JPS6163696A - Novel peptide - Google Patents

Abstract

NEW MATERIAL:A peptide expressed by the formula (J represents residue of methionine or H). USE:An antiviral agent or anti-malignant-tumor agent. PREPARATION:For example, a compound expressed by the formula Boc-Ser(Bzl)-OH(Boc is t-butyloxycarbonyl; Bzl is benzyl), which is an alpha-amino- protected c-terminal amino acid, is reacted with a chloromethyl resin, which is prepared by chloromethylating polystyrene crosslinked with divrinyl-benzene, in the presence of a basic substance, followed by elimination of alpha-amino- protecting groups. Then, procedures for reaction thereof with an alpha-amino- protected amino acid in the presence of a condensing agent and elimination of alpha-amino-protecting group are repeated according to an amino acid sequence of the peptide to obtain a peptidyl resin. Then the peptide is removed from the carrier resin and all protecting groups thereof are eliminated to obtain the peptide expressed by the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION (1) Purpose of the Invention The object of the present invention is to provide a novel peptide gold having excellent physiological activity such as antiviral action and cell proliferation inhibiting action.

More specifically, Formula (1): %Formula% ,,iet A311 Val Lys Ph
e Phe Asn Ser Asn Lys
LysLLI Arg Asp Asp Phe
Glu Lys Leu Thr Asn TyrS
er Val Thr Asp Lcu Asn V
al Gln Arg Ly+s Allalle)
iis Glu Leu Ile Gin
Val Met Ala Cr1u LeuS
er Pro A13, Ala Lys
Thr Gly Lya Arg Lys Arg
Ser Gin #Iet Leu Phe Arg
Gly Arg Arg Ala 5er (However, G
ln+Aqp, Pro, Tyr, Vals Lys*G
1u*Ala+Asr+, Leu, Phe, Gly+H
ia, Ser, Thr, IIs, Trp, Arg and biet are glutamine, asnozolagic acid, proline.

tyrosine,) serine, lysine, glutamic acid, alanine, asparagine N, leucine, phenylalanine, glycine, histidine, serine, threonine, inleucine, tryptophan, arginine and methionine residues: and J is the methionine residue zl'; or hydroxyl The purpose of the present invention is to provide a peptide having the following atom.

(2) Bottom of emitting groove (means) The i) r-regular peptide of the present invention can be produced by a conventionally known method,
That is, use the method described in "Synthetic Chemistry Series - Peptide Synthesis" by Nobuo Izumiya et al., 1975, published by Maruzen Co., Ltd.: Edited by the Biochemical Society of Japan, "Biochemistry Experimental Course / Chemistry of Proteins 1 - IVJ, published by Tokyo Kagaku Doujin, 1977. It can be manufactured by

As one of the embodiments, the solid phase method will be specifically explained below. After the carmenic acid group of amino fa having an α-amino protecting group is bonded to a support such as a polystyrene resin crosslinked with divinylbenzene, the α-amino protecting group is selectively removed. The free amino group is reacted with a second amino acid having an α-amino protecting group. This α-
Removal of the amino-containing i group and introduction of an amino acid having an α-amino protecting group are repeated to obtain the desired peptide chain. The covalent bond between the cardanic 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-amino protecting group or the side chain protecting group can be removed simultaneously with the cleavage of the covalent bond.

In the above-mentioned in-phase method, 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 there is no need for the reaction. This method is more advantageous than the so-called liquid phase method because the appropriate amount of reagent used and the J-"11 product can be removed by 5 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 styrene crosslinked with divinylbenzene and has a degree of crosslinking of 2. I
J Mama - A size of 200 to 400 mesh is desirable. 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, 5nC12f f”, j!
A method is used in which the aromatic ring of styrene used as f7 is reacted with chloromethyl methyl ether by Friedel-Krach reaction, and the aromatic ring of styrene is chloromethylated.

;As the α-amino protecting group used in the method of Calendar 1,
Preferably, it is stable upon coupling between the support and the amino ρ having an α-amino group and can be easily removed without adversely affecting the side chain preservation group or the peptide chain. A preferred α-amino protecting group as described above includes a tertiary-butoxycarbonyl (hereinafter abbreviated as rBocJ) group. This Boc structure is I N HCl/acetic acid mixture, 4N HCl/Geo=? ? 7 Mixed U or 50% (W/V) TfOH (tetrafluoroacetic acid) /CHaCA! z It can be easily removed from the niheptide chain using a mixed solution.

Side chain protecting groups include coupling and α-amino protection groups.
Groups that are stable upon removal of the group and that can be easily removed at the stage of peptide synthesis are preferred.

Such preferable side chain protecting groups include No2 or tosyl group for protecting the side chain of arginine, benzyl group (hereinafter abbreviated as rBzlJ) group for protecting the side chain of aspartic acid, and cysteine and glutamic acid. For side chain protection, a Bzl group or O-methylbenzyl (hereinafter abbreviated as "0BzJ") group is used, and for side chain protection of histidine, a Bzl group, benzyloxycarbonyl (hereinafter abbreviated as rZJ) group, or Two Boa groups are used to protect the side chain of lysine,
Tosyl group or diimprobyl methyloxycarzenyl (
A sulfoxide group is used to protect the side 1 of methionine, and a Bzl group is used to protect the side chains of serine, threonine, and tyrosine.

+br, bright peptides can also be obtained by the following method.

First, a genetic code corresponding to each amino acid in the amino acid sequence of the peptide of the present invention to be obtained is selected.

Based on each of the extracted genetic codes, a double-stranded DNA fragment having the genetic information of the peptide of the present invention is chemically synthesized. The obtained double-stranded DNA fragment is incorporated into a vector, which is a replicable expression vehicle. Next, the vector containing the DNA fragment is introduced into the host.

