JPS6097999A - Novel active peptide and its preparation - Google Patents

Abstract

NEW MATERIAL:A compound shown by the formula I (R<0> is H-Thr-Asp-Ser, H-Lys-Thr-Asp-Ser-, or H-Arg-His-Lys-Thr-Asp-Ser-). USE:A drug having hypotensive action and contraction action on smooth muscles. PREPARATION:A peptide derivative shown by the formula II [R<1> is R<2>-Thr(R<5>)- Asp(R<4>)-Ser(R<3>)-(R<2> is H, or alpha-amino-protecting group; R<3>, R<4>, and R<5> show presence or absence of protecting group of side-chain functional group of amino acid), or R<2>-Arg(R<8>)-His(R<7>)-Lys(R<6>)-Thr(R<5>)-Asp(R<4>)-Ser(R<3>)-(R<6>, R<7>, and R<8> are as shown for R<3>-R<5>), etc.; Rz is -NH2, or benzhydrylamine type resin; Y is (absence)presence of sulfoxide] is deprotected to give a compound shown by the formula I .

Description

[Detailed Description of the Invention] More specifically, the present invention relates to a physiologically active peptide having a number of constituent amino acids r to // and a method for producing the same.

Substance P is an undecapeptide amide found in both the mammalian brain and gastrointestinal tract. Blood pressure lowering effect. On the other hand, it has a smooth muscle contraction effect. It is attracting attention as a neurotransmitter in the central and peripheral nerves. A series of peptides with similar structures and actions to Substance P are called nukikinins (tach
ykinins), and most of them are obtained from amphibians. physalaemi
n), eledoisin', kassinin, and the like.

In order to elucidate the biosynthetic mechanism of substance P, the present inventors isolated mRNA encoding the substance P precursor from the corpus striatum of the bovine brain. The base sequence was analyzed. the result. There are at least two types of mRN A that code for substance P, and one of them is ``mRNA'' other than substance P. It was found that an amino acid sequence similar to substance P but clearly different from substance P was encoded. Because this amino acid sequence is similar to cassinin.

The present inventors have identified this as Substance K (Substan
ce K (hereinafter abbreviated as SK). The inventors are S.K.
We synthesized octa to undeca peptides suggested by. We investigated its physiological activity. We confirmed that these have kukikinin-like effects. The invention has been completed.

In this specification. Depending on the chain length of each peptide, SK-za,
Described as SK-9, SK-/θ and SK-17.

8 Among these peptides, the peptide corresponding to SK1/θ is. It was isolated from pig dorsal spinal cord around the time the present invention was completed. The structure was determined (Sadao Kimura et al. Proa Japan
Acad. 3W, Ser, B (/9f3)/θ/-
/θg).

The peptides and methods for producing the same according to the present invention are shown below. All amino acids described herein refer to the L-form.

The peptides of the present invention have the following amino acid sequences.

R-Phe-Val-Gly-Leu-net-NH.
2/SK-, f L=H-Thr-Asp-Ser
-SK-9 R= H-Lys-Thr-Asp-Se
r-SK-/θ R= FI-Hi's=Lys-Th
r-Asp-Ser -SK-// L=H-Arg-
His-Lys-Thr-Asp-8or The present invention relates to H-Ar g-Hi a-Ly s-Th r-As having the following amino acid sequence.
p-Ser-Ph e-Va l -G ly-/
．． 234L! Ft719 Leu-Met-NH.

lOll Octapeptide amide at position t~// (SK-f).

nonapeptide amide (SK-9) at position 3 to // and
It includes undecapeptide amide (SK-/) at positions 4-7 to 77 and pharmaceutically acceptable acid addition salts thereof (for example, addition salts of hydrochloric acid, sulfuric acid, succinic acid, citric acid, tartaric acid, etc.). That is, it includes peptides represented by the following general formula and acid addition salts thereof.

R'-P h e -Va l -G I y-L
e u -Me t -Nu. 2 4' [wherein BO is H-Thr-As p-Ser-, H-L
y s -Th r-Asp-Ser- or H-Ar
g-Hi a-Lys-Thr-As p-Ser-
represents. ] The peptides represented by the above formula / can be synthesized by organic chemical and enzymatic chemical means. In the former, a liquid phase method and a solid phase method can be used alone or in combination. That is,
It may be synthesized by stepwise introduction of amino acids according to the above sequence.

After dividing into appropriate fragments and synthesizing them, each fragment is condensed. It can also be a peptide of interest.

The method for producing peptides according to the present invention is shown below.

(a) Peptide derivative R' represented by the following general formula
-Phe-Val-Gly-Leu-Met(Y)-R
, 2 [where H/ is R'-Th r (f)-As p
(R'')-8e r (R')-, R'-Lys(R
”)-Thr(R”)-Asp(R″)-8e t (
R3)-, R'-Hls(R7)-Lys(R'
)-Tb r (R')-As p (R'')-8er
(R3)- or RJ-Ar g (R')-Hi
s (R7)-Ly s (R')-Th r (R
'')-As p (R'')-8et(R'')n, where p represents hydrogen or a protecting group for the α-amino group, and p
3. R''a', RA, R7 and R' each represent the presence or absence of a protecting group for the amino acid side chain functional group), Rz represents -NH,2 or benzhydrylamine type resin, and Y represents represents the presence or absence of sulfoxide] is subjected to a deprotection reaction to obtain peptides represented by the formula /.

What is the protecting group for the a-amino group in the above definition?

It is a protecting group commonly used in peptide synthesis.

For example, benzyloxycarbonyl (2), θ-chloropenzyloxycarbonyl ((A-Z), t-butoxycarbonyl (Boc), 41-methoxybenzyloxycarbonyl (Z(OMe)), t-amyl Oxycarbonyl (Aoc), /-methylcyclohexyloxycarbonyl (Mlxoc), /-methylcyclopentoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc), formyl, trifluoroacetyl, etc. can be used.

