JPS59130258A - Novel physiologically active peptide - Google Patents

Abstract

NEW MATERIAL:A peptide of human interferon gamma shown by the formula (J is H). USE:An inhibitor against cell multiplication and an antiviral agent. Having not only improved antiviral activity but also improved inhibitory action on cell multiplication and enriching action on NK cell, useful for remedying malignant antitumor, etc. PROCESS:A carboxylic acid of an amino acid containing an alpha-amino-protecting group is linked to a substrate such as a polystyrene resin crosslinked with divinylbenzene, the alpha-amino-protecting group is eliminated selectively, the liberated amino group is reacted with the second amino acid containing an alpha-amino- protecting group, removal of an alpha-amino-protecting group and introduction of another amino acid containing an alpha-amino-protecting group are successively repeated, to give the desire piptide chain, and covalent bond between carboxylic acid group of C-terminal amino acid of peptide and the resin is cleaved.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides antiviral effects, cell growth inhibitory effects, and
This invention relates to a novel physiologically active peptide with excellent physiological activity such as K cell activity enhancement, its production method, and its uses.

Conventionally, antimetabolites, alkylating agents, antibiotics, etc. have been used as antiviral agents or cell growth inhibitors, but these are highly toxic and undesirable. Therefore, the development of antiviral agents or cell proliferation inhibitors with low toxicity is strongly desired.

Recently, interferon-α (hereinafter referred to as “1FN-α”) and interferon-β (hereinafter referred to as “XFN-β”)
These studies have progressed because of the effectiveness of antiviral agents (referred to as antiviral agents) with low toxicity. However, T.F.N.
The cell proliferation inhibitory effects of -α and TFN-β are not satisfactory. On the other hand, interferon-γ (hereinafter “■F
The study of IFN-α and IFN-γ
-Compared to research on β, not much progress has been made.

However, in October 1981 the United States F7
7 The 5th International Conference on Interferon Research (7j) held in Cisco City
ual TnternaLionalCongress
for Ir+jerferon R55arch
), there were scattered reports that TFN-γ was isolated;
, C) oeddel) [Genentech (Ge
Pro-IFN- γ, IFN-
The amino acid sequences of r and IPN-γ-like peptides have been published.

Its amino acid sequence is represented by the following formula (I).

Formula (I): J Oys Tyr Oys Gin Asp Pro
Tyr Vat Lys, GluAla Glu
Asn Leu Lys Lys Tyr Phe A
sn Ala GlyHis Ser Asp Val
A18Asp Asn Gly Thr Leu P
heLeu Gly Ile Tjeu Lys As
n Trp Lys Qlu Glu 5erA, sp
A, rg Lys Ile Met Gln Ser
Gln Ile Val 5erPhe Tyr P
he Lys Leu Phe Lys Asn Ph
e Lys AspAsp Gln Ser T
le Gln Lys Ser Val G
lu Thr l1eLys Glu Asp M
et Asn Vat TJys Phe Phe A
sn 5erAsn Lys Lys Lys
Arg A, sp A, sp Phe Gl
u Lys LeuThr A, sn Tyr
Ser Val Thr Asp Leu
Asn Val GlnArg TJys A
la Tle His Glu Leu Ile Gl
n Val M+IAla GIIJ Leu Ser
Pro Ala Ala Lys Thr Gly
Lys 5- A, rg Lys Arg Ser Gln
Met Leu Phe Arg Gly
A, rg Furthermore, at the above-mentioned conference, a culture solution of cells containing a gene containing information for IFN-γ-like peptide has antiviral activity, but a culture solution of cells containing a gene containing information for pro-TFN-γ does not have antiviral activity. It was also announced that the country does not have a On the other hand, unlike IFN-γ-like pezotide, rFN-r does not have an artificial methionine residue added to its N-terminus, so it is more desirable to produce a culture solution for cells containing the gene containing IFN-γ information. I haven't been successful yet.

In view of the current situation as described above, the present inventors have developed a drug that not only has high antiviral activity but also has cell proliferation inhibitory effect and NK
We conducted intensive research in order to develop a bioactive agent that also has an excellent effect on enhancing #I cells.

Therefore, an object of the present invention is to provide a physiologically active peptide that not only has excellent antiviral activity, but also has excellent cell proliferation inhibiting action and NK cell enhancing action.

Other aspects and advantages of the invention will become apparent from the detailed description provided below, along with the claims.

According to the present invention, a novel physiologically active peptide represented by formula (CI) (wherein, J represents a hydrogen atom in formula (I)) is provided.

The novel bioactive peptide r of the present invention can be obtained by a conventionally known method, namely, Nobuo Izumiya et al. [Synthetic chemistry series - peptide synthesis j, published by Maruzen Co., Ltd.・Grind Chemistry T-TVJ
It can be produced by using the method described in L9'17, published by Tokyo Kagaku Dojin.

As one of the embodiments, the solid phase method will be specifically explained below. After the cardic acid group of an amino acid having an α-amine protecting group is bonded to a support such as a polystyrene resin crosslinked with divinylbenzene, the α-amine protecting group is selectively removed. The free amine group is reacted with a second amino acid having an α-amine protecting group. The desired Vepti P chain is obtained by repeating the removal of the α-amino protecting group and the introduction of the amino acid having the α-amine protecting group. The covalent bond between the carzene acid group of the C-terminal amino acid of the peptide and the resin is then cleaved. At this time, depending on the reagent used to cleave the covalent bond, the α-amine protecting group or side chain protecting group can be removed simultaneously with the cleavage of the covalent bond.

