been extracted from the brain and gastrointestinal tract of mammals. Null polypeptides and endogenous peptides that exhibit morphine-like effects
Since the discovery of some peptides with physiological activities such as β-endorphin, methionine enkephalin, leucine enkephalin, α-neo-endorphin, and dynorphin, research on the physiological effects of peptides has been actively conducted. .

[Problem that the invention seeks to solve]

However, on the other hand, there has been a desire to develop new peptides with physiological activity.

[Means for solving problems]

Under such circumstances, the present inventor extracted various physiologically active peptides from pig chains and investigated their secondary structures and biological activities. We have discovered the existence of a new compound shown by the following formula and succeeded in producing it synthetically.

That is, the present invention is based on the general formula % formula % (wherein, X is H or 1d H-Phe-Lya-val-
Asp-Glu-Glu-Ph e-Gl n-G1
y-Pro o-I 1 e -Va 1-8e r-G
l n-As n-Ar g-Arg-) [hereinafter, gold peptide (1) where X is H;
peptide (2)] and salts thereof.

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

Tyr; L-tyrosine, Phe; L-phenylalanine LeuHL-oyl, ArgHL-arginy/Pro; L-proly, Asn; L-
Asparagine Lys; L-lysyl, ValHL-valine Asp; L-aspartic acid, Glu:
L-glutamic acid Gin; L-glutamine, Gly
; Glycine 11e ; L-ia0ish: / ,
Ser: L-SelJy The novel physiologically active peptide of the present invention can be obtained by extraction and isolation from pig marrow or by synthesis.

To extract and isolate the peptide of the present invention from the pig marrow, for example, the pig marrow is homogenized in an appropriate acidic solvent, such as a formic acid aqueous solution or an acetic acid aqueous solution, and an extract is obtained by centrifuging the insoluble matter. Then, this extract was subjected to a method known per se for peptide purification, such as fractional precipitation with an organic solvent, solvent extraction, dialysis, ultrafiltration, gel filtration, ion exchange chromatography, adsorption chromatography, and high performance liquid chromatography. A substance having a corresponding molecular weight can be obtained using the following methods.

However, from an industrial perspective, obtaining it by synthesis is more advantageous in terms of availability of raw materials, mass production, and price.

The peptide of the present invention can be synthesized by a liquid phase method, which is a phase method commonly used for peptide synthesis. [Peptide Synthesis J 1984, published on September 11, by Nobuo Senian et al.; Edited by the Chemical Society of Japan, “Biochemistry Experiment Course (I)/Chemistry of Proteins”
4, pp. 208-495, 1977, published by Tokyo Kagaku Dojin].

For example, when a solid phase method is applied to the present invention, benzhydrylamine resin (polystyrene) is preferably used as a support for obtaining a peptide amide. In addition, the α-amino group of the amino acid used is ter
Protected with t-butyloxycarbonyl group (BoC group),
The β and γ carboxylic acids of aspartic acid and glutamic acid have a be/zyl group (Bzl group), the guazino group of arginine and glutamic acid has a tosyl 4 (Tos group), and the hydroxyl group of tyrosine has a 2,6
-Cyclolpe/zyl group (CL, -Bzl group), ε-amino group of lysine is benzyloxycarbonyl group (2 groups).

It is preferable that each of the hydroxyl groups of serine be protected with a benzyl group (Bzl group). Furthermore, condensation of protected amino acids can be carried out using dicyclohexylcarbodiimide (D-CCI method or D
By the acid anhydride method by CC, Boc-Aan-OH
, BoC-Gin-OH, it is preferable to use the active ester method.

For example, in the peptide of the present invention, the C-terminal amino acid asparagine is first introduced into a benzhydrylamine resin using its protected derivative Boc-asparagine-p-nitrophenyl ester, and then the amino acids are sequentially extended to synthesize a protected peptide resin. After death, this was mixed with hydrofluoric acid (HF).
) to obtain a crude synthetic peptide, which can be further purified if necessary to construct its structure.