The host is transformed by inserting the above vector incorporating the DNA fragment into the host, resulting in the formation of a transformant. Next, by a conventional method, this trait 1'
, r, transmutation is carried out from the original host depending on the presence or absence of gene expression. The peptide of the present invention is produced by culturing the transformed transformant.

Examples of the host used in the above method include microorganisms, microorganisms, J+M cells, and plant cells. Among these Yd sources, it is preferable to use microorganisms and mammalian cells. Desirable microorganisms include bacteria such as Bacterium cerevisiae, Bacillus subtilis, thermophilic bacteria, actinomycetes, and yeast.

The replicable expression vehicle or vector used in the present invention is one that is capable of infecting the host described above.

Such vectors include plasmids, phages,
Examples include viruses. The above vectors and hosts are
It is also used in the production of peptides from physiologically derived genes derived from biotechnology.

The genetic code corresponding to each amino acid residue shown below is used to synthesize the gene for the physiologically active peptide of the present invention (however, in the genetic code shown below, A represents a deoxyadenylate residue, and G represents a deoxyadenylate residue. Phanyl acid residue is C
represents a deoxycytidylic acid residue, T represents a thymidylic acid residue, the left side of the genetic code represents the 5'-hydroxyl group, and the right side of the genetic code represents the 3'-hydroxyl side): Phenylalanine The genetic code for is TTT or TTC
, the genetic code for el i'77% is 'rTA, TTG
.

CTT, CTC, CTA or CTG, i'l code for inleucine is ATT, ATC or FiATA
, the R genetic code for methionine is ATG, and the genetic code for valine is GTT, GTC, GTA or G
TG, the genetic code for serine is TCT, TCC,
TCA, TCG, AGT or AGC.

The genetic code for proline is CCT, CCC, CCA
Also, Ir1CCG, the genetic code for threonine is AC
T.

ACC, ACA or ACG, the genetic code for alanine is GCT, GCC, GCA or GCG, the genetic code for tyrosine is TAT or TAC, the genetic code for histidine is CAT or CAC, the genetic code for glutamine is CAA or CAG, as The genetic code for A Ragin is AAT or 7VAC%! The genetic code for J-gin is AAA or AAG, the genetic code for aspartic acid is GAT or GAC, the genetic code for glutamic acid is GAA or GAG, the genetic code for cysteine is TGT or TGC, the genetic code for IJ butophane is TGG, the genetic code for arginine is The code is CGT.

The genetic code for CGC, CGA, CGG, AGA or AGG 1 and glycine is GGT 1GGC
, GGA or GGG are used. In addition to the above genetic code, ATG is the genetic code for methionine and is also used as the starting code for peptide synthesis, and TAA.

TAG and TGA ff are used as termination codes for peptide synthesis. The gene consists of the four types of deoxyribonucleotides mentioned above linked at the 3' and 5' sites by phosphodiester bonds. Therefore, peptide genes can be chemically synthesized using protecting groups and binding agents. The protecting group is one that can selectively protect a predetermined position of the deoxyribonucleotide, is stable during the nucleotide synthesis reaction, and can be easily removed without breaking the bond between nucleotides. It's desirable. Furthermore, the reaction intermediates must be easily separated and purified.

For example, the triester method described below can also be used to synthesize a peptide gene.

That is, a trimer block of deoxyribonucleotides corresponding to the amino acid code and a dimer block f
? /J to synthesize. The resulting blocks are then condensed to form oligomers of about 12-23 nucleotides. This condensation reaction is carried out by an in-phase method using a support such as a polymer. That's 'm,'! Yes. Convert the obtained oligomer into DNA! By elongating the resulting DNA with water and adding water, the desired peptide's N, C1 butsuji double order DNA is obtained.
Form A.

The amino groups of deoxyribonuclease are
A benzoyl group is attached to the deoxyguanosine group, and an isobutyl group is attached to the molecule.As for the retention of hydroxyl group, trityl group, acyl group, silyl group, and the derivatives of -tJ, et al. In the case of these hydroxyl groups, derivatives of trityl groups are preferably used. Sul7onyltetrazolide (
A self-condensing agent such as rTPsTeJ (hereinafter often abbreviated as rTPsTeJ) is preferred. The retained Q& of oligonucleotides obtained by deoxyribonuclease condensation can be removed by the method described below. First, 3'-hydroxyl protecting groups such as amine δ groups and acyl derivative groups are removed with aqueous ammonia, and then 5'-hydroxyl protecting groups such as trityl derivative groups are removed. Remove with 80 volumes of acetic acid.

In the presence of dATP, oligo(deoxyadenosine-phosphate) [hereinafter often abbreviated as "oIJ:r(dA)J"] is added to the 3'-end of the double-stranded DNA obtained above by the action of terminal transferase. let

On the other hand, plasmid pBR322 (F, Zelino etc.,
``Construction, and, Characterization, Of, Niney, Kroninck, Guihikulus■;
A, multi-purpose, cloning.

System'', Gene, November (1977) [F.

Bolivar at al, “constructi”
on and characteri -zatLon
of new cloning v@hicles
(1: Amulti-purpose cloni
ng system”, Gone, November
(ls77)]) is cleaved with the restriction enzyme EcoRI and then treated with λ-exonuclease. Then, dTT
In the presence of P, oligo(dA) is added to the 3'-end of the cleaved plasmid pBR322 by the action of terminal transferase. The above oligo(dT)-added linear plasmid pBR322 is mixed and annealed. The plasmid obtained by annealing was used in Escherichia coli strain X1776 [American, Rockville, Maryland, USA, type. Culture Collection (ATCC Deposit No. 31244) K to transform the strain. The quality of the juice and the strain 4 are selected based on the properties of ampicillin and other drugs.

