JPS5896052A - Preparation of highly active h-pth (1-34) amide - Google Patents

Abstract

PURPOSE:To prepare the titled high purity substance useful as a remedy for parathyroid insufficiency, by amidating a C-terminated phenylalanyl group, condensing protected amino acids in liquid phase in a specific sequence, eliminating the protective group of the N-terminal, and purifying the product by gel filtration, etc. CONSTITUTION:The carboxyl group of the C-terminated phenylalanyl group is converted to amide group, and the protected amino acids and/or protected peptides are condensed by the liquid-phase synthesis in the order of formula (Ser is serine; Val is valine; Glu is glutamic acid; Ile is isoleucine; Gln is glutamine; Leu is leucine; Met is methionine; His is histidine; Asn is asparagine; Gly is glycine; Lys is lysine; Arg is arginine; Trp is tryptophane; Asp is aspartic acid residue), and the protective group of the N-terminal amino group is eliminated at the final stage of the condensation by acid decomposition. The product is separated and purified by the column chromatography using a gel filtration agent and an adsorbent.

Description

[Detailed description of the invention]

The present invention provides human parathyroid hormone (+1-PTH) (1
-J4) Relating to a method for producing an amide. More specifically, the present invention provides a method for producing a peptide represented by the formula % or a salt thereof.
The carboxyl group of the C-terminal phenylalanyl group is converted to an amide group, and individual protected amino acids and (-! or) protected lower peptides are combined in the amino acid order of formula [1] by a liquid phase synthesis method. In the final step of the combination reaction, the protecting group for the N-terminal amine group and the protecting group for the side chain functional group are removed by acid decomposition, and the resulting h-PTH(1-84) amide is treated with a gel filter and a This is a method for producing highly active h-PT)J(1-84) amide, which is characterized by separation and purification by column chromatography using an adsorbent. h-P'l"I-1 (1-84) represented by formula CI)
The amide is 1.5 to 2 times smaller than the original h-PTH (1-84).
It has twice the activity of h-P TH, and antibodies obtained using it as an antigen have immunological cross-activity with h-P TH. Therefore, this peptide is useful as a therapeutic agent for hypoparathyroidism and as a peptide for preparing antibodies for testing parathyroid function. The peptide CI) of the present invention is produced by converting the carboxyl group of the C-terminal phenylalanyl group into an amide group, and converting the amino acid order of the formula CI) into individual protected amino acids and (
It is obtained by condensing protected lower peptides (not yet available) using a liquid phase synthesis method, and removing the protecting group for the N-terminal amine group and the protecting group for the side chain functional group by acid decomposition in the final step of the condensation reaction. It will be done. The condensation reaction itself is carried out by repeating the attachment and detachment of a protecting group and the condensation reaction in accordance with conventional methods for peptide synthesis. That is, the various protecting groups used in the production of the raw materials of the present peptide [I] and all intermediates are those known in peptide synthesis, and therefore, hydrolysis, hydrolysis,
Protecting groups are used that are easily removable by known means such as acid hydrolysis, reduction, amine rinsing, or hydrazine. Such protecting groups are described in the literature and reference books in the field of peptide synthetic chemistry. For example, protective groups used for amine groups include formyl group, l-lifluoroacetyl group, phthaloyl group, p-1
luenesulfonyl group, o-nitrophenylsulfenyl group, aryl group, benzyloxy7carbonyl group, θ(
However, p)-promobenzyloquine carbonyl group, 0(
or p)-chlorobenzyloxycarbonyl group, p-
Benzyloxycarbonyl groups such as nitrobenzyloxycarbonyl group and p-methoxybenzyloxycarbonyl group, aliphatic oxycarbonyl group such as trichloroethyloxycarbonyl group, t-butyloxycarbonyl group, and 1-amyloxycarbonyl group group, 2-phenyl-isopropoquine carbonyl group, 2-tolyl-isopropoxycarbonyl group, 2-p-aralkyl-isopropoxycarbonyl group, and the like. These amino groups can be protected by formation of enamines obtained by reaction with 1,3-diketones such as benzoylacetone, acetylacetone, etc. Carboxyl groups are protected by amide formation, hydratide formation or esterification. That is, the amide group is 3,