Purification of the crudely synthesized peptide can be carried out according to conventional methods such as gel filtration, ion exchange chromatography, reversed phase high performance liquid chromatography (RP-HPLC), and the like.

Further, the salt of the peptide of the present invention can be obtained by dissolving the peptide in about equimolar amounts of acetic acid, citric acid, tartaric acid, fumaric acid, maleic acid, etc., and then freeze-drying the solution.

The peptide thus extracted or synthesized is peptide (1)
Or, to confirm that the peptide has the structure of peptide (2), the amino acid composition was determined using an amino acid analyzer, and the amino acid sequence was subjected to shed fluorolysis using a gas-phase protein sequencer. (P.T.
This was done by analyzing HI amino acids using reverse phase high performance liquid chromatography. The C-terminal amide was verified by digesting the purified peptide with trypsin, dansylating it with dansyl chloride, and confirming the formation of dansyl-as-laginamide by two-dimensional thin layer chromatography analysis using a polyamide sheet.

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

Summer This shows an example of amino acid analysis results, and the numbers indicate molar ratios.

[Effect]

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

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

(Test Method) The myometrium of a female rat (Wistar) was removed and immersed in Rock-Ringer solution using a 3Wll organ pass. A 95% 02-5% CO gas was passed through the Rotsu Ringer solution in the organ path to maintain the temperature at 32°C. 1 for muscle specimens? After applying static pressure of - E-Commercial (manufactured by MECl). Immediately after the measurement, the organ path was washed, and this was repeated every 20 to 30 minutes. The test substance was dissolved in physiological saline at a predetermined amount and administered.

(Results) As is clear from FIG. 2, peptide (1) with 8 amino acid residues has 1. It showed activity in QnM. On the other hand, peptide (2) with 25 amino acid residues has stronger activity than (1), exhibiting activity at 0.35 nM, and moreover, Q, 7
At nM, the activity was characterized by a significantly long duration. This feature can be effectively used to elucidate physiological effects.

〔Effect of the invention〕

Many physiologically active peptides have been known to have the activity of contracting or relaxing smooth muscles in the gastrointestinal tract, uterus, vas deferens, gallbladder, blood muscles, and the like. For example, substance P, neurotensin, and cholethystokinin have a strong effect on the contraction of the guinea pig ileum.

Neuromethin includes neuromethin L, neuromedin N, katsushinin, etc., and oxytocin has a strong effect on rat uterine muscle contraction.

Pradykinin, gastrin-releasing peptide, neuolgin B, neuromedin C, bombesin, etc. are known. These substances also cause smooth muscle contraction.

It is known that in addition to relaxation, the peptide has unique physiological effects in vivo, and it is thought that the peptide of the present invention also plays a new physiological role in vivo.

Furthermore, the amino acid sequences of conventionally known physiologically active peptides include, for example, Substane P, H-Arg-2ys-P
ro-Leu-Gly-NH,1,j,5-)+5-y
TItifl-Arg-Pro-Pro-Gly-P
he-8s r-Pro-Phe-Arg-OH, which 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 also useful for elucidating physiological effects.

〔Example〕

Next, the present invention will be explained with reference to Examples.

Example 1 The internal protease was inactivated by boiling 20 kl of pulp extracted from pigs immediately after slaughter for 10 minutes in IN-acetic acid containing twice the amount of 20 mM hydrochloric acid. After cooling, extraction was performed by homogenizing at 4° C. with a Polytron mixer. The resulting extract was centrifuged at t12,0OOXG for 30 minutes to obtain about 40% of the supernatant. Further, floating fat was removed using a GF/B glass filter (manufactured by Whatman), and the mixture was desalted and concentrated by ultrafiltration (UM-2, manufactured by Amico Co., Ltd.) to a strict liquid volume of 3.3 tons. Acetone was added dropwise to this prepared solution at 4° C. to a final concentration of 75 to perform acetone precipitation, and this was then centrifuged to obtain a supernatant of about 114 g. The obtained supernatant was concentrated to dryness under reduced pressure, and the residue was dissolved again in IN-acetic acid and freeze-dried. This dried product was dissolved in 2 t of 1-l acetic acid, and IN-
sp-Sephadex C-2540 equilibrated with acetic acid
The acidic fraction (SP-
i, neutral weakly basic fraction eluted with 2M-pyridine solution (
SP-[), a basic fraction (SP-1+) eluted with 2M-pyridi/-acetic acid (pH 5,0) was obtained.