Next, a 300 base pair DNA containing the promoter, operator and liposome binding site of the large JV to tryptophan operon, with EcoRI-truncated VJr ends in both ends.
The A fragment is connected to the 'F:coRI cut V4j site of the DNA incorporated into plasmid pBR322, and fIl is inserted into the plasmid.

Next, the plasmid that was swallowed in this way was added to the plasmid.
'1 into human cells.

ifi DNA lυr fragment or after the above promoter, sales to mRNA, if connected correctly to the force direction, Pi in E. coli 1, qa, r = eLINA) III
mRNA K is transcribed by the promoter of the above operon. Furthermore, the transcribed mRNA is translated into amino acids from the starting triplet code to create the desired peptide.

In this way, 0.17179 of the target peptide can be extracted from 1 liter of cultured E. coli cells η).

Next, A-m cells of E. coli that produce the desired peptide are collected after culturing and lysed. After decomposing the nucleic acid in the solution containing the 60-cell cultured cells with ribonuclease and deoxyribonuclease, the peptide of interest is salted out with ammonium sulfate of 659 J#. The obtained salting-out precipitate fraction was washed with chondral pore glass beads (Controlled pore glass beads).
ore glass beads).

The method for determining the amino acid sequence of the novel peptide of the present invention is as follows. First, the methionyl bond of the novel physiologically active peptide of the present invention is cleaved with bromo cyanide. Peptide Dr piece obtained by cutting? i7 Fatex G-100 (Pharmacia).

Then, the amino acid sequences of the '6 fragments were determined sequentially from the N-terminus of each fragment by a well-known high-resolution amino Q sequence analysis. The biologically active peptide of the present invention was partially digested with trypsin, and the resulting peptide fragments were separated using Sephadex G-100 Gym. The amino acid sequence of each fragment is determined from the N-terminus of each fragment using I. The sequence of each fragment of the peptide is determined by comparing the amino acid sequences of each fragment of the present invention.
Determine the amino acid sequence of a peptide.

Inhibition of i'LllI hypertrophy proliferation! 11j1 For example, the peptides of the present invention are generally used in 5. '4-Can be administered by injection intravenously, intracutaneously or subcutaneously. The daily dosage of the inventive peptide varies, of course, depending on the age, medical condition and weight of the patient, but it is administered by injection at 1X10' to 1X10' units per adult per 7 days.

To cure viral diseases, Autumn ET IF'1
An ointment containing 10 g of the peptide lXl0' to lXl09 of the present invention can be applied to the skin.

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

The daily dosage of the ointment containing the peptide of the present invention will of course depend on the age and medical condition of the patient, but it is usually applied in divided doses of 1X10' to 1X10' based on the peptide of the present invention. be able to.

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.

Explanation of abbreviations used in the examples a-2-lysine: A benzyloxycarbonyl group (2-) bonded to the lysine amino group G N (h Fulginine: A guanidino group (G-) of arginine imBzl-Histidine: imidazolyl of histidine &
(im) with a benzyl group (Bzl-) bonded to 0-1<zi-serine: serine water 6L (0-) with benzyl M (Bzl-) bonded to Boc-nophrine: α-amino of parin An ortho-methylbenzyl group with 6g tributoxycarzenyl excitation (Boc-) attached to the group uB z l 2&'lZj+ m1i-% VSV: Vesicular Stomatitis Virus
Virus) AMV2, IJ- to osteoblastosis virus (Avi
an Myeloblastosis Viru3) BSA: Morning serum albumin SEA: Staphylococcal, which is 11 times of mytonoene, Enterotonycin A 1, ↑jl”j CBio-Bead 5-Xz *
200-400 mesh (2nd division divinylbenzene)
, trade name of Ustyrene Bolimer, manufactured by Bio-Rad,
(United States of America)] was added to 11 distilled benzene with stirring. After 30 minutes, remove the 1.rl embryo using a glass filter. Next, add the dried resin to the methanol of 11 with stirring, and after 30 minutes remove the and dry. 1L side layer obtained by drying methanol, alcohol,
Wash sequentially with methanol/water and water. Washed I±L
(Fat is 11 1NNao)! Add to aqueous acid.

The resulting mixture was stirred in a boiling water bath for 1 hour, and the resin was removed and washed with water. Next, 14 to 7', l'J
Add Ita Purple 11 to an IN aqueous solution of hydrochloric acid and stir in a boiling water bath for 1 hour. The resin is then filtered off and washed with water.

The resin obtained by washing with water was suspended in distilled water for 1'+4 and then allowed to stand. 1 juice f430 minutes later, a floating political f
Remove fat A by decantation. ) to obtain cloud fat as a precipitate. The precipitated 100 g fat is collected by filtration on a glass filter and washed with methanol. The washed resin is added to a small amount of dimethylformamide (hereinafter abbreviated as "shu") and stirred at 80° C. for 30 minutes for 12 minutes, and then the stirred resin is filtered and washed with Dh and methanol. Furthermore,
The mixture was stirred for 1 hour in 21-g of methanol and then filtered. Dry the applied resin (fc area) and further heat at 100℃ for 2 hours.
11 times, 1 time, 1 time.

Nitoso 2 (forgery of chloromethyl oil) 1 侭IC(ff (rofl, da;; 4 bile 25,9 and distilled chloro, /F methyl tate/l/l OO
rd tcl 1 round bottom two-headed flask, put it in,
Stir quietly at 25°C for 1 hour.
After cooling, cool to 0°C. This volume has been published;, +, -, IEJ↑fat (τ1.F18+d
50 rtE glue containing 5 nCJ4 [;J Methyl Methyl Ether] After heating for 30 minutes with stirring, and then heating for an additional 30 minutes 1>;I
The product thus obtained was filtered through a gasine filter, and then 5θOi water → non-water (1:1), then 50Q, Te, 7. Oxa 7-3 N salt 12 (3:1) quiet K 6'
e, υ°. Further, the product was washed with water, dioxane, and dioxane, and then washed under reduced pressure for 1 hour.
Rub at 00℃.