In the solid-phase method described above, all reactions can be carried out in one container, so the production of peptides is easy in terms of operation, and the obtained product is soluble in the solvent used in the reaction, so the reaction process is easy. This method is more advantageous than the so-called liquid phase method because the appropriate amount of reagents and byproducts used in the method can be removed by filtration, and the desired peptide can be produced in a short time and in high yield.

The support used in the first method is generally a styrene polymer crosslinked with divinylbenzene with a degree of crosslinking of 2% and preferably has a size of 200 to 400 meshes.

Before reacting and bonding the resin with amino acids, functional groups are introduced into the resin to impart reactivity. Generally, in order to introduce a functional group into a resin, the aromatic ring of styrene is reacted with chloromethyl methyl ether by the Friedel-Krach reaction using S n O12 as a catalyst, and the aromatic ring of styrene is chloromethylated. A method is taken to do so.

The α-amine protecting group used in the first method is stable and easy to couple with the support and the amino acid having the α-amine protecting group without adversely affecting the side chain protecting group or the pezoti 1 chain. It is preferable to use one that can be removed quickly. Preferred α-amine protecting groups as described above include tertiary-butoxycarzenyl (hereinafter abbreviated as “BoC”) group. This BoC group was added to the INHae/acetic acid mixture, 4
NHCl/-)oxane mixture or 50% (W/V
) T r or-r (tetrafluoroacetic acid) /
It can be easily removed from the peptide chain using a 0H20/2 mixture.

The side chain protecting group is preferably a group that is stable during coupling and removal of the α-amine protecting group and that can be easily removed at the stage of peptide synthesis.

Such preferable side chain protecting groups include NO2 or tosyl group for protecting the side chain of arginine, benzyl group (hereinafter abbreviated as "Bzl") group for protecting the side chain of aspartic acid, cysteine and For protecting the side chain of glutamic acid, a Bzl group or 0-methylbenzyl (hereinafter abbreviated as "OBz") group is used, and for protecting the side chain of histidine, a Bzl group or pendyloxycarzenino (hereinafter referred to as "Z") is used. ) group or BoC group for protecting the side chain of lysine, Z group, tosyl group or diisopropylmethyloxycalzenyl (hereinafter abbreviated as "DipmocJ") group for protecting the side chain of methionine. Sulfoxy P groups and Bzl groups for side chain protection of serine, threonine and tyrosine are included.

The method for determining the amino acid sequence of the novel physiologically active peptide of the present invention is as follows. First, the methionyl bond of the novel bioactive pezothi 1 of the present invention is cleaved with bromo cyanide. The resulting peptide fragments were separated using Sephadex O-1OO (Pharmacia Fine Chemicals, Sweden), and the amino acid sequence of each fragment was determined using known high-precision amino acid sequence analysis. N-
We will decide in order starting from the end. On the other hand, the novel physiologically active peptide of the present invention is partially digested with trypsin, and the obtained peptide fragments are separated using a Sephadex (]-'100 column. Each separated fragment is subjected to the same method as described above. Then, determine the amine sequence of each fragment in order from the N-terminus of each fragment. The sequence of each fragment of the peptide is determined by comparing the amino acid sequences of the peptide.

In this way, the amino acid sequence of the novel bioactive peptide r of the present invention is determined.

When the purpose is to suppress cell proliferation, for example, suppress the proliferation of malignant tumor cells, the peptide of the present invention can generally be administered by injection intravenously, intramuscularly, or subcutaneously. The daily dosage of the peptides of the present invention will of course vary depending on the age, medical condition and weight of the patient, but is usually I x 10' per adult.
It is administered by injection in an amount of ~I x 109 units/day.

To treat viral diseases, ointments containing 10' to 109 units of peptide IX of the invention per tog of ointment can be applied to the skin.

Ointments containing the peptides of the present invention can be prepared using conventionally known pharmaceutically acceptable ointment bases.

The daily dosage of the ointment containing the peptide of the present invention will of course vary depending on the age and medical condition of the patient, but it is usually applied in divided doses of 1X10' to 1x109 units based on Bezoti 1 of the present invention. can do.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to examples, but the present invention is not limited to the following examples.

OBZ I: (Or)-methylbenzyl group, which is one of the flll chain fLJ groups vsv: Vesicular Sjomal Yamas Viru
s) AMV: Avian myeloblastosis virus (Avian
Myeloblastosis Virus)BSA
: Bovine serum albumin Example 1 Step 1 (Washing of resin) Resin r Bio-Bead 8-X2, 200-40
0 mesh (trade name of 2% divinylbenzene cross-linked styrene polymer, manufactured by Ioratsu R, USA)
] 150g is 11! of distilled benzene with stirring. After 30 minutes, filter the resin through a glass filter and dry.

Next, the dried resin is added to 1/2 methanol with stirring, and after 30 minutes, it is dried using a glass filter. The resin obtained by drying is washed successively with methanol, methanol/water and water. The washed resin was diluted with 1p of lNNa.
Add to OH aqueous solution. After stirring the resulting mixture in a boiling water bath for 1 hour, the resin is removed and washed with water. Next, the resulting resin is added to a 1/1 IN aqueous solution of hydrochloric acid and stirred for 1 hour in a boiling water bath. Next, the resin is washed with water.