Substituted with 4-dimethoxybenzyl group, bis=(p-methquinphenyl)methyl group, etc. The hydratide group includes a benzyloxycarbonyl group, a trichloroethyloxycarbonyl group, a trifluoroacetyl group, a 1-butyloxy7carbonyl group, a trityl group, and a 2-p-diphenyl group. The methyl group is alkanol such as methanol, ethanol, t-butanol, cyan methyl alcohol, benzyl alcohol, p-yromogbenzyl alcohol, p-chlorobenzyl alcohol,
Alkanols such as 2,6-cyclobenzyl alcohol, p-methoxy/hensyl alcohol, p-nitrobenzyl alcohol, benzhydryl alcohol, benzoyl methyl alcohol, p-promope/zoyl methyl alcohol, p-chlorobenzyl methyl alcohol ,2,
4.6=trichlorophenol, 2,4. ．． It is substituted with phenols such as 5-trichlorophenol, pentachlorophenol, p-nitrophenol, and 2,4-dinitrophenol, and thiophenols such as thiophenol and p-nitrothiophenol. The hydroxyl group of the serine can be protected, for example, by esterification or etherification. Groups suitable for this esterification include, for example, acetyl group, hensoyl group,
These include benzyloxycarbonyl group, ethyloxycarbonyl group, etc. Groups suitable for etherification include, for example, benzyl group, tetrahydronalanyl group, and 1-butyl group. The protection of these hydroxyl groups does not require 2,2.2-toIJfluoro-1-t-butyloxycarbonylaminoethyl group or 2,2.2-1refluoro-1-be. Examples of the group used to protect the amine group in the guanidino group of arginine include a nitro group, a tosyl group,
Examples include benzyloxycarbonyl group and me/ethylene-2-sulfonyl group, but it is not necessary to protect this guanidino group. Examples of the group used to protect the imino group of histidine include benzyl group, 1-IJ thyl group, benzylox7carbonyl group, to/tyl group, and 2,2°2-trifluoro-1-t-butyl group. oxycarbonylaminoethyl group, 2,2.2-fluoro-1-pendylaminoethyl group, etc., but it is not necessary to protect the imino group. The thiomethyl group of the methionine is preferably converted into a methylsulfokind group to prevent side reactions, but it is not necessarily necessary to protect it by oxidation. In the present invention, a t-butyloxycarbonyl group and a p-methoxybenzyloxycarbonyl group are used to protect the α-amino group, and a penzyloxysulfonyl group is used to protect the N-terminal α-amino group and the side chain amine group. wo use, α-
A methyl ester group or a benzyl ester group is used to protect the carboxyl group, a benzyl ester group is used to protect the side chain carboxyl group, and when the hydroxyl group of serine is completely protected, a benzyl group is used as the protecting group, and arginine It is preferred to use a mesitylene-2-sulfonyl group to protect the amine group in the guanidino group. In the synthesis of the present target compound (1), the condensation of individual amino acids and/or lower peptides is carried out, for example, with an amino acid or peptide having a protected α-amine group and an activated terminal carboxyl group, and a free +amine group and Either reacting an amino acid or peptide with a protected terminal carboxyl group, or reacting an amino acid or peptide with an activated α-amino group and a protected terminal carboxyl group with a free terminal carboxyl group and a protected α-amino acid group. - It can be carried out by reacting an amino acid or peptide having an amine group. In this case, the carboxyl group is, for example, an acid azide, an acid anhydride, an acid imidazolide or an active ester, such as cyanomethyl ester, thiophenyl ester, p-nitrothiophenyl ester, p-nitrophenyl ester, 2
, 4-dinitrophenyl ester, 2,4.5-t+)
Chlorophenyl ester, 2,4.6-t+)chlorophenyl ester, pentachlorophenyl ester,
It can be activated by converting it into N-hydroxysuccinimide ester, N-hydroquinphthalic acid imide ester, etc. Also, carbodiimides, such as N,N'-dicyclohexylcarbodiimide, N-ethyl-N'-8-dimethylaminocarbodiimide, N,N'-carbonyl-diimidazole, and isoxolium salts, such as Woodward reagents. It can be activated by reacting with a condensing agent such as. Preferred condensation methods in the present invention are the AND method, the active ester method, and the carbodiimide method. At each stage of condensation, it is desirable to use a method that does not cause racemization or a method that minimizes racemization, preferably the azide method, active ester method, Wünsch method, (Z
, Naturforsch, , 21b, 42
6 (1966)) or the Geiger method (Chem Ber.