F°23 A, (5-Z-Lysine) To L-lysine copper salt of 11.9, add 1/4 of 1oI NaHCOs and 1/4 of 12-Z-C1 every 10 minutes under water cooling (
d Add with stirring and stir for an additional 15 hours.

The precipitate thus obtained is filtered off and washed successively with water, ethanol and acetone/ether. The washed precipitate is dissolved in 250-ml water for 1 ttl f'j4, and then a 412-ml 6N aqueous hydrochloric acid solution is added to this suspension to dissolve the precipitate. Then, the resulting solution 1c
Bubble with Hz8 gas for 1-2 hours.

The resulting CuS is filtered off using a Hyflosuit 9-cell (Wako Tsumugi Co., Ltd., Japan), and the CuS is washed with an IN aqueous solution of hydrochloric acid. The filtrate and wash obtained from the above procedure are collected and air is passed through to remove H and S. Thereafter, this solution was cooled with water, and concentrated aqueous ammonia was added to adjust the pH to 6.5, and the solution was left in the refrigerator for 2 hours.

The resulting precipitate is filtered off and washed successively with water, ethanol and ether. In this way, melting point 254
10 g of -Z-lysine are obtained.

The yield of 1-2-lysine produced is 65%.

The obtained g-1+) gin is recrystallized by dissolving it in dilute hydrochloric acid and then neutralizing it with aqueous ammonia.

B, (G-No, -arginine) A mixture of 2120 mg of fuming glass G and 75 mg of oleum is cooled with water, and this mixture is mixed with 110 mg of oleum. Gradually add L-arginine (I) while stirring.Additionally, add 5d of L-arginine.
Add to the above mixture. The resulting mixture was stirred for 1 hour, poured into ice cubes, and adjusted to pH 8 with concentrated aqueous ammonia. Next, after adjusting the mixture to pH 6 with acetic acid,
Leave it in the refrigerator for about 4 hours.

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

In this way, H: 1 point G -No, -arginine 60,9 at 251°C is obtained. G N (yield of h-arginine is 50%.

Co(imBzl-Histidine)20.9 Histidine hydrochloride H20 was cooled to -30°C in a dry ice-acetone bath to give 200 rpJ! of liquid ammonia and add 1.0% gold to this solution. Add a small piece of Sodium X while stirring until the color of the bath liquid remains intact. Next, small J, t histidine 'E
, Mf6 solution until the ff color disappears, then add benzyl chloride to the solution. This mixed solution tl-30
After stirring for 2 minutes, the NH3t in this mixed bath was evaporated at room temperature under atmospheric pressure, and then the NH3t in this mixed bath was evaporated under reduced pressure using a water jet.
3fi - remove. NH3 + total 3 + leftover 100 m
Pour into 1 liter of ice water. The resulting precipitate is filtered off and recrystallized from 7oI % ethanol. Melting point 248℃
imBzl-histidiD, (0-Bzl-7) 237I acetyl-L-serine 235 rnt r
Leave the tE at 36°C for 10 days. The resulting precipitate is directly removed, filtered iH is added to the solution, and then Architon gold is added to the filtered solution to obtain crystals. In this way to
og O-nz 1-7 phosphorus is obtained. Following a similar method, 240 g of acetyl-L-threonine Kara 100
, q of 0-Bzl-threonine (; is added.

E, (0-11zl-tyrosine) 1.89 tyrosine and 1.2 g -CuSO4,5
H1OS: Suspend in a solution of 5 me of 2N Na0I and Iot4 of water and stir for 2 hours. To this suspension was added 60 methanol, followed by 1.2 mbenzyl bromide and 0.7 methanol of 2 N Na.
Gradually add the OH aqueous solution.

After 15 minutes 0.3 rnl of benzyl bromide and 0.8 rnl. Add more 2N NaOH water and Tani liquid. The resulting mixture was stirred for 1 hour, and after step 7, the resulting precipitate was collected by filtration and washed with methanol-water (1:3). In this way, 2 g of the copper salt of 0-13zl-tyrosine are obtained.

This copper salt of 0-Bzl-tyrosine and 20-IN E
DTA and water F8 liquid are kneaded in a mortar to obtain a mixed filtrate of 1.5 ml of solid matter, which is collected by filtration, and this solid matter is washed with water.

By washing 7 and recrystallizing the product from hot water containing a small amount of EDTA, 6O-Bz with a mark of 223 °C was obtained.
1 g of l-tyrosine is obtained. The yield of 0-Bzl-tyrosine is 30%.

F, (S-Bzl-cystine) 157 g of cysteine hydrochloride was dissolved in 21 2N NaOH
Dissolve in an aqueous solution, add 25βg of benzyl bromide to this solution while stirring vigorously 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. The generated precipitate is collected by filtration and washed 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 70
%.

15 from 160 g of aspartic acid in the same manner as above.
0I of 0-Bzl-asnozolagic acid and 1509 of 0-Bzl-glutamic acid are obtained from 160 g of glutamic acid.

G, (Boa-amino
+; (Set as '+7-. After adding all 150 me of tetrahydrofuran to this solution, add 1 OO+rri of Boa-C to 1j 7if while stirring vigorously at 10°C.
Add 1/5 of I every 10 minutes, while 2 N
NaOH is added to the solution each time the Boc-C1 is added until the pH of the solution is between 8 and 91 Ci.