The resin obtained by washing with water was suspended in distilled water of 41.13
− Afterwards, let it stand. After 30 minutes of standing, floating fine resin is removed by decantation. In this way, the resin is obtained as a precipitate. The precipitated resin is placed on a glass filter and washed with methanol. The washed resin was added to a small amount of dimethylformamide (hereinafter abbreviated as "DMF") and stirred at 80° C. for 30 minutes, then stirred several times and washed with DMF and methanol. Further, the resin was stirred in 2/2 methanol for 1 hour and then dried. After the resulting resin is air-dried, it is further vacuum-dried at 100° C. for 2 hours.

Step 2 (Manufacture of chloromethyl resin) 25 g of the resin obtained in Step 1 and 100 ml of distilled chloromethyl methyl ether! was placed in a 1/2-inch round-bottom two-headed flask, stirred gently at 25°C for 1 hour to swell the resin, and then cooled to 0°C. 1.88m in this swollen resin
50m7 containing 5n014 of g! of chloromethyl methyl ether was added over 30 minutes while stirring while maintaining the temperature at 0°C, and the mixture was further stirred for 30 minutes to obtain a reaction product. In this way, 14- The obtained product was offshored using a glass filter and 500 m
Wash gently with dioxane-water (1:1) and then with 500 ml dioxane-3N hydrochloric acid (3:1). Further, the product is washed successively with water, dioxane-water and methanol, and then dried at 100°C under reduced pressure.

■Procedure 3 A, (ε -Z -9 U2 N) 11g of lysine copper salt, 10g of NaHCO3 and 12ml! A quarter of Z-0/ was added every 10 minutes while stirring on water cooling, and the mixture was further stirred for 35 hours.

The precipitate thus obtained is separated and washed successively with water, ethanol and acetone/ether. 250m/! of washed sediment! of water, and then add 4127! to this suspension. The above precipitate is dissolved by adding 6N aqueous hydrochloric acid solution. The resulting solution is then bubbled with H2S gas for 1-2 hours. The resulting O u S is removed using a Hycross-Purcel (Wako Tsumugi Co., Ltd., Japan), and the CuS is washed with an IN aqueous solution of hydrochloric acid. The filtrate and washing liquid obtained in the above procedure were collected and passed through air to add H2.
Remove S.

Then, add water-cooled ammonia water to this solution and p1
16.5 and leave it in the refrigerator for 2 hours. The resulting precipitate is separated and washed successively with water, ethanol and ether. In this way, ε-Z with a melting point of 254°C
IJ Log is obtained. The yield of ε-Z-lysine produced is 65%. The obtained ε-7IJ lysine is recrystallized by dissolving it in dilute hydrochloric acid and then neutralizing it with aqueous ammonia.

B, (G-NO2-Arginine) A mixture of 120 ml of fuming nitric acid and 75 rnl of oleum is ice-cooled, and 110 g of .--arginine hydrochloride is gradually added to this mixture with stirring. Furthermore, concentrated sulfuric acid 45me is added to the above mixture. After stirring the resulting mixture for 1 hour, it is poured into ice cubes and the pH is adjusted to 8 with concentrated aqueous ammonia. Next, the mixed solution was adjusted to pH 6 with acetic acid, and then left in the refrigerator for about 4 hours.

The resulting precipitate is offshored and recrystallized from hot water. The obtained crystals are washed successively with ethanol and diethyl ether and dried.

In this way, G -NO2-7ruki= with a melting point of 251℃
This yields 60 g of tonne. The yield of G-No2-arginine is 50%.

C, (imRzl-histidine) 20g of 1. - Histidine hydrochloride H20 was cooled to -30°C in a dry ice-acetone bath at 200+++
/ in liquid ammonia and to this solution small pieces of sodium metal are added with stirring until the blue color of the solution remains. A small amount of histidine is then added until the blue color of the solution disappears, and then benzyl chloride is added dropwise to the solution. After stirring this mixed solution for 30 minutes, NH3 in this mixed solution is evaporated at room temperature under atmospheric pressure, and then NH3 is removed under reduced pressure using a water jet pump. 100m7 of residue after NH3 removal! Pour into ice water. After extracting the resulting mixture with diethyl ether, insoluble matter is removed.

Dilute sulfuric acid is added to the resulting solution to adjust the pH to 3, and the solution is left in the refrigerator for 3 hours. Offshore the resulting sediment;
Recrystallize from 70% by weight ethanol.

13 g of imBzl-histidine with a melting point of 248°C are obtained. The yield of imBzl-histidine is 50%.

p,, (0-Hzl-serine) 237g of acetyl-L-serine in 235ml! , 5
Dissolve in NNaOH aqueous solution and adjust pH to 7.3.

Add 4g of Takajustase (manufactured by Sankyo Co., Ltd.) to this solution.
of 25 ml of 0.1 M citrate buffer (pt-r 6
．． 7. A solution obtained by dissolving OaO/20.025M) was added, and several drops of toluene were added dropwise, and the resulting mixture was left at 36° C. for 10 days. After removing the resulting precipitate and concentrating the filtrate, acetone is added to the concentrated tear fluid to obtain crystals. 100 g of Q-Bzl-serine are thus obtained. Following a similar procedure, 240 g of acetyl-L-threonine or t) 100 g of Q-Bzl-threonine are obtained.