The protecting group for the amino acid side chain functional group is a protecting group generally used in peptide synthesis. For example, t-butyl (But) or benzyl (Bzl) is
) group, guanidino group with nitro or tosyl (To
s ) group and a tosyl group for the imidazole group, the side chain functional groups of the constituent amino acids in the peptide derivative represented by the above formula (S) are protected as necessary.

Methionine may also be protected as a sulfoxide.

For each protecting group, see E., Gross et al. [The
Peptidea.

Analysis, 5 synthesis, Biolog
y, VoL 3. Protection of Func
tional Groups in Peptide
5ynthesis J (/91/year.

Academic Press), Dysentery Volume 4 and others ed.
Protein chemistry 1jgθj page (1930, Kyoritsu Publishing)
and 7-11ii, Bodanszky et al. “Peptide
5ynthesi81 (/ 976, JohnWi
Iey & 5ons Inc.).

The hydrylamine type resin referred to herein includes all benzhydrylamine or carriers for peptide synthesis having chemical properties similar thereto.

For example, the resin described in Ohem, Commun, /97θ, Otsujθ-Otsuj/ is used.

Deprotection reactions follow methods used in the peptide synthesis field. i.e. catalytic reduction, hydrogen bromide/acetic acid.

Acid decomposition with hydrogen chloride/dioxane, trifluoroacetic acid, hydrogen fluoride, etc., and treatment with a base such as sodium hydroxide, potassium hydroxide, piperidine, diethylamine, ammonia, etc. are used as appropriate.

When the peptide derivative represented by Formula 2 is bonded to a benzhydrylamine type resin, treatment with hydrogen fluoride or the like can cleave the bond with the resin and simultaneously remove the protecting group.

Conversion of methionine sulfoxide to methionine is performed by coexisting thioanisole, mercaptopyridine, etc. during deprotection with hydrogen fluoride, or by converting the deprotected peptide into mercaptoethanol.

This can be achieved by treatment with thioglycolic acid, thiomalic acid, N-methylmercaptoacetamide, and dithiothreitol.

(b) Peptide R-Phe- represented by the following general formula
Val-Gly-OH3 [wherein R/ represents the same meaning as above. ] Leucyl-methionine amino is condensed and then subjected to a deprotection reaction to obtain peptides represented by the above formula /.

The condensation of the peptide of formula 3 and leucyl-methionine amide is carried out by conventional methods such as the azide method, mixed acid anhydride method, active ester method, and carbodiimide method. Note that a method using an enzyme may also be used, for example, the method described in Japanese Patent Application Laid-Open No. 5TT-6≠69.2.

The deprotection reaction is carried out according to the method described in (a).

The peptides thus obtained are subjected to gel filtration.

Purification is performed by ion exchange chromatography, partition chromatography, high performance liquid chromatography, countercurrent partition method, and electrophoresis method.

The raw material peptides for the synthesis methods (a) and (b) are produced according to methods commonly used in the field of peptide synthesis.

As mentioned above, the peptides/peptides of the present invention have an amino acid sequence extremely similar to cassinin, and exhibit physiological activities similar to substance P and cassinin, so they are useful as reagents for experiments or clinical tests. be.

The results of physiological activity tests of these peptides are shown below.

(Left below) (1) Test method Blood pressure lowering effect Using SD male and female rats, Urethane 739
Under 7kQ (subcutaneous) anesthesia, a polyethylene tube was inserted into the carotid artery, one end of which was connected to an electronic blood pressure monitor, and blood pressure was recorded on a polygraph. The test drug was previously inserted into the femoral vein, placed in place, and administered via a polyethylene tube.
Measure changes in diastolic pressure.

Smooth muscle contraction effect G. using guinea pig ileum, hamster bladder, rat difatid intestine and vas deferens.

The method of Erspamer et al. [N, -8, Arch, P
harmac+r+1.279゜Otsu/-7guC/973
), same magazine V? , gu/-3/(/97ta)] and C1M
, Lee et al.'s method [Ibid. 3ii, 2/-2f7 (79
and 2)]. Each organ specimen extracted
Aerate with a gas consisting of 9% oxygen and 1% carbon dioxide, and use Krebs or Tyrod.
e) Suspend in an organ bath containing 30 ml of liquid, connect one end to a contraction force transducer, and record the tension generated by the contractions isometrically on a polygraph. ileum.

In the duodenum and bladder, it was caused only by each drug.11
- The contractile force was measured, and in the case of the vas deferens, the potentiated response to contraction by electrical stimulation (0/Hz, pulse width/m5ec supramaximal voltage) via an electrical stimulator was measured.

(2) Test results table/hypertensive lowering effect Note * Dose that lowers blood pressure by /3yrtyttHg (
i, v, ) (blank below) 12- As is clear from the experimental results of the old translation, the peptides according to the present invention exhibit a blood pressure lowering effect and a smooth muscle contracting effect, and in particular, SK-// has a smooth muscle contracting effect. is similar to cassinin, and exhibits unique properties such as being comparable to substance P in its blood pressure lowering effect.

These peptides can be used as experimental reagents, and an immunoassay system using them can also be established and used for measuring kukikinins.

Furthermore, it is also possible to use it as a medicine or veterinary drug based on the above-mentioned physiological activity, and the dosage, method of administration, and duration of administration are determined depending on the purpose and subject of administration.

Next, embodiments of the present invention will be shown in Examples, but these Examples are not intended to limit the present invention in any way.

