JPS5976043A - Novel polypeptide - Google Patents

Abstract

NEW MATERIAL:The polypeptide of formula (X is L-norleucine residue or L- norvaline residue). USE:It has ACTH secretion accelerating activity and hypotensive activity comparable to corticotropin-releasing factor (CRF), and is useful as a diagnostic agent or a drug. The chemical structure of the polypeptide corresponds to that of CRF provided that the 21-methionine residue of CRF is substituted by norleucine residue or norvaline residue. It has high oxidation stability compared with CRF. PROCESS:The objective peptide can be prepared by bonding amino-protected alpha-amino acids and carboxyl-protected alpha-amino acids in the order of the amino acid sequence of the objective paptide by the conventional method for the synthesis of peptide (e.g. active ester process, mixed acid anhydride process, etc.). A solid-phase synthesis based on a synthetic resin (e.g. benzhydril synthetic resin) is most preferable as the bonding process. The resultant protected peptide resin is deprotected and purified to obtain the objective peptide.

Description

[Detailed description of the invention] The present invention relates to a novel polypeptide.

More specifically, the present invention relates to derivatives of corticothrobin release promoting factor (CRF).

It was already suggested more than 25 years ago that the secretion of corticotropin (ACTH), a hormone that promotes the secretion of adrenal cortical hormones, may be regulated by a chemical released from the hypothalamus of the brain. But what this chemical is has long been a mystery. In 1981, Veer et al.
Vol. 213, p. 1394, 1981). This CRF
is a polypeptide consisting of 41 amino acid residues, AC
It has been shown that it promotes the release of not only TH but also β-endorphin.

Although research into the physiological role of CRF has just begun and most aspects remain unclear, it is believed that its role is extremely important, and as this is elucidated, CRF may be used as a diagnostic agent. Of course Ar,
We can fully expect that it will become important as a medicine.

By the way, CRF has a methionine residue at the 21st position counting from the N-terminus, and this methionine is relatively easily oxidized and turns into methionine sulfoxide, but oxidized CRF promotes the release of ACTH. It is known that the activity is reduced to about ten times less than that of CRF itself, and there is a serious problem in its preservation.

As a result of intensive research to develop a technology to stabilize CRF, the present inventors found that a derivative in which the methionine residue of 21-1 was replaced with norleucine or norvaline, while maintaining ACTH release promoting activity, It was discovered that it is extremely stable against oxidation, and based on this finding, the present invention was accomplished.

That is, the present invention provides a novel polypeptide represented by the following formula:

H-Ser-Gln-G Iu-Pro-Pro-
I Ie -3er -Leu -Asp -Leu-
Thr -Phe -Hj s-Leu-Leu-Ar
g (l;Iu-Vat-Leu-Glu-X-
Thr-Lys-Ala-Asp-G1. n-Leu-
Ala-Gin-Glu-Ala-His-S e
r-Asn-Arg-Lys-Leu-Leu-
A novel polypeptide represented by Asp-Ile-Aha-NHl, where X in formula 1 is an L-norleucine (Nle) residue,
or L-norvaline (Nva) residue.

Each other symbol means the following amino acid residue.

Ala: L-alanine Arg: L-arginine Asp: L-aspartic acid Asn: L-asparagine Glu: L-glutamic acid Gln: L-glutamine His: L-histidine 11e: L-isoleucine Leu: L-leucine Lys: L- Lysine Phe: L-phenylalanine Pro: L-proline Set: L-serine Thr: L-threonine Val: L-valine The polypeptide of the present invention can be prepared using a protected amino group according to a conventional method used in peptide synthesis. It can be produced by sequentially peptide bonding an α-amino acid and an α-amino acid with a protected carboxyl group in the above structural order. Peptide bond formation at this time can be carried out by appropriately selecting from known methods such as the active ester method, mixed acid anhydride method, azide method, and methods using condensing agents such as DCC, but particularly advantageous methods include This is a solid phase synthesis method based on synthetic resin. According to this method.

First, a synthetic resin having a reactive group 2, for example, a benzhydrylamine resin, is protected with an amino group and reacted with an alanine derivative to produce a synthetic resin with an L-alanyl group bonded to it. The protecting group of the amino group of was removed, and this was reacted with an inleucine derivative having a protected α-amino group.

By repeating this operation, the 41 α-amino acids can be combined to obtain the desired polypeptide.

The thus obtained polypeptide of the present invention.

(Nle”) -CRF (I) and (Nva2I
) CRF (III.