B, (0-Bzl-tyrosine) 1.8 g L-tyrosine and 1.2 g Ou S 04
- 5H20 is suspended in a solution of 5-2N NaOH aqueous solution and 10-water and stirred for 2 hours. This suspension contains 60-
of methanol is added, followed by the gradual addition of 1.2-ml of benzyl bromide and 0.7 ml of 2N aqueous NaOH. 1518-min after 0.3 me benzyl bromide and 0,8-2N
Further add Na'o T-1 aqueous solution. After stirring the resulting mixed solution for 1 hour, the resulting precipitate is taken off and washed with meta/-ru water (]: 3). In this way, 2 g of the copper salt of O-Bzl-tyrosine is obtained.
The solid content is removed from the mixture obtained by kneading the above INEDTA aqueous solution in a mortar, and the solid content is washed with water. Recrystallization of the washed product from hot water containing a small amount of ET)TA gives 1 g of 0-Bzl-tyrosine with a melting point of 223[deg.]C. The yield of 0-Bzl-tyrosine is 30%.

F, (S-Bzl-cystine) 157 g of L-cystine hydrochloride was dissolved in 21 2N NaOH
Dissolve in an aqueous solution, add 256 g of benzyl bromide to this solution with vigorous stirring under water cooling, and stir in a cold room for 5 hours. The solution is then adjusted to pH 5 with acetic acid to form a precipitate. Collect and wash the formed precipitate with water. In this way, 5-Bzl-cysteine 16 with a melting point of 212°C
0g is obtained. The yield of 5-Bzl-cysteine is 7
I am in charge of 0.

1 from 160 g of as-ξlagic acid in the same manner as above.
50 g of OBzl-aspartic acid and 150 g of O-B z I-glutamic acid are obtained from 160 g of glutamic acid.

0, (Roc-amino acid) 29g ■,-250m7 of thumarin! lNNa01
1 Add water to the solution obtained by dissolving it in an aqueous solution and make 400 m
Let it be a solution of e. After adding 150 mff of tetrahydrofuran to this solution, while stirring vigorously at 10°C, 1/5 of 100+++e Roc-Ol was added to the solution for 10 minutes.
Add every minute. On the other hand, 2N NaOH was added to the Boc-0
/ is added to the solution each time to increase the pl-1 of the solution to 8.
Try to keep it at ~9. After 2 hours, the mixture is extracted with diethyl ether, the resulting aqueous layer is made acidic with a 0.5M aqueous citric acid solution, and the precipitated oil is extracted with ethyl acetate. The resulting extract was washed with a small amount of water and N B2
After drying with S04, concentrate under reduced pressure. Add petroleum ether to the concentrated extract and leave it in the refrigerator to form crystals. The resulting crystals are dried several times.

30 g of Boc-valine with a melting point of 78 DEG C. are thus obtained. The yield of Boc-/2-phosphorus is 55%.

I(, (Other Boc-amino acids) By performing the same method as above, 75 g of Boc-glycine, 16 g of Boc-alanine, Boc
−.

Isine 15g, Roe-inleucine 15g, Boc-
Serine (B21) 14 glBoc-threonine (B
zI) 14g, Boc-cysteine (Bzl) 15
g, Boc-methionine 15g5 Boc-proline 12g1 Boc-aspartic acid (OBzl) 15
g, Boc-glutamic acid (OBzl) 15
g, Boc-Glutamine 1"gs' Boc-Histidine (BZI) 12 gSBoc-Leucine (Z)
13 g, Boc-arginine (NQI) 15
g, Boc-phenylalanine 15g, Roc-
15 g of tyrosine (BzI) and 10 g of Boc) lysotophan, 30 g of l'+) gs, 30 g of alanine, 30 g of isine, 30 g of isoleucine, 30 g of serine (Bzl).

Threo = /, (Bzr) 30 g, cysteine (Bz
l) 30 g.

Methionine 30 g1 Zoroline 30 g1 Asnoξlagic acid (OBzl') 30 g s Glutamic acid (OBz
I) 30 gs 21- Glutamine 30g, Histidine (, nzl) 30
gs Lysine (Z) 30g, arginine (NO2)
301Zs Phenylalanine aog, Tyrosine (Bz
I) obtained from 30 g and tryptophan 30 g.

Step 4 (Coupling of Boc-glutamine and resin) 2 g of chloromethyl resin (CI!at amount 4 mmol), 2 mmol of Boc-T, -glutamine, 18 mmol of triethylamine and 12 ml! Dimethylformamide P
The mixture was heated at room temperature for 24 hours.

Next, the solid content is removed from the mixture using a glass filter, and the resulting product is washed sequentially with 7'OOme dimethylformamide P1 ethanol, vinegar #/ethanol, and methylene chloride, respectively.

The product is then dried under reduced pressure at room temperature. To determine the amino acid content of the product, weigh 50w1 of the product,
The mixture was placed in a mixture of 12N hydrochloric acid and dioxane at a volume ratio of 1:1 and hydrolyzed for 24 hours, and then measured using an amino acid analyzer. The amino acid content of the product is 0.2 mmol/g.