(Leaving space below) Example / (Synthesis by solid phase method) / Preparation of resin Benzhydrylamine type resin (amino foundation amount θ3mm
The hydrochloride (ol/f) is placed in a reaction vessel of a solid-phase synthesizer, and dichloromethane is added to sufficiently swell it.

1) 5% diisopropylethylamine (DIE
A)-2 times for 2 minutes with dichloromethane, ii) 3 times for 2 minutes with dichloromethane, 1ii) 2 times with coprobanol.
The free amine resin is obtained by washing three times for 2 minutes with iv) dichloromethane for 2 minutes.

2. Synthesis of protected peptide resin 3 equivalents of t-butoxycarbonyl-L-methionine sulfoxide (B
oc -Met (0)) in dichloromethane and an equimolar amount of dicyclohexylcarbodiimide (DCC
Add a dichloromethane solution of ) and allow to react at room temperature for 2 minutes. After repeating this coupling operation once more,
Add 75% acetic anhydride-dichloromethane and /j% DIEA-dichloromethane and react for 15 minutes. Then, the Boc group is removed by adding 50% trifluoroacetic acid (TFA)-dichloromethane and reacting for 2e minutes. Note that after each reaction, the washing operations i) to iv) described in the previous section are performed.

Similarly, Boc-L-o Ishi:/ (Leu), B
oc-glycine (Gly), Boc-L-)<'
) :/ (Val), Boc-L, -phenylalanine (Phe), Boc-0-Hensyl-L-Seri:
/ (Ser(Bzl)), Boc-β-benzyl-
L-77, paragic acid (Asp(OBz 1)),
and Boc-0-pendyrules leonine (Thr(
Bzl)) The protected octapeptide, Boc7hr-(Bzl)-Asp(O
Bzl )-8er(Bzl )-Phe-Val-G
] y-Le u-Met-NHQ((C,H,)C
, H,-resin (,1') 3.731 is obtained.

Add Boc-N'-2-'y to 212f of I obtained above.
lolobenzyloxycarbonyl-lysine (Lys(
CI-z)), Boc-NIn-tosyl-L-Hy, and tidine (His(Tos)) were sequentially introduced to protect the decapeptide, Boc-His(Tos).
)-Lys(CI-z)-Thr(Bzl) 16-Asp(OBzl)-5e r(Bzl)
-Phe -Va 1-Gly-Leu-Me t-
NHCH(C,H,)C, ester resin (II) is obtained.

■2 obtained above. /'If is further added with t-amyloxycarbonyl-NG-tosyl-arginine (AocmA
rg(Tos)) to introduce the protected undecapeptide, Aoc-Arg(Tos)-His(T
os )-Lys (CI-z)-Thr (Bz
l )-Asp(OBz l )-3e r (Bz
1)-Phe-Va IGly-Leu-Me t-
NHCH(C,H,)C,H4,-resin (ill), 2
．． Obtain 3/g.

Note that the introduction of Val and Phe requires the equivalent amount. In all other cases, 3 equivalents of reagents were used.

Take the peptide resin Iθ1.7f and add anisole/
Add 1ml of hydrogen fluoride/θml and boil at 3'EJ"C.
Stir for 0 minutes. After removing hydrogen fluoride under reduced pressure at the same temperature,
The residue was purified with 0θ23M ammonium hydrogen carbonate (NH,HCO).
, ), and the extract was lyophilized to obtain ~72 g of the substance 1,5-Met(0)-analog (hydrofluoride, crude standard). This is expressed as θθ23 MNI(,H
DEAE dissolved in several ml of CO3 and equilibrated with the same buffer
- Sephadex A-23C (trade name, Pharmacia)
Column (l Otsu 3x23; C1n), put on, 0023
11. having a concentration gradient of ~θ2.3M; HQ) Elute with J buffer (2, θθθl111). The eluate was fractionated into 3 ml portions using a fraction collector, and 23θnm
Measure the absorbance at Collect test tube number 3 and freeze-dry to obtain 72~.

Met(0)-analog 72119 obtained above was dissolved in M acetate, and 7M niemercaptoethanol was added thereto.
The solution was added to the solution so that the amount of the solution was adjusted to 100.degree. C. and left at 37.degree. C. for 3 days.

Then, the residue obtained by drying under reduced pressure was again applied to a column of DEAF, -Sefazonx A-2J- (/Otsu 3x, 23; on
) with a concentration gradient of 003 to 01M.
Elute with O3 buffer. The target octapeptide 3 (2) is obtained by fractionating into 3 ml/3 ml portions, collecting the portions with test tube numbers O6 through 3 ml, and freeze-drying them.

Amino acid analysis values of acid hydrolyzate (M hydrochloric acid, //θ°C 92θ hours): ASp10 (1), Thr105 (7
)SerQ9θ(/), GIYo,9'l(/
) ,, Val/θ, 2(/ ) , Met(1),
Leu/, 00 (1), Phe09g (1).

Furthermore, the octapeptide obtained in this step 5 was shown to have satisfactory purity by high performance liquid chromatography (HPLC). The HPLC conditions used were: Column, Nucleosil 3C, (trade name, Makezie Nagel), θ! X! jα flow rate, /ml/min:
Detection, 22θnm.

The peptide resin ■/θ31 obtained above is treated with hydrogen fluoride-anisole in the same manner as in the case of Ni-S-. After distilling off hydrogen fluoride, the residue was extracted with M acetic acid, and the extract was applied to a column C13x2θCIn of Amberlite CG-IAOO (trade name, Rohm & Haas Co., Ltd.) (acetate type) and eluted with water. . The solvent was distilled off under reduced pressure = 19, and the residue was further freeze-dried to obtain substance K.
-/θ Met(0)-analog (acetate, crude preparation) 2
Otsu/Da Tokuharu. Next, this was mixed with 0.3M ammonium acetate buffer (I) 1. A column of carboxymethyl cellulose (trade name: CIV [-, 5-, 7, Whatman Co., Ltd.) equilibrated with -X, 2';
ammonium acetate buffer (pHA,!;
.