,, 108.788 (1970)) etc. are suitable. If the condensation order is the amino acid order shown by formula (I),
Although it can be synthesized in any order, it is preferable to connect amino acids and/or peptides sequentially from the C-terminal side. For example, it is preferable to condense a C-terminal fragment consisting of amino acids 29-34 with a peptide fragment consisting of amino acids 25-28. The AND method is suitable for condensing this C-terminal fragment with tetrapeptide 25-28. A peptide fragment consisting of amino acids 22 to 24 is linked in front of the obtained C-terminal fragment 25-84, which is suitably carried out by the Geiger method. Obtained C-
Terminal fragment) Peptide fragment consisting of amino acids 19-21 sequentially before 22-84, 16-1
Peptide fragment r-1 consisting of eight amino acid sequences
A peptide fragment consisting of the order of amino acids 1, 8 to 15, a peptide fragment consisting of the order of amino acids 8 to 12, a peptide fragment consisting of the order of amino acids 4 to 7, and a peptide fragment consisting of the order of amino acids 1 to 3. It is light and heavy to connect. These condensations are suitably carried out by the azide method. The above C-terminal fragment) 25-34 is the C-terminal fragment) 29-434, and the remaining amino acid sequence 2
It is preferable to link peptide fragments consisting of 5-28 by the azide method. C-terminal fragment 29-8
4 is a dipeptide fragment 5l-
82 is preferably linked by the azide method, and before that, each of the remaining amino acids in sequence is linked by the active ester method. The protective group for the α-amine group in the condensation reaction described in -12, such as the t-butyloxy/carbonyl group and the p-7 benzyloxy/carbonyl group, is removed with hali-lifluoroacetic acid. Protecting groups for the α~carboxyquine group, such as methyl ester, can be decomposed with dilute sodium hydroxide solution to form a protected hydratide such as hydrated hydratide or trichloroethoxycarbonyl hydratide; It can be decomposed by decomposition, hydrogenolysis, or converted to hydratides. The tetratriacontapeptide protecting group with the N-terminal α-amino group, ξ~amine group, -1 side chain carboxyl group, guanidino group and (yet) hydroxyl group thus protected is preferably subjected to acid decomposition, e.g. anhydrous hydrogen fluoride. The target compound of formula (1) is obtained by elimination in one step by sieving or by a method using trifluoromethanesulfonic acid. When reducing the methyl sulfoxide group of methionine to a thiomethyl group, dithiothreitol, thiocricholic acid, mercaftethanol, ethanedithiol, etc. may be used. The peptide [1] thus obtained can be separated and purified by known means for purifying peptides or proteins. For example, gel filtration using a gel filtration agent such as Sephadex G-25, Sephadex G-50, Sephadex LH-20, column chromatography using carboxymethyl cellulose, ion exchange resin, etc., high performance liquid chromatography, etc. It can be carried out. The peptide (1) of the present invention can be obtained in the form of a polybasic or a salt thereof depending on the conditions of the method. Salts include inorganic acid salts,
Formic acid, acetic acid, propionic acid, glycolic acid, succinic acid,