, After 2 hours, extract the mixture a with diethyl ether,
The resulting aqueous layer is made acidic with a 0.5M aqueous citric acid solution, and the precipitated oily i'r is extracted with ethyl acetate. After washing the obtained extract with mild water and drying with NIL2SO4,
7. Under reduced pressure. , 'VI. Add petroleum ether to the 4-condensed extract and leave it in a refrigerator to form ash crystals. Take the formed crystals and dry them.

In this way, Boa-valine 30y with a melting point of 78°C is obtained. The yield of Boc-valine is 55%. By performing the same method as for H8 (other Lloc-amino acids) promotion, BoC-glycyl:/15g, Boc-alanine 16 fi, B
oa-.

Ishin 15. ! 9, Boc-inleucine 15,9. B
oa-serine (Bzl) 14.5', Boa-threony (Bzl) 14.9, Boc-cysdine (Bz
l) 15 g, Boc-methionine 1 sg, Boc-proline 12. ! ? , Boc-asparagine rR (OBz
l) 15 g, Boa-glutamic acid (OBzl)
15 jJ, Boa-glutamine 15 g, Boc-histidine (Bzl) 12 g, Boc-0 isine (Z)l
3,9SBoa-Arginine (Not) 15.9
XBoc-Phenylalanine 15g, Boa-Tyrosine! Bz1)159 and Boc-)Liptophan10
g is so, y lysine 30!9, alanine 30J, leucine 30g, inleucine 30.9. Serin (BZI
) 30g 1 Threonine (Bzl) 301% Cystine (Bzl) 30g, Methionine 30g, Proline 3
011 As, e Ragic acid (OBzl) 301 s Glutamic acid (OBzl) 309, Glutamine 30g1 Histidine (Bzl) 309, Rishi7 (Z) 30 p
Step 4 (Boa-serine (Bz
l ) and resin bonding) 2 g of chloromethyl resin (C1
! A mixture of 4 mmol total content), 2 mmol BoC-L-serine (Bzl), 1.8 mmol triethylamine, and 12 me dimethylformamide was prepared at room temperature for 24 hours.
Time distribution - 4 - 勺. Take 8 portions of the solid mixture from the above mixture using a thin filter, and add the resulting solid product to it (77t70.0'rrtl) of dimethylformamide, ethanol Wash sequentially with methylene chloride. After step 7, dry the product under reduced pressure.

Main:, PAζ′:′J amino r:, the content is 1liit
To determine the generation 'l? J 5 Q Weigh Ih9 (
), and after hydrolyzing for 24 hours in a mixture of 12N hydrochloric acid and dioxane,
Measure with an amino acid analyzer. The amino acid content of the product was 0.2 mmol/g.

Engineering j45 (Boa -Ala -5er(Bzl)-
Resin) processing 1. 2 g of Boa-serine (B
zl) (hereinafter abbreviated as F rBoc-GlnJ) resin i
In 160m of ot-acid: ・Put it in and leave it at the lowest temperature for 6 hours. This mixture is then filtered through a Gance filter. The obtained solid mushrooms were placed in 160 ml of acetic acid and shaken.
Don't take it. Repeat this operation three times. i1 above in the sky
Form;, 'l k 16 C1r=t's IN salt ridge water bath version/
I put a person in acetic acid and left it in the room/route for 30 minutes. Take 5 pieces of the product thus obtained and wash each portion three times for 1 m with vinegar, ethanol and dimethylformamide. The purified product was placed in 160-10-fold λ-width triethylamine/trimethylformamide.
The neutralization reaction was carried out for 0 minutes. As a result, we get n
Wash the Otsu-generated enzyme with Uratori Shite dimethylform/L-mamide,
It is then immediately washed with methylene chloride. 611: L2mi methylene chloride for Josei DZ stuff! , 2 mmol of BoC-
Place the alanine in the evaporated solution and remove from the fan. 10
After a few minutes, add 2 Q to 60 ml of methylene chloride to this mixture.
IJ moles of dicyclohexyl ruzedimide are added to a solution and the system is stirred at room temperature for 2 hours to carry out coupling. The resulting product is filtered off and washed with methylene chloride and ethanol.

Step 6 (Boc-peptidyl resin) Repeat the same operations as Step 5 to lengthen the entire amino acid to obtain the following Eoc-peptidyl resin: 0Boa Gin
Asp (OBzl) Pro Tyr (Bzl)
Val Lys (Z)Glu (OH2,1)Al
a Glu rOBzl) Asn Leu Lys
(Z) Lys (Z) Tyr (Bzl)
Phe Asn Ala Gly His (Bz
13Scr (Bzt)Aap (OBzl)Va
IAl+1IAap (OBZl) AsnGly
Thr (By, 1) Lcu Phe Lea G
ly IIs Lau LyafZ) Al11
n Trp Lys (Z) Glu (OTh
zl) Glu (OBzl)Ser fRzl
) 8sp (OBy, 1)Arg fNO2)C
15(z) IleMet Can Ser
(BZI) GIII Ile Val Srr
(Bzl)PheTyr (87,1) Phe
Lys (z＞Leu Phe Lys
(Z) AsnPhe Lys (Z) Aap
(OBzl) Asp (OBzl) Gi
n 5er(BZl"l 11cGin Lys
(Z)Set (Bzl)Val Glu (L
)BZI)Thr (lzl) Ilc Lyr
; (,Z) Glu (CIlzl) As
p (OBzl)8let ASn Val
Lys (Z) Phe Phe Asn S
er (Bzl)AanLys (Z)Lys(
Z) Lys (Z)Arg (NO2) ASl)
(OBy, 1) Asp (OBzl)Phe
Glt+ (ORzl) Lys (Z)
Leu Thr (BZI) Asn Tyr [h
zl) Sar (BzlJ Val 'l'hr
(Bzl)Asp (OBzl)LeuA3n V
al G11l Arg (Now)LyS <z
)Ala Ile His(Bzll Glu
<OH2,1)T,eu Ila Gin Val
Met Ala Glu(L'13yi) Lau
Ser (Bzl) Pro Ala Ala
f,ys (Z) Thr(13zll Gly
Lys (Z) Arg (DJOa) L
yes (Z) Arg (NO2)Ser
(!(zll Gln ;・!et Leu Ph
e Arg (NO23Gly Arg(Now)
Arg (Not) Ala Set (Bzl)
Using the above Boa-peptidyl resin and Boa-methionine, the following 11oc.
- Obtain methionyl peptidyl tfij fat.