2, θθ01IIl). Fractionate /3W11, collect the portion with test tube number 2'l-'I/, concentrate to dryness, and lyophilize to obtain /9θ.

6+V of the Met(0)-analog obtained above was dissolved in M acetic acid/l.
111, 2-mercap, toethanol a11t
After adding sygl, let stand at 37°C for 3 days. This was transferred to CM-32, (7) column (2,2X 27 C1B
) on top.

θθ23-〇/M ammonium acetate buffer (p■t.

The mixture is eluted with θθθtgl) and fractionated by /3tttl to obtain the desired decapeptide j from the portion of test tube number 22-gλ. [α]1)-32,θshino,θ0C,,cO
, 30'lM acetic acid) Acid hydrolyzate (gM hydrochloric acid, Ilo''0.2θh) -2
〇- (1), Letl/: (7θC, /),, Phe091
(1), Ly8099(1).

Hiso, 5’7 (1).

The decapeptide thus obtained was shown to have sufficient purity by HPLC. The HPLC conditions used were as described in 3. It is the same as in .

The peptide resin m2.291 obtained above is treated with hydrogen fluoride-anisole in the same manner as in -f.

After the hydrogen fluoride is distilled off, it is treated with Amberlite CGIθθ (acetate type) as in the case of 1/θ to obtain Met(0) analog (acetate, crude sample) j63■ with substance 1//. This was applied to a CM-52 column (,2,/
3×23m) and eluted with 0θj-03M ammonium acetate buffer (pH 6, 3, 2, θθθ). / 3.
Fractionate into each tit, collect the portion of test tube number 31-7.15'', concentrate to dryness, and lyophilize to obtain 3 Otsuj~.

Met(0)-analog 0)27/'*I obtained above is M
Dissolve in acetic acid 'Awl, add 2-mercaptoethanol/ml, and leave at 37°C for 3 days. This was added to CM-3;2 (D column (2,/!; x, 21cm
) d(7)(t.

θ-02M ammonium acetate buffer (pH Otsuj, 2.θθθ
ml) and fractionate by elution/Etttl. Target undecapeptide 22 from test tube number //θ-730 part
Get Gu Cape. [α] D='l'A3±/2°(Cθ6
θ 07M acetic acid) Amino acid analysis values of acid hydrolyzate (gM hydrochloric acid, //θ°C22θ hours): Asp /// (1), Thro5'j
(1).

Ser, 0.90 (/), G1y094Q-/'),
Val/:θ/ (/), Met0? f(1), L
eu/θ0(1), Phe091(1), Lyso? J
'(1).

Hisθ97(i), 'Atg09g(1)
The peptide thus obtained is 3. HPLC was performed under the same conditions as in t-butoxycarbonyl-L-methionine (Boc-net t-OH) to ensure satisfactory purity.
l, 2,! 07:/(TH
F) 'Itttl, tri-n-butylamine l
Add 3/ml. This is cooled to -2θ to -3θ°C, and 073 nl of isobutyl chlorocarbonate is added dropwise.

Continuing stirring for 3θ minutes at -10 to -2θ°C was also -30°C.
Cool to ~-Vθ°C, then heat to 21°C with vigorous stirring.
% ammonia water Sml. Stirring was continued for 2 hours at 30°C, THF was distilled off under reduced pressure, and the gel-like substance formed was extracted with ethyl acetate. The obtained ethyl acetate solution was washed successively with ice-cooled M hydrochloric acid, water and 9M sodium bicarbonate, and then dried over magnesium sulfate. The solvent was distilled off under reduced pressure and the resulting crystalline residue was recrystallized from ethyl acetate-petroleum ether to obtain compounds 1/3-9; yield 92%.
”P//9-120°C.

I-α]63. To t-? , 9±θ'10 (CIO, ethyl acetate).

[α]23) Juzaza ±03°CC'lθ, ethyl vinegar pongee)
.

61. 2. 'BOC-Leu-Met'-NH(If) compound I'i/llQ with trifluoroacetic acid ('TFA)
Dissolved in about 11tttl, 2! ;'C to react for 3θ minutes. D. 93- Add one telugu θ and separate the resulting precipitate, wash with ether and dry. Next, this was dissolved in N,N-dimethylformamide (DMF) and cooled with water.
-Le'umO8u) l j 2 'i added and 2
Wash with 3°C thorium. Further, after drying over magnesium sulfate and distilling under reduced pressure, the resulting crystals are recrystallized from methanol-ethyl acetate-ether to obtain a compound (1)/3θg; yield: 7♂%;

[a] i '119±θl0 (CIO, , +1 tanol).

3, 1E(-Leu-1'Vfet, -NH2-
Saturated hydrogen chloride-acetic acid solution was added to the compound m130f obtained on HCl' (I[[), and 2! ;'C for 30 minutes. The precipitate formed by adding ether is collected, washed with ether, and dried to obtain 7 g of compound II; yield ioθ.
%.

[α]7244♂±060 (clO9 acetic acid).

IA BoC-Val-Gly-OBz 1 (IV)
24- t-b) Kishi M # Ho = JL/ =L-)flJ
, :z'(Boc-Val 1-G() 3.2 otf and tri-n-butylamine 3.!; 7 ml were dissolved in dichloromethane 3θtttl, cooled with water, and added with glycine benzyl ester p-toluenesulfonate. jθ
Add g and dicyclohexylcarbodiimide (9).
DCC) 3.099 dichloromethane solution and <z
Leave to stand between 1 and 2. The precipitated dicyclohexyl urea (DCU) crystals were separated from the furnace, the solvent was distilled off, and the residue was dissolved in ethyl acetate and cooled with water.