In a system using rats, AC is approximately the same as CRF.
It exhibited TH secretion promoting activity and blood pressure lowering activity. In addition, CRF was dissolved in 5% hydrogen peroxide solution (1N4 acetic acid solution) at 0.
While the 21st methionine was almost completely oxidized and inactivated at °C for 5 minutes, polypeptides ① and ② were completely unchanged under the same conditions.

Next, the present invention will be explained in more detail by way of examples.

Abbreviations shown in the examples have the following meanings.

CRF: fluticotropin release promoting factor A1a:L
-Alanine unit Arg: L-arginine unit Asp: L-aspartic acid unit ASn: L-
Asparagine unit Glu: L-glutamic acid unit Gln:
L-glutamine unit J-Iis: L-histidine unit 11e:
L-isoleucine unit J,eu: L-leucine unit Lys: I,-lysine unit phe: L-phenylalanine unit Pro
: L-proline unit Ser : L-serine unit Thr : L-threonine unit Val : L-valine unit Nle : L-norleucine unit Nva : L-norvaline ■1st position DCC : Dicyclohexylcarbodiimide TFA :
l-Lifluoroacetic acid TEA: Triethylamine DMF: Dimethylformamide Boc: t-butoxycarbonyl group 3zl
: Benzyl group CI-Z: O-chlorobenzyloxycarbonyl group T
os: ) syl group ONp: p-nitrophenyl ester Example A) Preparation of Boa-Ala-resin 7.00 g of benzhydrylamine resin hydrochloride were suspended in chloroform <L, after neutralization with TEA.

Add 1.09 g of Boc-Ala -OH dissolved in 30 ml of dichloromethane, leave for 5 minutes, and then add DCC1.
, 18 g of dichloromethane solution (30 ml) was added and stirred for 1 hour. The resin was collected on a glass filter, washed with methanol and dichloromethane, and then a dichloromethane solution of N-acet7-tylimidazole was added.

Unreacted amino groups were acetylated. This resin was washed with dichloromethane and methanol on a glass filter, and dried under vacuum to obtain 7.50 g of Boc-Ala-resin. The Ala content of this resin is 0 per gram of resin.
．． 40 mi! It was J moles.

B) Boc -Set (Bzl) -Gln -
Glu (Bzl) Pro-Pro-11eSer
(Bzl) -Leu-Asp (BZ+) -Le
u-Thr (Bzl) -Phe -His (To
s) Leu -Leu -Arg (Tos) -G
lu (Bzl) -Val -Leu-Glu (B
zl) -Nle-Thr (Bzl) -Lys (
CI-Z) Ala-Asp(Bz I)-G
In -Leu -Ala -G In -GIn-A
l a-His (Tos)-8er (Bzl)-As
n-Arg(Tos)-Lys(CI-Z) Leu
-Leu-Asp(Bzl)-11e-Ala- Resin was synthesized by solid phase synthesis using an automatic peptide synthesizer. For the protecting group of α-amino group.

Boc was used, and the following was used to protect the amino acid side chain. Asp, Glu, Ser, Thr:
Bzl; Arg+His: Tos; Ly
s: C1-Z, The condensation of each amino acid was performed according to the steps shown in Table 1. Amino acid derivatives and DCC
The condensation was repeated two or three times using 2.5 times the equivalent of Ala attached to the resin - 8=. A
The active ester method was used to introduce sn+ Gln.

It was carried out according to the steps in Table 2. Boc-Asn-
ONp and Boc-Gln-ONp were used in an amount equivalent to 8 times that of Ala.

After completing the entire condensation process, the resin was collected on a glass filter, washed with DMF, methanol, and dichloromethane in this order, and dried under vacuum. By this method.

6.48 g of protected peptide resin were obtained from 2.61 g of Boc-Ala-resin.

1st Table 1. Dichloromethane, cleaning 222.50
%'rFA (dichloromethane solution), deprotection 3013
, dichloromethane, wash 234, chloroform, wash 235.10% TEA
(chloroform solution), neutralization 216,〃〃51 7, chloroform, washing 238, dichloromethane, washing 239, Boc-amino acid 510, DCC, condensation 6011, dichloromethane, washing 2312, Boc-amino acid
513, DCC, 300 14, Dichloromethane, cleaning 211
5, methanol, washing 2116,
Dichloromethane, washing 2117° Methanol, washing 2118, dichloromethane, washing 21 No. 2
Tables 1-7 Same as Table 1 & DMF, washing 2310,
DMF 2111,
Dichloromethane, washing 2112, methanol, washing 2113, dichloromethane, washing 2]14, methanol, washing 2115, dichloromethane, washing 21C) HSer -
Gln-Glu-Pro -Pro-11e-3er-
Leu -Asp -Leu -Thr -Phe -
His-Leu-Leu -Arg -G lu Va
l -LellG Iu -Nl e -Thr -
Lys-Ala-Asp-Gin-Leu-Al
a-Gln Gln-Ala-Hjs-3er-As
n-Arg-Lys-Leu-Leu-Asp-11e
-Ala-Nru(II) production.