By freeze-drying this SP-seal fraction, dried product 1
0.5 Li- was obtained, which was dissolved in IN-acetic acid and gel-filtered with Sephadex G-50 (445X 140
cm: flow rate 40'nl/h; fraction size 5
0R1/tube) was divided into 5 times.

Here, rat myometrium was assayed, and both fractions with contractile activity were lyophilized and dissolved in IN-acetic acid, followed by gel filtration through Sephadex G-25 (7.5X 135
Tan; Flow rate 60rnt/hour; Fraction size 100r
nl/tube). Rat myometrium was assayed here as well, and fractions A to F were obtained as shown in FIG.

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

The F fraction was further subjected to cation exchange HPLC.

Conditions [Color A: TSK gel CM-2s w,
4. Q x250 B (manufactured by Toyo Soda): Flow rate 1.0
rnl/min; solvent system (A110 mM) ICOONH
, (pH 6,6) Niacetonitrile = 90:
10, (B) 1. OM HCOONH
, (pH 6,6) niacetonitrile = 90:10, (A
): (B) = 100: From Q (Al: (B) = 85
: Linear gradient of 48 minutes to 15 (A)
:(B) =85:15 to (A):(131=: 5
A 48 minute linear gradient was applied to 0:50.

] Retention time 103-105 with this HP LC
I got both minutes with myometrial activity at the minute. This fraction was further subjected to reverse phase HPLC. Conditions [Column Chemcoaorb s 0DS-H, 4
, 6x250 (manufactured by Kemco); flow rate 1 d/min, solvent system (
A) Water Niacetonitrile = 10 Titrifluoroacetic acid (T
FAl=90:10:1, CB) Water Niacetonitrile=10TFA=40:60:1, (A): tB
)=100: O to (A): (Bl=0: 1
A linear gradient of 80 minutes to 00 was applied. ] The main peak with a retention time of 39 was fractionated and the substantially pure peptide (1) was collected in approximately 1μ? Obtained.

Peptide (2) is obtained from both sides of Sephadex G-2 empty B. Fraction 2.6y- of B was added to 101 nl IN
-Dissolved in acetic acid and subjected to reverse phase HPLC in 7 portions. Conditions [Column, TSK gel OD S-12OA.

° 20x250 Dragon (manufactured by Toyo Soda); flow rate 5-/min;
Solvent system (A) water Niacetonitrile: 1O%TFA=90
:10:1. (B) Water niacetonitrile; 10% TFA
=40:60:1, (Al:(B)=
From 90:10 (Al: (Bl=30:70
A 250 minute linear gradient was applied. ]This 1 (P
A fraction with myometrial activity was obtained at a retention time of 128 to 136 minutes in LO. This minute was then subjected to cation exchange HPLC. Conditions [Column; TSK gel
CM-2sw 4.6X250 (manufactured by Toyo Soda);
Flow rate 1 rttl/min Solvent system (At 10 mM HCO
ONI′i4 niacetonitrile = 90:10,
(B) t, oMHcOONH, niacetonitrile = 90: 10. (Al: (B) = 100
: 0 to (Al : (Bl=50 : 10 to 50
A 0 minute linear gradient was applied. ] Ririn/John Time 68 to F1 showed both uterine myometrial activity. Furthermore, both of these aliquots were subjected to reverse phase HPLC.

Conditions [Column; Chemcosolp 7-diphenyl.

4, 6 x 250 Tsuru (manufactured by Kemco); flow rate 1.511L
/! /min.