Step 5. (BO(: Ser-Gl n-resin)
22- 2 g of Boc-glutamine obtained in step 4 (hereinafter referred to as “L”)
The resin (abbreviated as loc-Ol nJ) was placed in 160me acetic acid and left at room temperature for 6 hours. This mixture is then filtered through a glass filter. 160% of the solid material obtained
Pour into 1 ml of acetic acid, shake, and weigh.

Repeat this operation three times. Next, add 160% of the above solid matter.
+++e IN aqueous solution of hydrochloric acid/acetic acid for 30 minutes at room temperature.
Shake for a minute to remove Boc groups from the resin.

The product thus obtained in [7] was separated and washed three times each with acetic acid, ethanol, and dimethylformamide. 160++y/! of cleaning products! 1
The mixture was placed in triethylamine/dimethylformamide having a weight ratio of 0 and was shaken for 10 minutes to carry out a neutralization reaction. The resulting product is washed several times with dimethylformamide and then with methylene chloride. 1 wash product
2 mmol of Roc-serine (
Bzl) into the solution and shake. After 10 minutes, a solution of 20 mmol of dicyclohexylcarbodiimide in 60 g of methylene chloride is added to this mixture, and coupling is effected by shaking at room temperature for 2 hours.

The resulting product is washed sequentially with Okitori 17, methidichloride/and ethanol.

Step 6 (Boc-peptidyl resin) The same operation as Step 5 is repeated to elongate the amino acids to obtain Boc-peptidyl resin.

Step 7 (cleavage of Vepti 1 from resin) 1.4 g of Boc-peptidyl resin obtained in Step 6 and 1 me of anisole were placed in a reaction vessel containing a stirring bar heated with polytetrafluoroethylene. Then add 20m of hydrogen fluoride while cooling with dry ice-acetone.
was introduced into the reaction vessel, and after stirring at 0°C for 1 hour, the hydrogen fluoride in the reaction vessel was removed under reduced pressure at 0°C. A 1% by weight acetic acid aqueous solution is added to the resulting residue, and after stirring, Bezoti 1 is extracted.The extract is placed in a separating funnel, washed with ether, and freeze-dried. In this way, peptides without protecting groups are separated.

Step 8 (Physical properties of synthetic peptide) The molecular weight, amino acid conjugation, and amino acid sequence of each peptide obtained in Step 7 are measured.

The molecular weight of each peptide is determined from the mobility measured by SNS gel electrophoresis.

Amino acid synthesis (molar ratio) involves adding a certain amount of each peptide to 6N
It is hydrolyzed in an aqueous hydrochloric acid solution at 110°C for 24 hours, and the resulting mixture is subjected to two-dimensional penoξ-chromatography for determination. Tryptophan, cystine and cysteine are not detected by this method. Therefore, by decomposing another fixed amount of each peptide with a protease, the composition of tryptophan and cystine or cysteine is determined. Note that it is not possible to distinguish between glutamic acid and glutamine, and between asno-ξlaginic acid and as-ξlagin.

The amino acid sequence of each peptide is analyzed by an automatic amino acid analyzer manufactured by Hitachi.

Step 9 (Measurement of antiviral activity) The antiviral activity of each peptide obtained in step 7 was determined by B.C. Merrigan - Human interferon using a monolayer of human neonatal dermal fibroblasts and bovine varicella stomatitis virus. Plaque formation 25- Evaluation of inhibition - “In vitro method in cell-mediated immunity”
E.D.B.R. Bloom and B.R. Grade (eds.) Academic Press, New York 1971, p. 489 CP, O, McCrigan
+Plaq,ue Inhibition As5ay
For Human Tnjerferon Emp
loying HumanNeonaLe 5kin
FibroblasL Monolayers & B
ovine Vesticular Stomatiti
s Virus+ ” In-vijro M
method in Ce1l-Mediate
aImm un i ty"t edited by
E, D, B, R, Bloom & P, l't, Gra
de+AcademicPress+ N, Y, 'l'
971 + pp489].

Specifically, each PeptiP obtained in step 7 is diluted in various ways into a growth medium containing 10 volumes of fetal calf suppressant. FSL4 cells (human neonatal skin fibroblasts) are cultured in a monolayer in this medium. After 18 hours, the cells were infected with varicella stomatitis virus (VSV), which has the ability to form 20 plaques per cell, for an additional 1 hour.
Incubate at 7°C. Next, the cells are washed twice with the above growth medium, and then cultured again in the growth medium at 37°C for 24 hours.

Whether or not a virus has occurred can be determined by checking the cells after culturing.26
− be examined by microscopic observation; The resulting cell damage is due to the virus. The peptide exhibits antiviral activity at a concentration of 1 μg/ml.

Step 10 (Measurement of cell proliferation inhibitory effect) 2X105FS-4 cells are transplanted into the growth medium described in Step 9 in a Petri dish with a diameter of 60III+. 5
After an hour, remove the growth medium and replace it with 0.1 μg/m7
! Pour the fresh medium containing bezotir obtained in step 7 at a concentration of . After culturing, the medium was removed, and a medium containing 3 I-thymidine at a concentration of 5 μCI/ml was added.
Incubate for 2 hours at ℃. After culturing, the cells are washed with phosphate buffered saline, and then 5% by weight of trichloroacetic acid is added to the washed cells. Drying an aliquot of the resulting precipitate;
Liquid scintillation counter () ξ Manufactured by Tsuker Corporation,
The 3H radioactivity of the dried precipitate is measured using a method (United States of America).