It is a salt with organic acids such as malic acid, tartaric acid, and citric acid. The present peptide ('l) can be added with certain inorganic or organic substances to form a complex. By complex is meant a compound that is formed upon addition and gives a sustained effect to the peptide. Similarly, the abbreviations described in this specification have the following meanings. Phe; L-phenylalanine Tyr; L-tyrosine Asn; L-asparagine His; L-histidine Mel + L-methionine Vat; I-no-valine Asp; L-aspartic acid (lln; L-glutamine ■ ) eu; L-leucine Lys; L-lysine Arg; L-argini/Trp; L-tryptophan Glu + L-glutamic acid 1e; L-inleucine Ser; L-serine o+y; glycy/Z; benzyloxy carbonyl Boc; t-butquine carbonyl Z (OMe); 1-methoxybenzyloxycarboxylic M+s; N-mesitylene-2-sulfonyl 0Bzl; benzyl ester OMe; methyl ester 0TCP; 2,4.5-t+)chlorophenyl ester ONp: Grand-nitrophenyl ester O8; N-hydroxysuccinimide ester NHN
HTroc; t') Chloroethyloxycarbonyl hydrazide NHNH, ; hydrazide TFMSA; 1,1 fluoromethanesulfonic acid MSA; methanesulfonic acid TFA; trifluoroacetic acid TosOH; ;l-ethylamine CHA;cyclohexylamine DCHA;zinchlorohekifluamine EDT;enone>saol DCC;NN'-dicyclohexylamine lupodiimide ED
'I'A; Ethylenediaminetetraacetate DMIi'; Dimethylpolamide EtOH; Ethanol BuOI (; %-Butanol CIJCI3; Chloroform TI (F''; Tetrahydrofuran ether; Diethyl ether TLC; Thin layer chromatography; Production examples of the present invention will be specifically explained.
The carrier and developing solvent system of T i,C), conditions for amino acid analysis, and method for measuring P T H activity are as follows. <TLC) Carrier;/Licagel G Solvent system; 1, CI-ICl3-MeOII (10:0.5
)2, CHCl3-MeOH-water (8:8
:l) Lower layer a Cl-ICl3-MeOH-acetic acid (9:1:0.5)4, BuOH-pyridine-acetic acid-water (4,:l:l:2) Support; Cellulose (manufactured by Merck & Co., DC -AIufolien) Solvent system; 5 BuOH-pyridine-acetic acid-water (5:8:0,
l:11) Upper layer (Amino acid analysis) Unless otherwise specified, the sample was incubated with 6N hydrochloric acid at 110°C, 24-4
Hydrolysis was carried out in a sealed tube for 8 hours. (PTI-1 activity measurement method) (1) Preparation of PTH receptor Male and female SD rats (body weight 200-2509) were frontally exsanguinated, the kidneys were removed after laparotomy, the surface membrane was removed, and the kidneys were Cut out the cortex and cool it on ice. The following operations are carried out at the lowest possible temperature (0 to 4°C).
Lys hydrochloride buffer containing MEDTA (10 mM) (pH
7,5) The wet weight (g) of the renal cortex was immersed in liquid A (hereinafter referred to as solution A), and the kidney cortex was immersed in a glass tube made of Teflon perasl.
Add 3 times the volume (mg) of Solution A to pomogenize. This homocyne-1. is centrifuged at 150XiX for 10 minutes, and the supernatant is further centrifuged at 2200X for 15 minutes. Discard the supernatant and add the cloudy colored part of the layer to the precipitate.
This suspension was washed by centrifugation for 15 minutes at 2% 00×9.
Freeze at ℃ and store at -20℃. (2) Reaction between PTH and PTH receptor h-'PTH (1-34) N1-12 of the test sample was added to 2 μ9
A to obtain a concentration of /ml and l OμfJ /ml.
T P゛Mg2mM, MgCl210mM, KCl6
0mM, GTP 20μM. Isobutylmethylxanthine 1mM, creatine phosphate 8mM and bovine serum albumin (BSA)
) 100mM Tris-HCl buffer (pH 7) containing 0.2%
, 5) (hereinafter referred to as B solution), and add this to standard beef P.
This is also done for TH(1'-84). Dispense 50 μl of each of these four solutions into glass test tubes, and prepare 8 tubes each. Samples were kept in ice water and ATP
suppresses the decomposition of other substances such as P stored at −20°C
Thaw the T H receptor preparation at room temperature, add creatine kinase pre-dissolved in solution A, and add creatine kinase 0, 1 my/rul, PTI in solution A.