This was cooled on ice and washed successively with 6M hydrochloric acid, water and 9M sodium bicarbonate. This solution was dried over magnesium sulfate, concentrated under reduced pressure, and added with petroleum ether to precipitate crystals. This is recrystallized from ethyl chloride-petroleum ether to obtain the compound ``A21f''; yield 7♂%, desired 7727F 'C,
”[α],3','-213±07° (CIO, methanol).

Yotansu upper formula l me small θ aim frog Ωσ Compounds tv, r+f were dissolved in TFMθml under water cooling.

After standing at L2j°C for 3θ minutes, TFA was distilled off under reduced pressure. After thoroughly drying the residue under reduced pressure, it was dissolved in TM wax θtel and cooled on ice. In addition, t-butoxycarbonyl-L-phenylalanine N-hydroxysuccinimide:), 5
-L(Boc-Phe-O3u) i'l/f
After distilling off the solvent under reduced pressure, the residue was dissolved in ethyl acetate, and after washing in the usual manner, it was crystallized to form compound (1). Recrystallize from ethyl acetate to obtain /θg
; Yield: 7%, desired/37-/! ；za°C, [α昂95-
3'AI±07 (C7O,) Evening/-JL/).

Boc-3er (Bzl) -Phe-Val-G
Compound V9.ly-OBZI(VI) ．． 2. O
The fl is treated with TFAT at 25°C for 10 minutes, and ether is added. The resulting precipitate is separated and washed with ether.

This is the ear tree n −5e r (Bz 1 ) −0
Su) 7θty and reaction sacell. The crude product obtained by treating the reaction solution in a conventional manner is recrystallized from ethyl acetate to obtain compound 7721/; yield λ%, jI#)/♂6
-/f♂°C1[α]ji1' -/, 2. /±03(c
lθ, chloroform).

7 Z-Asp-(OBut)-5er (Bz I
)-Phe-Va 1-Gly-OBzl (■) Compound ■7 g of compound ■ is treated with TFAT-, 25θ°C for 9 minutes, ether is added and the resulting precipitate is collected (777
9). This was added to the ear tree n-butylamine (,2 tails θy
benzyloxycarbonyl-β-t- in the presence of
It is reacted with 3 g of butyl-thi-aspartic acid N-hydroxysuccinimide ester. After evaporation of the solvent, the residue was digested with hot water, and after cooling with water, the solid matter was weighed and placed in a 2jθg column (11×39
Place it on m).

This was mixed with chloroform-methanol (,2θθ:/~2θ
:/) and further recrystallized from chloroform-methanol to obtain compound 13/f;
Yield f3%, "P2O2-203"C.

[α] 22,/±θ 0 (C70, chloroform).

Compound ■2. After catalytic reduction of θgy in DMF using palladium black as a catalyst for 20 hours, the solvent was changed to 60% acetic acid, and catalytic reduction was further carried out for 200 hours.

27- The solvent was distilled off under reduced pressure and the residue was treated with ethyl acetate to form compound ■
13θg was obtained; yield was 9t%, a single spot was obtained by TLC (silica gel plate, ethyl acetate-acetic acid-water (R:/:/), ninhydrin coloration), and the results of nmr spectrum measurement showed that benzyl groups remained. was not recognized. [
α],,'-/,5: 6±θot0 (C70, acetic acid).

5! Z-Lys-(Boc)-Thr-OMe
(IX).

Y-benzyloxycarbonyl-N”-t-butoxycarbonyl-L-lysine N-hydroxysuccinimide ester (Z-Lys(Boc)-0SuM, 7Og
and L-threonine methyl ester (free base)/za0
g in the ear at 23°C for 20 hours.

The reaction solution is treated in a conventional manner to obtain a crude product. This was placed on a column of silica gel (Silica Gel H, Merck & Co., Ltd.)/θθf, and eluted with chloroform-methanol (9f:2). The eluate (7tttl/both parts) was examined by thin layer chromatography, and both parts containing only the main product (test tube number 3) were examined by thin layer chromatography.
-70) was obtained from crystalline compound (2); yield %, θ-O/°C[α]fi-73. /±030C
ciO,) evening)-11/).

28- [α No.:, -3! ;, g±θza0 (C70, methanol).

/θZ-Hi 5-Lys (Boc)-Thr-C
The compound obtained on Ne (X)
It was dissolved in methanol together with tnl, and catalytically reduced using palladium black as a catalyst for 3 hours. The solvent was distilled off under reduced pressure to obtain an oily residue.
Acetate is obtained.

On the other hand, benzyloxycarbonyl-L-histidine hydrazide (Z-His-NHNH) was added to DIV
IF, dissolved in 2 f tttl and cooled to -30'C.

To this was added 2.1M hydrogen chloride-dioxane. fml and then 19 ml of isoamyl nitrite al are added and stirred for 30 minutes. The azide solution obtained in this way is -3θ to -+θ°C.
After cooling until and adding the dithylamine tnl,
It is combined with the above H-Lys(Boc)-Thr-OMe acetate. The reaction solution was stirred at 20°C for 20 hours, then evaporated under reduced pressure, and the residue was dissolved in ethyl acetate and dissolved in 1M hydrochloric acid.
After washing with saturated saline (/:/), M sodium bicarbonate-saturated saline (/:/), and saturated saline in this order, it is dried over magnesium sulfate. The residue obtained by distilling off the solvent was treated with ethyl acetate-ether (/:/) to form a gel precipitate,
Further, reprecipitation is performed from methanol-ethyl acetate to obtain the target compound
1-/g 2°C9[α], 3'-#: wo±060(Cl
θ, methanol).