For comparison, David Bui Gödel (David
V.

Goeddel) Well elucidated Zorro IFN-γ, I
Peptides having the same amino acid sequences as FN-γ and IFN-γ-like bezotide are prepared by the solid-phase synthesis method described above, and their cell proliferation inhibitory effects are measured by the method described above.

The results are shown below.

Zoro IFN-γ, 1FN-γ and IN- obtained in step 9
The results of the antiviral activity of the γ-like peptide are shown below.

A case where antiviral activity is observed without dilution but not observed at 1:2 dilution is defined as 1 unit, and a case where antiviral activity is observed at 0 times dilution but not observed at 21 times dilution is defined as n units.

From the above, ZoroTFN-r has no ability to suppress cell proliferation, and
It can be seen that FN-γ and IPN-γ-like bezotide have a 20-degree cell proliferation suppressive ability.

Boc-peptidyl resin obtained in step 6: 1, Bo
c Oys (Bzl) Tyr (Rzj) Oys
(+'3zl) Gln Asp(OBzl) Pr
o Tyr (Bzl) Val Lys (Z) G
lu (OBzl) Ala Olu (OBzl) A
sn Leu Lys (Z) Lys (Z) Ty
r(Bzl) Phe Asn Ala Gly Hi
s (Bzl) Ser (Bzl)Asp ((H3
zl) Val Ala Asp (OBzl) As
n Gly Thr(821) Leu Phe Le
u Gly Ile Leu Lys (Z) Asn
Trp Lys (Z) Glu (OBzI) Gl
u (OBzl) Ser (Bzl)Asp (OI
3zl) Arg (NO2) Lys (Z) Ij
e Met G1n8er (Bzl) Gln rl
e Val 8er (Bzl) Phe Tyr 2
9- (Bzl) Phe Lys (Z) Leu
Phe Lys (Z) A, sn PheL
ys (Z) Asp (OBzl) Asp (
OBzl) Gln Ser (T3zl)11e
Gln Lys (Z)Ser (BZI)V
at Glu (OBzI) Thr (Bzl)
Ile Lys (Z)Glu (OBzl)A
sp (OBzl)Met Asn Val Ly
s (Z) Phe Phe Asn Ser
(Bzl) Asn Lys (Z) Lys
(Z) Lys (Z) Arg (NO2) A
SI) (OBzl) Asp (OBzl) Phe
Glu (OBzl) Lys (Z)Leu T
hr (Bzl) Asn Tyr (BZI)
Ser (Bzl) ValThr (Bzl) As
p (,0Bzl)Leu Asn val Gi
n Arg (NO2) Lys (Z) Ala
Tle His (Bzl) Olu (OBzl)
Leu Ile Gln VaIMet Ala
Glu (Or3zl) Leu8er (T
3zl) Pro Ala Ala Lys (Z
) Thr (Bzl)Gly Lys (Z)A
rg (NO2)Lys (Z)Arg (NO2
)8er (BZI) Gln Me4 Leu
Phe Arg (NO2) GlyArg (NO2
) Arg (NO2) Ala Ser (Bzl)
Gln-resin, 2, BocMet Oys (B
zl)Tyr (Bzl)Oys, (Bzl)Gln
Asp (OBzl)Pro Tyr (Rzl)
Val Lys (Z)Glu(,0Bzl)Al
a Glu (OBzl) Asn Leu L
ys (Z) Lys (Z) Tyr (Bzl)
Phe Asn Ala Gly His
(Bzl)8er (Bzl) Asp (OB
zl) Val Ala Asp (OBzl) 3
0- Asn Gly Thr (Bzl)Leu
Phe Leu Oly rle LeuLy
s (7,) Asn Trp Lys (7,)
(llu (OBzl) Glu(OBzl)Se
r (Bzl)Asp (OBzl)Arg (N
O2) Lys(Z) Tle Met Ol
n Ser (Bzl) Gln Ile
Val 5er (Bzl) Phe Tyr
(Bzl)Phe Lys (Z)Leu P
heTJys (Z)Asn Phe Lys
(7,)Asp (OBzl)Asp(OBzl)
Gln Ser (Bzl) Tle Gl
n Lys (Z) 5er(Ilzl) Va
t Glu (013zl) Thr (Tlz
l) Tle Lys (Z) Olu (OB
zl)Asp (OBzl)Met Asn Val
Lys (Z)Phe Phc Asn S
er (By, l) Asn Lys (Z)
Lys (Z) Lys (Z) Arg (NO
2) Asp (OBzl) Asp (OBzl)P
he Olu (OBzl)Lys (Z)Le
u Thr (Bzl) AsnTyr (Bzl
)Set (Bzl)Val Thr (Bzl
)Asp(OBzl) Leu Asn Vat G
in A, rg (NO2) T, ys (Z)A
la Tle His (nzl)()lu (O
Bzl) Leu Ile GlnVat Met
Ala Glu (OBzl) Leu Ser
(BZI) Pr.