] Prepare the receptor preparation so that the protein content is 1.4 my/ml and keep it in water cooling. After soaking the above dispensed sample solution in a constant temperature bath at 37°C for several minutes,
Add 50 μl of TH receptor-creatine kinase solution and incubate at 87° C. for 10 minutes. then 0
．． Add 100 μl of 1M acetate buffer (pH 4,0) and immediately place the test tube in ice water. Immediately immerse the test tube in boiling water.
Heat for a minute to stop the reaction. (3) Measurement of produced C-A M l) The above reaction-stopped sample was diluted 10 to 30 times with distilled water,
Protein removal is performed by centrifugation at 2000XG for 15 minutes. The amount of C-AMP in the supernatant is measured using an R, TA kit (manufactured by Yamasa Soy Sauce Co., Ltd.). (4) Measurement of PTH titer The measured value of C-AMP is P M / m9P T Hv
(!Convert into units of pig protein/min, use this as the reaction value, and test the test product against the value obtained by the Muku standard using the parallel line test 2 x 2 point method. Example 1 h-P'l'H(1-84) NH,, ;H-8er
-Vat-8er -QGlu-Tie -Gln -
Leu-Met-His-Asn-Leu-Gly
-Lys-His-Lys-Leu-(lln-Asp
-Vat -Hls-Asn -Phe-NH2 1)F (84); Z (OMe)-Phe-NH2
(1) Z (OMe) -Ph e-OH82,93
4g (0゜IM) was dissolved in 200ml of THF, 15.29ml of E13N (0,IIM) was added thereto, and 14.45ml of isobutyl chloroformate (0゜l1M) was added with stirring while cooling to -20°C. ) was added dropwise. Crystals precipitated and stirring became difficult, so THF20
Added 0 ml and continued stirring. After 15 minutes, 20.9 ml of concentrated aqueous ammonia was added, and the mixture was stirred for 4 hours while cooling under a salt-ice base. The precipitated crystals were collected. The tear fluid was concentrated under reduced pressure, and the resulting crystals were combined with the previous crystals, washed three times with 5% aqueous ammonia and three times with water, and then recrystallized with dioxane-methanol-ethyl acetate to obtain [l]. Ta. Yield 28.186g (yield 85.8%) Melting point: 180-1
82°C 'l'Lc: Rf2=0.74 [α]20-17.89' (c=0.92.DMF) Elemental analysis (as C+s H+rN204) 0%
14% N% Calculated value 65.84 6.14 8.58 Measured value 65,76 6,28 8.442)
F (88-84); Z (OMe) -Asn
-Phe -NH2 [2] (1) 5.8789 to Anino fiv8.89m1, T
After adding 15.56 ml of li'A and stirring at 0°C for 1 hour, TFA was distilled off at room temperature under reduced pressure. The residue was treated with hexane and the resulting precipitate was separated by a decanting method.
ml. Add 2.51 ml of E t 3N, then add Z (O
Me)-Asn-ONp7.4719 (17,9m
M) and 2.51 ml of E t 3N were added, and the mixture was stirred at room temperature for 20 hours. After neutralizing the reaction solution with a small amount of acetic acid while cooling,
It was concentrated under reduced pressure. The residue was crystallized by adding 5% citric acid water and ethyl acetate to obtain [2]. Yield 6.019 (Yield 75.9%) Melting point: 243-245°C [α]D-19.3° (c = 0.9.DM80)'rL
C; 1-tf2=0.60, Rf3=0.15 elemental analysis (as C2□H26N40B) 0% N%
N calculation value 59.72 5.92 12.66 foot value 59.4'l 5.98 12.68
8) Ii'(81-84); Z
(OMe) -Val-His-A s n -P h
e-NH2(8)(2) 7 to 6.00g = :/-
After adding 4.48 ml of 'l' p A and 17.72 ml of 'l' p A and stirring at 0°C for 60 minutes, TFA was distilled off under reduced pressure at room temperature. Hexano was added to the residue and the resulting precipitate was counted. Add DMSO-DMF (1:1) 50
m1XE13Nl. Add 90ml of H-'-As
A solution of n-Phe-NI2 was obtained. On the other hand, Z (OMe) −V a l −IT i
Dissolve 27,049 g of 5-NHNH in 80 ml of DMF, and add L 809N-HCl/D to this while cooling to -50°C.