//Susaratakashi danshu shuko] For ``Young Ji μ'' 730g of compound X was dissolved in 20πl of ethanol.

/θθ% Add 2ml of hydrazine hydrate and heat to 2θ at 23°C.
Let stand for a while. This reaction solution was dried under reduced pressure, and ethanol was added to the residue to form a gel-like precipitate, which was then reprecipitated from DMF-ethanol to obtain the desired hydraziF (XI).
/, :zo y wo getru; [α], 3″-711+0
! ;0(C70,DMF), [a]Skin:, -3i
3±07° (clθ, DMF).

The hydrazide M0 obtained above was treated with isoamyl nitrite to convert it into the corresponding azide as in the case of Z-Hi5-NHNHY in the synthesis of X above, and the DMF solution (/3; vsl) was converted into the corresponding azide. ■θ17g
The mixture was combined with a dimethyl sulfoxide solution (/θyptl) and stirred at Cc for 22 hours. Next, IIVF was distilled off under reduced pressure, and 30 trtl of water was added to the residue, and the resulting precipitate was poured into the furnace.

Reprecipitation from DMF-ethyl acetate followed by lyophilization from acetic acid yielded 170 g. This was suspended in θ/M ammonium bicarbonate jθtrl, left to stand overnight, and then dried to obtain the target compound θ71fl; yield t3%.

Compound ■θ709 and N-hydroxysuccinimide (H
O3u)731ng and DMF/! ; Dissolve in 2 ml and cool in water.

After adding DCC/3'1lfV to this and stirring for 3 hours,
Compound ■/7/fir/ and tri-n-butylamine θ/
9 mL of DMF was dissolved in DMF, and the reaction solution was further stirred at Cc for 20 hours. The solvent was distilled off under reduced pressure and water jθ111
The precipitate formed by adding DCU was dissolved again in DMF, and the insoluble DCU
After separating in a furnace, the solvent is distilled off. The residue was freeze-dried from acetic acid, further suspended in ethanol, and the poorly soluble precipitate was filtered to obtain compound X.
71 fl of l11θ is obtained; yield 92%.

Dissolve in 7 ml of TFA' and leave at 23°C for 30 minutes.

TFA% is distilled off under reduced pressure, ether is added to the residue, and the resulting precipitate is filtered. This was dissolved in about 1/3 ml of hydrogen fluoride together with 03 g of anisole θfm1.2-mercaptopyridine and stirred at 1/θ°C for 3θ minutes. Hydrogen fluoride is distilled off under reduced pressure at the same temperature, and the residue is thoroughly washed with ether. Next, when about Sθπl of water is added, a sparingly soluble gel-like precipitate (hydrofluoride of the target substance) is produced, to which Amberlite CG-tiθθ (trade name) is produced.

Rohm & Haas) (acetate type) approx./θm
1 and shaken, the target substance becomes acetate and dissolves. After the resin is separated in the furnace? Fi liquid is also Amberlite C
G-! Pass through a θθ (acetate type) column. The column was thoroughly washed with water, and the washing and eluate were combined and applied to a carboxymethyl cellulose (trade name: CM-52, Whatman Co., Ltd.) column (volume: jθ), and ammonium acetate buffer with a concentration gradient of θ-θ/M was added. Elute with solution (4 days 0θllll1). The eluate is fractionated by /θMl, and the absorbance at 23θ is measured.

The portion with test tube number /θj-/3θ is collected, concentrated, and lyophilized to obtain 370q. This was re-chromatographed using the same column, and among the three products obtained, 73q was further purified using partition chromatography. Sephadex G-, 2J-(M
) (Pharmacia) lAθ× Lycium (7), n in the solvent
-Butanol-acetic acid-water (l/l:/:2) was used. The eluate was aliquoted into 9 ml portions and placed on a cellulose plate (Merck & Co.).
TLC is performed using 3:3:/θ:/j) as a solvent, followed by coloring with ninhydrin. Collect both tubes containing only the main component (test tube number 33-IA7), evaporate the solvent, and freeze-dry to obtain the desired SK-/θ and Otsuden;
α]H-! ;, 2. /±2θ (cO!; , 0/M
acetic acid). Amino acid analysis value: Acid hydrolysis (gM hydrochloric acid, //
θ°c, 2θ time.

(in the presence of a small amount of phenol): Asp/θ/(1), T
hr09! ;Cl), 5erQ9θ(1), Gly/θ
2(1), Vallθθ(1), Met0? ? (1),
Leulθθ(1), Phelθ2(1), Lyslθ
J(1), His/θ! (1); Aminopeptidase M digest (37°C, 2θ hours): Asp0?3(/
), ThrO, 92(1), 5er091C/).

Gly/:0.2(1), Vallθ/(1)Meto
97(/')+Leu/θθ(1), Phe099(1
), Lys09j (1), Hisn,d,' (1). Cannot be measured due to large amount of NE (-) contained in the enzyme preparation (n, d,).

Compound ■(Z-Lys(Boc)-Thr-OMe
') Dissolve O'l'AI in 2 ml of ethanol, add 02 ml of 700% hydrazine hydrate, and let stand at 25'C for 2θ hours. The precipitated gel-like precipitate was separated and reprecipitated from ethanol to obtain the desired hydrazide (X■).
Obtain 2 g of 0IJt; yield 97%, /1I9-/3piC, [α] information-13±θ<<', [α],, -7
3±θ40<cy, θ, DMF).

The hydrazide (XIV) θg obtained above was converted into the above-mentioned X
As in the case of Z-Hi 5-NHNH2 in the synthesis of Z-Hi 5-NHNH2, it is converted to the azide by treatment with isoamyl nitrite.