After adding 6 ml of anisole to 3.14 g of the protected peptide resin produced in B) and leaving it overnight, 50 ml of HF was added to the 9 reaction container while cooling it with dry ice-ethanol, and the mixture was reacted at 0°C for 1 hour. The HF and anisole were removed under reduced pressure and the residue was thoroughly washed with ethyl acetate and diethyl ether.

This was extracted with 200 ml of 2M acetic acid, the resin was filtered off using a glass filter, and the remaining resin was washed with 100 ml of water. The filtrate and washing solution were combined and freeze-dried.

The resulting pale yellow powder was purified by gel filtration through Sephadex G-25 (eluent: 2M acetic acid), and the highest polymer peak was collected. This was further purified by semi-preparative high-performance liquid chromatography using a Nucleosil 5018 column (10X250) using a 0.05M triethylammonium phosphate-acetonitrile mixture as the eluent, and finally using the same column as above, 0.196TFA- Desalting was performed using an acetonyl-IJ mixture to obtain 70 mg of the desired purified peptide.

HPLC analysis conditions Two columns: Nucleosil 5 Cls (10x 2
50 rrm) Eluent: 0.05M) Diethylammonium phosphate, 39.5% acetonitrile Detection: UV 210 nm Elution position: 12.6 minutes (chromatogram shown in Figure 1) Hydrolysis with 6N hydrochloric acid (22 hours) Later amino acid composition.

Asp: 4.15 (4), Thr: 1.80
(2), Ser: 2.75 f3), Glu
Near, 30f7), Pro: 1.85 (21,A
la: 4.20f41. Vat: 0.95fl
, Ile: 1.87 f2), Leu: 8
．． 32 (81, Phe: 1.00 (1).

His: 1.93 (21, Lys: 1.95 (2
), Arg: 1.90f2), N1eO,93(
11 Values in parentheses are theoretical values D) H-3er-Gln Glu Pro-Pr
o-11e-3er -Leu Asp Leu-
Thr-Phe-Hls Leu Leu-Ar
g Glu -Val -Leu -Glu -Nv
a -Thr -Lys-Ala -Asp Gln
-Leu"Ala-Gln Gln Aha -H
ls -Ser -Asn -Arg -Lys -L
eu -Leu Asp-11e -Ala NH
Manufactured for 21.

Synthesize using the same method as for polypeptide +1+.

Purified. 8.85 g of protected peptide resin were obtained from 3.5 Og of Boc-Ala resin, of which 3 g was purified.

The desired polypeptide (III) of 65r was obtained.

HPLC analysis conditions Two columns: Nucleosil 5 C1s (10X 2
50 rrm) Eluent: 0.05 M) ethyl ammonium phosphate, 39.5% acetonitrile Detection: UV 210 nm Elution position: 9.9 minutes (chromatogram shown in Figure 2)
Amino acid composition after hydrolysis (22 hours) with 6N hydrochloric acid.

Asp: 4.08 (41, Thr: 1.75 (
2), Ser: 2.73 (3), Glu: 7.
13 (71, Pro: 1.97 (2), Ala
: 4.30 (4), Vat: 0.99 (1),
Ile: 2.03 (2), Leu: 8.2
5f81. Phe: 1.00 fl, His:
1.92f2), Lys: 2.02f21.
Arg: 1.90 (2), Nva: 0.95
(1)

[Brief explanation of the drawing]

Figure 1 is a chromatogram of Compound 1 of the present invention. FIG. 2 shows a chromatogram of the second compound (2) of the present invention. Figure 1 5 10 15 Time (minutes) Figure 2 5 10 Time (minutes) 315-

Claims (1)

[Claims] A novel polypeptide represented by Formula 1 (% Formula %) (where X in Formula 1 means an L-norleucine residue or L-norvaline residue, and the other symbols are as follows. Ala: L-alanine Arg: l, -arginine Asp: L-aspartic acid Asn: L-asparagine Glu: L-glutanic acid Gln: L-glutamine His: i, -histidine 11e: L - Inleucine Leu: L-- Leudino Lys: L-lysine Phe: i, - Phenylalanine Pro: L-Proline Ser: L-Serine '17hr: L-Threonine Val: L-valine