Ala A, Ia T, ys (Z) Thr (
Bzl)Gly Lys (Z)Arg(NO2)
Lys (Z) A-rg (NO2) Ser
(Bzl) Gln MetLeu Phe Ar
g (NO2) Gly Arg (NO2) Arg
(NO2)Ala Ser (Bzl) Gln-resin, 3, Boc Met Lys (Z)Tyr
(Rzl)Thr (Ilzl)Ser(Bzl)
Tyr (BZI) Ile Leu Ala
Phe Gln LeuOy's (Bzl)
Tle Val T,eu Gly Ser (I
lzl) LeuGly Oys (Bzl) Ty
r (Bzl) Oys (Bzl) Gln A
sp (OBzl) Pro Tyr (Bzl) Va
l T,ys (Z) Olu (Or(zl)A
la Glu (oBz+) A, sn Leu L
ys (7,) Lys (Z) Tyr(Bzl
) Phe Asn Ala Gly His (B
zl) Ser (Bzl)Asp (OBzl)
Vat Ala Asp (OBzl) Asn G
ly Thr(821) Leu Phe Leu G
ly Ile Leu T, ys (Z) A, 5n
Trp Lys (Z) Glu (OBzl) Gl
u (OBzl) Ser (Bzl) A, sp
(OT3z I) Arg (NO2) T, y
s (7,) Ile Mal GInSer
(Bzl)Gln Tle Vat 8er
(Bzl) T'he Tyr (Bzl) Phe T,
ys (Z) T,eu Phe Lys (
Z) Asn T'heLys (Z) Asp (
OR14) Asp (OT3zl) Gln Ser
(Rzl)IIe Gln T,ys (Z)
Ser (Bzl)Val (]llu (OBz
l)Thr (Bzl)Tie Lys (Z)
Glu (OBzl)Asp (OBzl)Met
Asn Val Lys (Z) Phe
Phe Asn Ser (Ilzl)Asn
T,ys (Z)Lys (Z)Lys (Z
)Arg (NO2)Asp(OBzl)Asp
(OBzl)Phe Glu (OBzl)Lys
(Z)Leu Thr (Bzl)Asn Tyr
(Bzl)Set (Rzl)ValThr
(Hzl)Asp (OBzl)Leu Asn Va
l Gln Arg(NO2) Lys (7,
) Ala lie T4is (Bzl) Gl
u (OBzl)Leu Tle Gln V
al Mej Ala Glu (OBzl)
Leu8er (Bzl) Pro Ala
Ala T, ys (Z) Thr (B
zl) Gly Lys (Z) Arg (NO
2) IJys (Z) Arg (NO2)8e
r (nzl) Gln Met Leu Ph
e Arg (NO2) GlyArg (NO2)
A, rg (NO2) Ala Ser (Bzl)
Gln-resin, (where the symbols are the same as defined above).

■Physical properties obtained from Culm 8.

1. Veptyl of TFN-γ Molecular weight ° approx. 20,000 g 1 mole Amino acid composition (mol%): Histidine (1,,4) Tricytophane (O3) Lysine (13,6) Arginine (S, S) Asnoξlagic acid/ As ξ Ragin (13,6) Serine (
7,6) Glutamine e/f Lutamine (11,3) Threonine (3,4) Glycine (34) Proline (
1,4) Alanine (S, S) Noζphosphorus (S,
S ) ≠ Cystine (1,4) Methionine (2,
7) Inleucine (4,8) Leucine (6,8) Phenylalanine (6,s) Tyrosine (3,4) Amino acid sequence: Oys Tyr Oys Gln Asp Pro T
yr Val Lys GluAla Glu Asn
Leu Lys Lys Tyr Phe Asn
A-1aGly l11s Ser Asp Val
Ala Asp Asn Gly ThrLeu Ph
e Leu Gly Tle Leu Lys Asn
Trp LysOlu Glu Ser Asp A
rg TJYS Tle Met Gln 5erGl
n Tle Val Ser Phe Tyr Phe
Lys Leu PheLys Asn Phe L
ys Asp Asp Gln Ser Tle Gl
nLys Ser Val Glu Thr
D1e Lys Glu Asp MetA
sn Val TJYS Phe P
he Asn Ser Asn Ti
ys LysLys Arg Asp Asp P
he Glu Lys Leu Thr AsnTyr
8er Val Thr Asp Leu Asn
Val Gln ArgLys Ala Ile )T
is Glu Leu Ile Gln Val Me
tAla Glu Leu Ser Pro
Ala Ala Lys 'Thr Gly
Lys Arg Lys Arg Ser Gin M
et Leu Phe A, rgGly Arg Ar
g Ala Ser G1n2, IPN-γ-like target r Molecular weight: approx. 20,000 g 1 mol = 34- Amino acid composition (molti): histidine (1,4')) liptophan (0: 3)
Lysine (13,6) Arginine (5,4) 7sinoξ
Laginic acid/Asunoξlagin (13,6) Serine (7
,5) Glutamic acid/glutamine (12,2) Threonine (3,4) Glycine (3,4) Zoroline (1,4) Alanine (5,4) Noronine (5,4)
μcystine (1,4) Methionine (3,4) Isoleucine (4,s) Leucine (6,s)
Phenylalanine (6,8) Tyrosine (3,4) Amino acid sequence: Met Oys Tyr Oys Gln A, sp
Pro Tyr Val LysGlu A, Ia G
lu A, sn Leu Lys Lys Tyr P
he A-snAla Gly His 8er As
p Val Ala Asp Asn GlyThr
Leu Phe Leu Gly Ile
Leu Lys Asn TrpLys Gl
u Glu Ser Asp Arg Lys Ile
Met G1n5er Gln Ile Val S
er Phe Tyr Phe Lys LeuPhe
Lys Asn Phe Lys Asp
Asp Gln 8er l1eGln L
ys Ser Val Glu Thr Ile Ly
s Glu AspMet A, sn Val
Lys Phe Phe Asn Ser
Asn LysTJYSLysArgA, spノ＼5
pPheGluLysLeuThrAsn Tyr
Ser Val Thr Asp Leu
A, sn Val OlnArg Lys
Ala Tle His Glu Leu
Tle Gln ValMet Ala Gl
u Leu Ser Pro Ala Al
a Lys ThrOly Lys Arg
Lys A-rg Ser Gln Met
Leu PheArg Gly Arg
Arg Ala Ser G1n3, pu0IF
N-r target IS Molecular weight: Approximately 20,000g 1 moleAmino acid composition (mol%): Histidine (t2) Tricytophane (0,3) Lysine (12,7) Arginine (4,8) 7Sno-lagic acid/Asnoξ Lagin (12,0) Serine (7,8
) Glutamic acid/glutamine (9,4) Threonine (36) Glycine (4,2) Proline (12) Alanine (s, 4) Tucrine (a4) Intestinal cystine (1, S) Methionine (3,0) Isoleusif (5, 4) Leucine (8,4) Phenylalanine (6,6) Tyrosine (4,2) Amino acid sequence: Met Lys Tyr Thr S
er Tyr Ile Leu A-
la 'PheGln Leu Oys T
le Val Leu Gly Ser L
eu GlyOys Tyr Cys Gln Asp
Pro Tyr Val Ly4 GluAla
Glu Asn Leu Lys
IJYS Tyr Phe Asn.
AlaGly His 8er Asp Val
Ala Asp Asn Gly ThrLeu
Phe Leu Gly Ile L
eu Lys Asn Trp Lys
Glu Olu Ser Asp Arg
Lys Ile Ml Gln 5erGln
Tle Val Ser Phe Tyr
Phe Lys Leu PheLys A
sn Phe Lys A, sp A, sp Gln
Ser rle GlnLys Ser Val
(]Iu'Thr Ile Lys Glu A
sp MetAsn Val Lys Ph
e Phe Asn Ser Asn
Lys LysLys Arg A-sp A,
sp Phe Glu Lys Leu Thr
AsnTyr 8er Val Thr Asp
Leu Asn Val Gln ArgLys A
La Ile His Glu Leu Ile
Gln Val MetAla Glu Leu
Ser Pro A-Ia Ala Ly
s Thr GlyLys A-rg TJYS
A, rg Ser Gln Met Leu P
he ArgGly Arg A, rg A, Ia
Ser Gln patent applicant Asahi Kasei Corporation = 37 =