The DMF solution (3 ml) was mixed with pentapeptide (■) 0
Mix with 97 g of dimethyl sulfoxide solution (3 ml).

Stir at 30°C for 2θ hours. Next, DMF was distilled off under reduced pressure, and water was added to the residue to separate the resulting precipitate, which was reprecipitated from IMF-ethyl acetate to obtain the target compound xv03♂f; yield: 6 g%.

Compound XVO2:H and HO5u II O f/V and D
MF /ltl Ni f8 melted and ice-cooled, and DCCf /
After adding flfl and stirring for 3 hours, compound 11 [(H-L
eu-Met-NH,-HCl) 90~ and tri-n-butylamine (007 ml) dissolved in DMF were added, and the mixture was further stirred with Cc for 2θ hours.

The solvent was distilled off under reduced pressure, 2θml of water was added, and the resulting precipitate was again dissolved in three tubes. After removing the insoluble DCU, the solvent was distilled off.

The residue is treated with ethyl acetate-ether (/:/) to obtain the target compound XVI03/I as an insoluble precipitate;
5 = dissolved in TFA 3 ysl with ml and incubated at 2j °C for 3
Allow to react for θ minutes. TFA is distilled off under reduced pressure and the residue is treated with ether to obtain an insoluble residue θ279. Next, use this as Anisole 02! ml and hydrogen fluoride approximately / 3;
ml and stirred at -70°C for 3θ minutes. After the reaction, hydrogen fluoride is distilled off under reduced pressure at the same temperature, and the residue is thoroughly washed with ether. Next, add water! ; Add about 0ml of Amberlite CG-+θO (acetic acid form)/θml and shake for a while (by this operation, the target substance changes from poorly soluble hydrofluoride to soluble acetate). The P solution is passed through a column of Amberlite CG-pθO (acetic acid type) in layers. The column was thoroughly washed with water, and all eluates were combined and lyophilized to reduce O'2θ.
get g.

The crude sample 02Of thus obtained is divided into λ degrees corresponding to θ/θf and purified by partition chromatography. Sephadex G-
2j (M) (Pharmacia) lAθ x 11.2cm,
Solvent: n-butanol-acetic acid-water (ll:/:, 2)
use. The eluate was fractionated into Ftttl.

Both parts containing only the main product were collected, the solvent was distilled off, and then lyophilized to θ/! ;3;(/ is obtained. This is further CM → Le 36
- Rose (CM-!; 2. Whatman Co.) column (/4
X/3a) and elute with ammonium acetate buffer (/θθθ#!l) with a concentration gradient of θ-θ/M. The eluate is fractionated into 7-proof fractions, and the absorbance at 23θnm is measured. Collect the portion of test tube number Hiro9-Lt7, concentrate and freeze dry to obtain the desired SKL? ://Get 3W; [aL4”!
;33±2, θ0CCO! ;,07M acetic acid).

Amino acid analysis value: Acid hydrolyzate (OtsuM hydrochloric acid, //θ'C
, 20 hours, phenol addition): Asp/θ0 (1).

Thrθ95(1), 5er09/(1), Glylo
, 2(1).

Va110θ(1), 'MetO? t(1), Leui
θθ(1).

PhelO/(1), Ly'slOθ(1);7 minopeptida-Met'09? (/), Le'ulθθ(1
), Pheθwa? (1).

Ly 5099 (’/).

Phe-■a1-G1y-OH (X■).

Compound XTl0.32. f is catalytically reduced in 60% acetic acid using palladium black as a catalyst for j hours. After evaporating the solvent under reduced pressure, the residue was filtered onto a column C11-, 2×lA2α) of Wosephadex LH-xo (Pharmacia), and ethyl acetate-
Partition chromatography is performed using acetic acid-water (!:/:/) as a solvent. Fractionate to 10 ml, check with TLC, and collect both fractions (test tube number O!-7θ) containing only the main component, evaporate the solvent, and freeze-dry from acetic acid to obtain compound X, 032f.

DIVIF /θml of the compound X02g obtained above
Add 009ml of tri-n-butylamine dissolved in the solution.

This was added to t-butoxycarbonyl-NG-nitro L-arginine pentachlorophenyl ester (Boc-
Arg (No2)-OPop) 02 Zaoto f.

2! Stir at i-"C. Once the reaction solution becomes clear, let it stand overnight. The solvent is distilled off under reduced pressure, and the resulting precipitate is collected by adding ethyl acetate to the residue (033θg).

This was applied to Sephadex LH-20 column CIl2X!
Partition chromatography was performed using ethyl acetate-acetic acid-water (g:/:/) as a solvent, and test tube number G3 lysate (
Amino acid analysis value of Asplθθ(1
), ThrO? g(1),5erO,? /(1), G1
y/θ/(1), Val(7,9?(1), Phe/:
oo(1), Lyslθθ(/ ), His0? ? (/
), Argo7" (1), 0rnO/f" (θ).

*Nitroarginine gives arginine and ornithine (Orn) when hydrolyzed.

Compound layer/10111fl was catalytically reduced in acetic acid using palladium black as a catalyst for j hours, then lyophilized, ethanol was added to this and the resulting precipitate was collected/Compound XI of θ3LQ
obtain X; [αL-3'-, 21A, 0+13;'
CCO Hiroshi.

acetic acid).

Il, H-Arg-Hi5-Ly5-Thr-A
sp -3er -Phe -Val Once Kuchitoyoko Ayuhimura was built, soil comforted, and heated.