Boc Met Gin Asp (OBzi)
Pro Tyr (Bzl) Val Lys (Z
) Glu (OBzl) Ala Glu (OBzl)
l) Asn Leu Lys (Z)Lys (Z)
Tyr (Bzl) Phe Asn Ala Gl
y Hls (Bzl)Sar (Bzl) Asp
(OBzl) V'al Ala Asp (ORzl
) AI! 1nGly Thr (Bzl) Leu P
he Lau Gly IIs Leu Lys (Z
) Aan Trp Lys (Z) Glu fOBz
l) Glu (OBzl) 5er(Bzl) As
p (OBzl) Arg (Now) Lys (Z
) Ile MetGin Ser (Bzl) Gi
n Lie Mal Ser (Bzl) Phe T
yr (Bzl) Phe Lys (Z) Leu P
he L12 (Z) Asn PheLys (Z)
Asp (OBzl) Asp (OBzl) Gin
Ser (Bzl) Its Gin Lya (Z)
Set (Bzl) Vat Glu (OBzl)
Thr (Bzl) Ile Lys (Z) Glu
(OBzl) A11l) (OBzl1M@t A
sn Val Lys (Z) PbePhe Aan
Ser (Bzl) Asn Lys (Z) Lys
(Z) Lys (Z) Arg (NO2) As
p(OBzl) Asp (OBzl) Phe Gl
u (OBzl) Lya (Z)Lcu Thr (
Bzl) Asn Tyr (Bzl) Ser (B
zl) VatThr (nzl) Asp (
OBzl) Leu Asn Vat Gin Ar
g(NO2j Ly+ (Z) Ala Ilc
His (Bzl) Glu (OBzl)Le
U Ila Gin Val? ,'1. et A
la Glu (OBzl) Leu 5er (B
zl) Pro Ala Ala Lys (Z
) Thr (Bzl) Gly Lys(Z)
Arg (NO2) Lys (Z) Arg(
Not) Ser (Bzl)G11", Let
Lqu Phe Arg (NO2) Gly A
rg (Now) Arg (Not l +'il
a Ser (Bzl) -;,'↑fat, Nisa 7 (:
:':r iE' peptide appeal) m in Epi 6
Boc-pezotidyl-j 1iir of L4,9
and 1.4 g of each of Boc-methionyl peptidyl resin and 1 m of anisole to 1 polytetrafluoroethylene 7
) with a stirrer in the reaction vessel Ilc,
Hydrogen fluoride 20% while cooling with dry ice-acetone
me was introduced into the reaction vessel and 1 ['-'p r
jJ 4 times, ζL (1, JT removed.
After stirring the acetate solution to /li[+Ete], extract the peptide, put the extract into a fraction funnel, wash with ether, and freeze-dry. In this way, each peptide that does not contain i& is separated.2. . The peptide obtained from Boc-peptidyl resin is called Peptide 1 # Boa-methionylpeptidyl-i, and the peptide obtained from fat is called Peptide 2.

(Binding of BoC-glutamine and resin) The following experiment was carried out in the same manner as in Engineering 84.
Synthesize r. That is, 2. chloromethyl resin (C1
A mixture of 4 mmol of total impurities), 2 mmol of Boa-L-glutamine, 1.8 mmol of triethylamine and 12-methylformamide is taken up for 24 hours at room temperature.

The solid content is then filtered from the mixture using a glass filter, and the resulting solid product is washed sequentially with 700-dimethylformamide, ethanol, acetic acid/ethanol, and methylene chloride, respectively. The product is then dried at room temperature under reduced pressure. To determine the amino acid content of the product, 50 da of Jisheng was weighed and placed in a rattan mixture of 12N hydrochloric acid and dioxane for 7 JO for 24 hours.
After water decomposition, it is measured using an amino acid analyzer. The amino acid content for production is α2 mmol/9.

I BoI+-8cr Gin '3::fat)? 4
2g of Boc-glutamine (rBoe-G)
1 fat (referred to as lnj) was placed in 160 ml of acetic acid and heated at Yτ1%' for 6 hours. Next, pass the mixture through a glass filter. Place the resulting solid in a 160-e lr'[V] and shake it.
, take lH. Return this operation to 31 ril (: l). Add the above solids to 160 μg of 1Nλ water, liquid/
Bo
C. Remove toads.

The product thus obtained was separated and washed three times each with acetic acid, ethanol or dimethylformamide. 1st clean beef product kz;oy I
Neutralization reaction is carried out by adding kC in Q23% triethylamine/dimethylformamide and stirring for 10 minutes. The kJ solution and the resulting product were collected by filtration and washed with dimethylboramide (7) and then washed with methylene chloride.
i' with millimoles of Boa-serine (L3zl) dissolved
, :r j6 sign person ζ and knee ; After 10 minutes, coupling is carried out by adding to this mixture a solution of 20 mmol of dicyclohexylcal-diimide in 50 =;'' a-ratio menalene and shaking for 2 hours at room temperature.The resulting product is f. Filter and wash with methylene chloride and ethanol.