15.41 ml of MF solution was then added, followed by 2.61 ml of isoamyl nitrite. After stirring at -20°C for 20 minutes, -
Add 2.51 ml of E t3N under cooling to 50°C, and add the above H-A sn -P he -NI-1 to this.
A solution of 2 was added, and the mixture was stirred at 4°C for 18 hours. After the reaction, the solvent was distilled off under reduced pressure, 3% aqueous acetic acid and ethyl acetate were added to the residue, and the resulting precipitate was collected and washed with 1114 of 3% aqueous acetic acid, 5% aqueous sodium bicarbonate, and water. . D M S O-M e
Recrystallization with OI-1 gave [3]. Yield 6.21
6.9 (yield 64.8%) Melting point; 204-207°C [α') 25-13.9' (C=1.1, DMSO
)'T''LC; R, f2=0.80 amino acid analysis; Val 1 02 (1), HisO,
95(1), Aspo, 98(1), Phel
, 00 (1) Elemental analysis [C33I] 42N608
・1% and 2■ (as 20) 0% N%
N% Calculated value 56.16 6.43 15.88 Measured value 55.90 6.14 15.704)
P (80-84,); Z (OMe)-
Asp(OBzl)-V a I-His-A
s n-Ph e-NI2 (4) (8) 6.00
7 = 7-le 2.88 m1. 'l'PA11.52
After stirring at 0° C. for 60 minutes, TFA was distilled off under reduced pressure at room temperature. Ether was added to the residue, the precipitated crystals were collected in F, and after drying, 1) MP 50 ml z
E 13N 2.46 ml, Z(OMe)-Asp
-(OBzI)-ONp5.3898g, Et3N1.
23ml was added and stirred at room temperature for 18 hours. After reaction, D
MF was distilled off under reduced pressure, and 3% acetic acid water and ethyl acetate were added to the residue. [
4] was obtained. Yield 6.00g (yield 76.8%) Melting point:
236-273°C [α) -16.8° (C=1.0.DMSO)T
L C; Rf2 = 0.89 Amino acid analysis; A sp 2-01 (2), V
a l l , 00 (1), Phel. 00 (1
), Hiss, 91 (1) Elemental analysis (C44H
5a As H901+]0% N% N% Calculated value 59.78 6.04 14.26 Measured value 60.06 6°25 14.375)
P (29-84); Z (OMe)-Gln-A
sp(OBzI)-Val-His-A
s n-Phe -NI2 (5) (4) 5.00
．． iii (5.66mM) with 8.08mg of anisole
, TPA12.82m1. After stirring at 0°C for 60 minutes, Tl''A was distilled off under reduced pressure. Ether was added to the residue, and the precipitate was dried several times.
-G l n-0Np2.92. ! 9, Et3N2.5
8 ml was added and stirred at room temperature for 48 hours. The reaction solution was neutralized with several drops of acetic acid under cooling, and 1) MP was distilled off under reduced pressure. 3% aqueous acetic acid and ethyl acetate were added to the residue, and the precipitated powder was collected and washed in the order of 3% aqueous acetic acid, 5% aqueous sodium bicarbonate, and water. Reprecipitation was performed twice with DMSO-methanol to obtain [5]. Yield 5. ill, 9 (yield 90.8%) Melting point: 234~
238℃ [α”I)' 22.6° (C=1.1.DMSO
) TLC; Rf3=0゜31 Amino acid analysis; Oful, 06 (1), Asp
2.06 (2), Vall, 00 (1), Phe
l, 00 (1), HisO, 8B (1) Elemental analysis (C49H61Nll 013・I] as 20) 0
% F1% N% Calculated value 57.18 6.16 14.96 Measured value 57.89 6.08 14.766)
F” (27-28) Z (OMe)-Lys (Z
) -1, e u-OMe (6) 1-1-Le u-OMe 2. ,91 jj DM
Dissolve in 50 ml of F, cool to 0℃, and dilute with Et3N2.