Claims (1)

[Claims] Formula (1) (■): J Oys Tyr Oys Gln Asp Pro
Tyr Val Lys GluAla Glu A
sn Leu Lys Lys Tyr Phe As
n Ala GlyHis Ser Asp Val
Ala Asp Asn Gly Thr Leu P
heLeu Gly Tle Leu Lys Asn
Trp Lys Glu Glu 5erAsp
Arg Lys Tle Met Gln
Ser Gln Monthe Vat 8erPh
e Tyr Phe Lys Leu Phe Lys
Asn Phe Lys AspAsp Gin
Set rle Gln Lys 8er
Val Glu Thr l1eLys Gl
u As'p Met Asn 'Val Lys'P
he Phe Asn 5erAsn TJys Ly
s Lys A, rg Asp 'A, sp Phe
Glu Lys LeuTbr A, sn Tyr S
er Val Thr Asp Leu Asn Va
l GlnArg Lys Ala Ile His
Glu Leurle Gln Val MetAl
a Glu Leu Ser Pro Ala A,I
a TJysThr G'ly LysArg Lys
A-rg Ser Gln Met Leu Phe
Arg Gly ArgArg Ala Ser G
in (However, Oys+'l'yr+Gln+Asp+Pro
+Val'+Lys+01u +AI a +Asn
+Leu +Phe +GI y +Ili s +S
er, Thr + r le +Trp+Arg and Met are cysteine, tyrosine, glutamine, asnoξlagic acid, proline, tyrosine,) serine, lysine,
Glutamic acid, alanine, asnoglagic acid, leucine, phenylalanine, glycine, histidine, serine,
Threonine, inleucine, tryptophan, arginine, and methionine residues; and J represents a hydrogen atom). A method for producing Vepti-P of human interferon-γ represented by the above formula (T), which is characterized by a reaction mechanism in which a derivative and a certain amino acid derivative react to form a Pepti-R bond (however, the amino acid derivative may contain a plurality of (3) Human interferon according to claim 2, which is formed by reacting a certain amino acid derivative with another amino acid derivative to form a bezotide bond. Process for producing r with heftyl (4) After bonding the carboxylic acid group of an amino acid having an α-amine protecting group to a support such as a polystyrene resin crosslinked with divinylbenzene, the α-amine protecting group is selectively removed. removing the amino acid, reacting the free amino group with a second amino acid having an α-amine protecting group, and then sequentially repeating the removal of the α-amine protecting group and the introduction of another amino acid having an α-amine protecting group. Claim 3 consisting of
A method for producing human interferon-γ bezoti-r as described in (5) A cell proliferation inhibitor comprising a human interferon-γ peptide, and (6) a human interferon-γ pezoti-P antiviral agent.