Compound XIX, /θoq obtained above was dissolved in DMF, l, l, cooled with water, and QM HC1θ/ml was added thereto.
ml) and count the precipitate. The hydrochloride of XD (obtained here) was dissolved in DMF2ysl again, cooled with water, and N-hydroxysuccinimide (HNSu
) 3j~, dicyclohexyl-carbodiimide (DCC
) Add 3q of Otsu one after another and stir at ℃°C for 2θ hours. This reaction solution was mixed with ethyl acetate-ether (/:/) (/θ
Drop the resulting precipitate into N of X
-Hydroxysuccinimide ester/θθq is obtained.

Next, this was dissolved in 2 m, l of DMF, and compound (H-Leu-Met-NH2.HCI) 23-s
yy and tri-n-butylamine θθ3ml in DMF.
After dissolving and adding the solution, it was left to stand at 70°C for 2θ hours. After distilling off the solvent under reduced pressure, the residue is treated with ethyl acetate and further dried to obtain //θq. The protected peptide was dissolved in 2 ml of TFA in the presence of Anisole 02 tttl;
React for 3θ minutes at 23□C.

40- After distilling off TFA under reduced pressure, add ether and collect the resulting precipitate, dissolve it in 3 tri of water and make Amberlite CG-
Place on a gθO (acetic acid type) column (approximately 3 ml).

The column is thoroughly washed with water, and all eluates are combined and lyophilized to obtain 10θ.

The crude sample/θθ obtained here was mixed with CM-cellulose (CM-
3:2, Whatman Co.) column (approximately θtR1) and eluted with ammonium acetate buffer (4000 ml) having a concentration gradient of θ-θ3M. The eluate is gmlml
The fractions, test tube number jθ-O, are collected and freeze-dried to obtain jθq. This medium-sized θ-shibari is further purified by partition chromatography. Sephadex G-2,t(M) for the column
(Pharmacia) 1Aθ x 2m, using n-butanol-acetic acid-water (2:2) as the solvent, fractionate to 9tttl. Collect the portion with test tube number <zt-73, evaporate the solvent, and freeze-dry to obtain the desired SK-//, 271
Obtain ftQ; [α]]S'-G'A2±/3 (C
θ Otsu, 07M acetic acid). Amino acid analysis value: acid hydrolyzate i
M hydrochloric acid, //θ°C 12θ hours, added to a small amount of '7E/-): Asp/θ, t(1), Thr099(1)
, 5erOJ3(1), GIy/θ/(1), Val/
θθ(1), Met09J'(1), Leuiθθ(1
), Phe/θ/(1), Lyslθot(1), His
lo, r(1), Arg/θg(1); aminopeptidase M digest (37°C, 2θ hours): ASplθ3(
1), Thrlθ3(1), Ser/θ, 2(1), G
lylO3(1), GluO,97”(θ), Va1/
,02(/),Meto,95'(1),Leulθ
θ (1), Phelo, 2 (1), Lys/θ Otsu (1)
, Hisn, d, "(1). * Arginine is converted to citrulline by arginine oxidase contained in the enzyme preparation. Citrulline is quantified as glutamic acid under these analysis conditions. * Existed in large amounts in the jar enzyme preparation. cannot be measured because of the group ζ+ (n, d, ).

(Margin below)

Claims (1)

[Claims] (]) A peptide represented by the following general formula and an acid addition salt thereof. R-Ph e-Va I-G I y-Le u-Me
t□-NH. [Wherein, RO is H-Th r-As p-8e r-;
H-Lys-Thr-As p-8er- or H-Arg-Hi5-Ly5-Thr-A
It represents sp-8e r-. ] (2) Peptide derivative R-P represented by the following general formula
l+ e-Va I-G ly-Leu -Me
t (Y)-Rz [where R/ is R'-Th r (
R'')-As p (R'')-8e t (R3)+. R'-Lys(R')~Thr(R)-Asp(R)-
8er(R")-. R'-His(R)-Lys(R)-Thr(R)-
Asp(R)-8er (R bar or R2-Ar g
(R')-His (R7)-Lys (R'
)-Th r (R')-As p (R'')-8e
t(RJ)- (where Hj represents hydrogen or a protecting group for the α-amine group, and R3,R″; H, t, R4, R7 and P each represent the presence or absence of a protecting group for the amino acid side chain functional group) ), R2 represents -NH or a benzhydrylamine type resin, and Y represents the presence or absence of sulfoxide.] is subjected to a deprotection reaction to obtain a peptide represented by the following general formula. R-Ph e -Va 1-Gly-Leu -M
et-NH. [Wherein, R is H-Thr-Asp-8er-, H-L
ys-Thr-Asp-8e r-, H-Hi 5-
Lys-Thr-As p-8e r- or H-Ar
g-His-Ly5-Thr-As p-8e
Represents r-. A method for synthesizing a peptide, characterized by obtaining the following. (3) Peptide R-Ph e represented by the following general formula
-Va 1. -G1 y-OH [wherein B/ is R2-T
hr(R')~Asp(R'')-8er(R3)-, R
'-Ly s (R')-Th r (R')-As
p (R'')-8er(R-, R'-Hls(R7
)-Ly s (R')-Th r (R')-As
p (R″)-8er(R3)- or B2-Ar g
(R')-His (R7)-Lys (R')
-Th r (R')-As p (E'')-8e r
(R3)-. However, R2 is hydrogen or a-
Represents a protecting group for an amino group, R3,R″H,t,
R'. R7 and R' represent the presence or absence of a protecting group for the amino acid side chain functionality. ] is condensed with leucyl-methioninamide, and then subjected to a deprotection reaction to obtain a peptide R-Phe-Va1-Gly=Leu-Me expressed by the following general formula.
t -NH,2 [wherein R is H-Thr-As p-
8e r-, H-Lys-Thr-Asp-8e
r-, THis T4s-Th r-As p-
8e r- or H-Arg-Hi 5-Ly 5-T
h r-As p-8e r-. A method for synthesizing a peptide, characterized by obtaining the following.