Solvent (A) Water Niacetonitrile = 10% TFA = 90:
10:1 (B) Water Niacetonitrile: 1O%T
F'A=40:60:i (A): (B)=80:2
From 0 to (Al: (B)=O: 100, a 128-minute linear gradation/t? was applied.) Retention time 27
Both minutes with myometrial activity were obtained at ~8 minutes. The active fraction was subjected to reverse phase HPLC for final nI production. conditions〔
Column: Chemcosolve 3 0DS-H, 4,6X75m
m (manufactured by Kemco); flow rate 1 d/min; solvent system (5) water Niacetonitrile: 10% TF'A = 90: 10: 1
(Bll Mizuko Acetonitrile 10T F A = 40:
60:1, (Al:(B)=80:
20 to (A): (Bl = O: 100 to 192
A linear gradient was applied. ] Retention time 3
The main peak at 4 minutes was fractionated and the substantially pure peptide (2
) was obtained about 3.5μ.

Fu Example 2 Pe/Zhydrylamine resin 1y-(0,4meqNH2
/1) was well swollen in dimethylformamide (DMF) 5a+J, and Boa-asparagi/-p-nitrophenyl ester 0.715' (2, Ommol
) was added to the mixture, and the mixture was stirred gently at room temperature and reacted for 6 hours. The mother liquor is removed and the resin is washed three times with DMF. Same reaction 1 [returned. Progress and completion of the reaction was monitored by Kaiser test with ninhydrin. Then DMF and methylene chloride.

The resin was washed with isopropyl alcohol and methylene chloride in that order and dried.

In the synthesis of the protected peptide resin, all the α-amino groups of each constituent amino acid are protected with Boa groups, and among the active side chains, the hydroxyl group of tyrosi/ is protected with a dichlorobenzyl group (C4-
In Bzll, the guazino group of arginine is replaced by a tosyl group (To
In sl, the ξ-7mino group of lysine is retained by the pen/zyloxycarbonyl group (Z)ff1l, and the β-7 of aspartic acid.
Carboxylic acid is benzyl group (Bzl), curtamic acid r
- Carboxylic acid is Bzl group and serine hydroxyl group is Bzl
protected with base.

When condensing the protected amino acid, the Boa group, which is the protecting group for the terminal amino group of the protected peptide bound to the resin, is almost completely removed by repeating the treatment twice with 50% trifluoroacetic acid in methylene chloride at room temperature for 20 minutes. did. The amino group liberated by the Boa removal is then condensed with the carboxyl group of the Boa-protected derivative of the next amino acid in the amino acid sequence of the target peptide. During this condensation of protected amino acids, Boc-Asn and Boa-Gin are p-
An operation of reacting for 10 hours using 5 equivalents of this as a nitrophenyl ester was performed twice. Other Boc-amino acids were condensed using dicyclocarbodiimide as a condensing agent. If the reaction was not completed by this operation, the same operation was repeated. The progress and completion of the reaction was monitored by Kaiser test using ninhydrin.

In this way, benzhydrylamine 1? Yoshi Boc-
Tyr (C4-Bzl) -Phe-Leu-Ph
e-Arg(Tos)-Pr.

-Arg(Tos)-Asn-NH-benzhydryl resin [hereinafter referred to as protected peptide resin (A)] was synthesized, and a portion (500 .mu.) of this resin was taken out. Boc-Ph
e-Lys (zl-Val-Asp (OBzl l
-Glu (OBzl 1LG1u (OBzl -
Phe-Gln-Gly-Pro-I 1e-Val-
8e r (Bz 11-Gl n-Asn-Arg
(To s l −Arg (To s ) −Tyr
(C4-Bz 1 )-Phe-Leu-Phe-A
rg(Tos)-Pro-Arg(Tos)-Asn
-NH-benzhydryl resin [hereinafter referred to as protected peptide resin (B)] 1460 rng was obtained.

Desorption and elliptical formation of the peptide in 11) from the resin were performed by the following method. Protected pegged resin (Al 300) was placed in a HF reactor with 1.5 ml of anisole.
8 ml of F f was introduced and reacted at 0° C. for 60 minutes.