(Boa-peptidyl resin) The same method as in step 5 is carried out by milling and peening to elongate the amino acids and obtain fat at the CB and C-peptidyl positions below J: B
oa Gin Asp (QBzl) Pro Tyr
(Rzl) ValLys (zl Glu (O
Ezl) Ala Glu (OBzl) AanL
au Lys (Z) Lya (Z) Tyr
(Bzl) Phe AsnAla Gly Hi
s (Bzl) Ser (Bzl) Asp
(OBzl) Val Ala Asp (Ollz
l) Agn Gly Thr (Bzl)Leu
PheLeu Gly Ile Leu Lys (Z
) Asn Trp Lys(Z) Glu(OBz
l) Glu (OBzl) Ser (Bzl) A
sp(OBzl) Arg (NO2) Lys (Z
) rle Mat Gin 5er(Bzl) Gi
n Ila Vat Ser (Bzl) Phe T
yr (Bzl) Phe Lys (Z)
Leu Phe Lys (Z) A
sn Phe Lys (Z)Asp (O
H2I) Asp (Ollzl) Gin Set
(Bzl) Ile GinLys (Z) Ser
(B7.L) Val Glu (OBzl) Thr
(Bzl)11e Lys (Z) Glu (08
y, l) Asp (OBzl) Mat AsnMa
l Lys (Z) I'he Phe Asn S
er (BzL) Asn Lya (Z)Ly3
(Z) T,yQ (Z) Arg (NO2)
Asp (OBzl) Asp (OBzl>
Phe Glu (OBzl) Lys (Z
) Leu Thr (Bzl) 83n Tyr
(Bzl) Ser (Byl) Val
Thr (Bzl)Asp(OBzl) Leu
Asn Vat Gln Arg (No2)
Lys (ZI Allalc Hi, q
f13zl) G1t+ (OBzl) Le
u Its Gin Val MetAla
Glu fOBzl'l Leu Scr (
ilzl) Pro Ala Ala Lys(Z)
Thr (Bzl) Gly Lya (Z')
Arg (NOx)Lya (Z) Arg(NO=
l Scr (Bzl,) GLn Met L@u
Pka Arg (Not) GIYArc (NO,
) Arg (NO2) Alt Set (Bzl)
Gin-(cleavage of peptides from Qt fat (X-・r fat)) was obtained, 7t was added with 1.4 g of BOC-pezotidyl resin and 1-anisole, and heated with polytetrafluoroethylene. Place the mixture in a 6-in-1 container containing a hemorrhoid stirrer, cool with dry ice and acetone, then add Q20 phi of fluorinated water to the oven reaction container for 5 people, and heat to 0°C.
After stirring for 1 hour at 0°C, the hydrogen fluoride in the reactor was removed under crossbar pressure at 0°C. Obtained/,) 1 on the residue! : Add acetic acid aqueous solution and stir 1. Extract Sibebugudo and put the extract into a separating funnel.1. Wash with ether and lyophilize.

In this way, peptides without protecting groups are separated.

The peptide obtained from Boc-peptidyl-resin is called interferon-γ.

Engineering a8 (Physical properties of synthetic peptides) The molecular weight, amino rr1 composition, and amino acid sequence of each peptide obtained in Engineering A8 are measured.

The molecular weight of each peptide was determined by SDS gel electrophoresis measurement.
#Determine from degrees.

The amino acid composition (mol%) is -6N of each peptide in the amount of
It is hydrolyzed in an aqueous hydrochloric acid solution for 24 hours at 110 DEG C., and the resulting mixture is subjected to two-dimensional P-zotalomatography 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 aspartic acid and asnozolagin.

The amino acid sequence of each peptide is analyzed using a new Hitachi automatic amino acid analyzer.

Peptide 1 molecule: approx. 20. θ0Oj 1 mole per 11 mole amino acid yield: Histidine (L4) Tryptophan (0,3>Lysine (1λ6) Arginine (5,5) 7 Sunogradon [3, / Asunozolagin (216) Serine (7,6
) group p mi;3//'ru l miy (lO,6
) threonine (14)/'glycine 3.4) -j'
Mouth+)n(1,47 alanine(s,s) Noronin(
s, s) ■ Cystine (0) Methionine (Z?) Isoloy77 (4°8) Leucine (a8) Phenylalanine (6,3) Tyrosine (λ5) Amino acid sequence: Gln Ago Pro Tyr Val Lys G
lu Ala Glu Aan LeuLys Lys
Tyr Phe Asn Ala Gly His
Ser Asp ValAla Aap A11n
Gly Thr Lau Phe Leu
Gly lie Leu! ,yq Asn T
rp Lys Glu Glu Ssr Asp Ar
g Lys IleMat Gin Scr Gln
Ile Val Ssr Phe Tyr Pho L
ysLou Phe Ly5Asn Phs Lys
Asp Asp Gin Sor l1eGin Ly
! 1Sar Val Glu Thr Ile Lys
Glu Asp rJet Asn Mal Ly
s Phe Phe Ajn Ser Asn
Lys LysLys Arg Aap
Asp Phs Glu Lya Leu
Thr AsnTyr Sar Val Th
r Amp Leu Asn Val Gi
n ArgLys Ala Ile His
Glu Leu Ile Gin Val
MetAla Glu Lau Ssr P
ro Ala Ala Lys Thr G
lyLya Arg Lya Arg Ser
Gin Met Lau Phe Arg
Gly Arg Arg Ala Ser Peptide 2 Molecular weight: Approximately 20-000,97 moles Amino acid composition (moles): Histidine (L4)) Liptophan (0,3) Lysine (13,6) Arginine (5,4) Aspartic acid/ Asparagine (1:L6) Serif (7,5
) Glutamic acid/Glutamine (10,4) Threonia ('14) Glycine (3,4) Florin (1
,4) Alanine (5,4) Nonorin (5,4)
A dimethine (0) Methionine (14) Isoleucine (4,a) Leucine C6,8) Phenylalanine (6,8) Tyrosine (Z5) Amino I:'2
, :Self 1j: Llat Gln Asp Pr+ Tyr
Mal Lys Glu Ala GluA
sn Leu [, ys Lys Tyr
Phe Aan Ala Gly H1sS1
3r Aap Val Ala A3P A
an Gly Thr Lau PhaLG+
J Gly Ile Lau Lys As
n Trp Lys Glu Glu Ser
Ajp Arg Lyn Ile Aiet
Gin Ser Gln ll5V31 S
nr Phe Tyr Pho Lys L
eu Phe Lys AanPhe Lys
Asp Asp Gln Ser Ile
Gin L) r+s 5erVal Glu
Thr ILe Lys Glu A:ap M
et Asn VaILys Phe Phe A
sn Ser Asn Lys Lys Lya A
rgAsp Asp Pho Glu Lys
Leu Thr Aan Tyr S@r
Va! Thr Aap Leu Asn
Val Gin Arg Lya AlrLl
le Hls Glu Leu Ile G
in Val Met Ala GinLeu
Sor Pro Ala Ala Lys
Thr Gly Lya ArgLys A
rg Ser Gin Met Leu P
he Arg Gly Arg Arg Ala
Ser Interferon-γ Gakoi Tsuji: Approximately 20,000,97 moles Amino 73,7 Moles: Histidine (1,41) Liptophan (0,3) Lysine (lλ6) Arginine (5,5) 7 Sunozo line acid/
Asparagine (116) SeI77 (7,6) Glutamic acid/Glutamine (9,9) Threonine (λ4)
Guijshin (λ4), ;”olJn (L4) Alanine (!L5) Valine (5,5) Cystine (O)
Methionine (L?) Inleucine (48) Leucine (6,8) Phenylalanine (a8) Tyrosine (z5) Amino acid sequence: Gin Asp Pro Tyr val Lys G
lu Ala Glu AsnLsu Lys Lya
Tyr Pha Asn Ala Gly Hls
5srAap Val Ala Asp Asn Gl
y Thr Leu Phe LeuGly Ile
Lsu Lys Asn Trp Lys Glu G
lu 5srAap Arg Lya IIs Met
Gin Ser Gin IIs ValSer P
he Tyr phe Lye Leu Phe Ly
8Asn pHeLya Asp Asp Gin S
ar Ile Gln Lys Ser ValGlu
Thr Ile Lys Glu Aap iνie
t Asn Val LysPhe Pha Aan
Ser Asn Lys Lya Lys Arg A
spAsp Phc Glu Lrys Leu Th
r Aan Tyr Sur ValThr Aap
Leu Asn Mal Gin Arg Lye A
la l1eFtis Glu Leu Ilc
Gin Val Met Ala Glu
LeuScr Pro Ala Ala
Ly, Thr Gly Lya Arg Ly
eArg Ser Gln Mst Leu
Phe Arg Gly Arg ArgΔl
a Ser Gin d9 (Measurement of antiviral activity) The antiviral activity of each peptide that is +q in 7 is measured by B. C., Merrigan - Human Neonatal Skin π] (,
Striped buds, +:' II cyst ■ Plaque formation of human interferon using sublayer and bovine varicella virus.
3, 7F City - [, "In Vitro Methods in Li'tl-Mediated Immunity" 4-, D. B., R. Bloom and B. R., Grade Academic Press, New York 1971, p. 489. C.P.

C11-murigan, Plaque Inh
ibition Aanay for H*rr+3
n Intsrferon T2ploying
Human NaonateSkin Fibrob
last Monolay@r3 & Bovin
a Vesttculax Stomatitia
Virus, “In-vitro ~ method”
in Ca1l −)+'*adi, 1tct
l lmn1unity”, editOd by E
, D, B, R.

Bl Oom &I', R+Grade
＋Acadenic Pres 31N
, “l”.

1971, pp. 489:].

Specifically, each peptide obtained in step 7 is diluted variously into 10 volumes of growth medium containing fetal microsera. In this medium, F S -4n4B cells (human neonatal skin FEI fibroblasts) are cultured in a monolayer. 1
After 8 hours, the cells are infected with varicella stomatitis virus (VSV) capable of forming 920 cells per cell, and incubated for an additional hour at 37°C.

Next, the cells are washed twice with the growth medium described above, and then cultured again in the growth medium at 37°C for 24 hours. Whether or not a virus has been generated is examined by microscopically observing the cells after culturing. The cell damage observed in the fruit is caused by the virus.

The peptide exhibits antiviral activity at 8 degrees at 1 pg/me.

The results of the antiviral activity of peptide 1, peptide 2, and interferon-γ obtained in step 7 are shown below.

Anti-tsu 1' Rusu Katsue is Kanazawa, 1 unit is the chorus that is recognized in the 1st episode and not recognized in the culture dilution, nl,, the agedai en unit is recognized in the i dilution, and is not recognized in the 2n times dilution. shall be.

Peptide 1 and peptide 2 have twice as much fI5 as interferon-r.

(3) The ineffective peptide of Hikari is a new peptide with antiviral activity.
It is a unique peptide.

Claims (1)

[Claims] Formula (I): [There is an amino acid sequence] (However, [There is an amino acid sequence] and Met are glutamine, aspartic acid, proline, tyrosine, valine, lysine, glutamic acid, alanine, asparagine, leucine, Phenylalanine, glycine,
Peptides represented by histidine, serine, threonine, isoleucine, tryptophan, arginine, and methionine residues; J represents a methionine residue or a hydrogen atom)