Excess HF was then distilled off, the anisole was removed by washing with 25 d'i of ether, and the product was extracted by adding 14.2 d'i of IN-acetic acid. Separate the resin and insoluble matter in a furnace, and further add water to
After o-fold dilution, 9QmJ of ODS resin [L C-5orb
(manufactured by Kemco)] packed column (2, OX 4
0 cm) and 0.0 cm). After thorough washing with IN acetic acid, elution is carried out with 200 g of 60 thiacetonitrile containing 0.1% trifluoroacetic acid (TF'AI).The eluate is evaporated under reduced pressure to remove the acetonitrile, and then lyophilized to obtain approximately 90 g of crude peptide (1 ) t-obtained. This was dissolved in 0.1% trifluoroacetic acid 6r!Ll and divided into 6 times and applied to the reverse phase) (PLO Nikon. Conditions'(: Column; TSK Gel 0DS120A
.

2,, OX 250 cm: Flow rate 5rnl/min; Solvent system TA) Water: 7ml) = lJle: 10% TFA = 90
: 10 : 1° [B) Water Niacetonitrile : 10t TFA = 40 : 60 : 1 , (A) ? (B
)=75: 25 to (A): (B)=25=7
A 100 minute linear gradient of 5 was applied. ] This operation was repeated six times and the main peak was fractionated. This fraction was lyophilized after distilling off the acetonitrile under reduced pressure to obtain the desired peptide 11)H-Tyr7Phe-Leu-Ph.
e-Arg-Pro-Arg-Aan-NH, 58.
I got 2η.

Desorption and purification of the peptide (2) from the resin was performed by the following method. Protected peptide resin (B1600 with anisole 2-) (in a F reactor, HF 104
The mixture was introduced and reacted at 0°C for 60 minutes. Then, after distilling off excess HF, washing with ether 301R1 to remove anisole,
24.211 Ll of IN-acetic acid was added to extract the product.

The resin and insoluble matter were separated and further diluted 10 times with water.
0d ODS resin [W-sorb (manufactured by Kemco)] was adsorbed onto a column (LOX40 cm). IN
- After thorough washing with acetic acid, elute with 60% acetonitrile 200M containing 0.1% TFA. The eluate was lyophilized after acetonitrile was distilled off under reduced pressure to obtain approximately 228 m9 of crude peptide (2).

This crude peptide 6Z2Ing was mixed with 0.11 TFA of 7-
The solution was dissolved in water and subjected to reverse phase HPLC in 5 portions. Tokyo [Column; 'I'SK gel, 0DS120A, 2. O
X 25 cmH flow rate 5 d/min; Solvent system (A) water Niacetonitrile: 10% TFA = 90: 10: 1
, (B) Water Niacetonitrile = 10 TFA = 40
: 60: 1, (Al:(B)=75:25 (
A 100 minute linear gradient was applied to A):IB)=25:75. ] This operation was repeated five times, and the main peak was collected. After distilling off the acetonitrile under reduced pressure, this fraction was lyophilized to obtain the desired peptide i2) H
-Ph e -Ly s -V'a 1-Aap-Gl
u-Glu-Phe-Gin-Gly-Pro-11e
-Val-8er-Gl n-Aan-Arg-Arg
-Tyr-Phe-Leu-Phe-Arg-Pro
-Arg-Asn-NH, 11. ! I got 1119.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the results of gel filtration of the basic fraction (sp-I) in Example 1 using Sephadex G-25, and is a diagram showing an absorption curve at 280 nm and relative activity. FIG. 2 is a diagram showing the change in contraction length over time when the peptide of the present invention was administered to a specimen of rat myometrium. that's all

Claims (1)

[Claims] 1. General formula X-Tyr-Phe-Leu-Phe-Arg-Pro
-Arg-Asn-NH_2 (wherein, X is H or H-P
he-Lys-Val-Asp-Glu-Glu-Ph
e-Gln-Gly-Pro-Ile-Val-Ser
-Gln-Asn-Arg-Arg-) and its salt.