．． After adjusting the pH to 7 with 24 ml, Z (OMe) −
Ly s (Z) −〇'l”CP l O, j9 to T
Solution dissolved in 50 ml of HF and 2.24 m of Et3N
1 was added and stirred at room temperature for 20 hours. The reaction solution was concentrated under reduced pressure, and the residue was mixed with ethyl acetate and 5% citric acid. The organic layer was washed with 5% citric acid, brine, 5% sodium bicarbonate, and brine in this order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Ether was added to the residue to obtain crystals. Me
Recrystallized from OI-J-ether [6) 7.8
8.9 (yield 80.1%) was obtained. Melting point; 107-108°C (a) D-21,2' (C = 0.9, MeOH) TL
C; Rf2=0.78, Rf3=0.63 Elemental analysis [C
As 3oH4103N8] 0% 1% N% Calculated value 68.08 7.28 7.85 Measured value 63.18 T, 28 7.26? ) F
(26-28) Z (OMe)-Lys (Z)
-Lys(Z)-Le u-OMe (7)
(6) 5.72.9 to 7 = :/ -le 2.17ml
, 8.68 ml of 'I'FA was added, and the mixture was stirred at 0°C for 1 hour. T I''A was distilled off under reduced pressure, and the residue was washed three times with n-hexano and dried.
Dissolve in ml, E

[After adjusting the pH to 7 with 4 ml of 3N1゜, Z (OMe)-Lys (Z)-OTCP
6.24 g and 1.4 ml of Et3N were added and stirred at room temperature for 20 hours. After the reaction, DMF was distilled off under reduced pressure and 5%
Citric acid and ether were added to obtain the resulting precipitate (%). Melting point; 153-154°C [α)n, , -15,8″ (C=1.l, DMF
) TLC; 1lLf, ~0.41 Elemental analysis (as C44H2O011N5) 0%
1% N exemption calculated value 68.37 7.18 8.40 Measured value 68.54 '121 8.888)F (
25-28) Z(OMe)-Arg(Mis)-L
ys(Z)-Lys(Z)-Leu-OMe (8
) [7] Anisole 2.83m1XTii on 6゜oog
'A9.33m/ was added and stirred at 0°C for 1 hour. 'I
After FA was distilled off under reduced pressure and ether was added to the residue, the resulting precipitate was collected and dried to obtain a powder. On the other hand, Z (OMe)-Arg(Mts)-0H-CH
A6°71.9 was dissolved in 20 ml of ethyl acetate and 8 mL of IN hydrochloric acid.
．． I mixed it with 64me. The organic layer was washed with 20 ml of water and then concentrated under reduced pressure. This residue is T Hi' 20 m
-io
1.86 ml of ibutyroxycarbonyl chloride was added at °C and stirred for 5 minutes. Add the above powder to this in DMF20m/! Dissolve in Et3
Add a solution adjusted to pH 7 with 1.00 mJ of N, and heat to 0°C.
The mixture was stirred for 4 hours. The reaction solution was concentrated under reduced pressure, and 55% was added to the residue.
78.9 (yield 68.5 mm) was obtained. Melting point; 151 to 152°C [α), -8,9° (c=o, 7, DMF) TL
C; Rf2. −0.76, Rf3=Q. 75 element analysis (as Ca9H8sO+4N9S) 0% 1%
N% Calculated value 60.44 6.96 10.75 Measured value 60.48 6.97 10.659)
F (25-28); Z (OMe)-Arg(Mis
) -Lys(Z)-Lys(Z) -Leu-Nl-
INH2 [9] (835,78g to M e OH50m
1.28 ml of hydrazine hydrate was added thereto, and the mixture was left at room temperature overnight. The precipitated crystals were counted and Et
OI]. M e OI-1-E 10 T-
Recrystallized from 1 [9] 55.25g Yield 91.3%
) was obtained. Melting point; 178-180°C [α)20; -10,1' (C=0.7, DMI
i”) TLC; Rf2=0.48 Amino acid analysis; Leul (1), Lysl, 9
4 (2), A r g 1 , 01 (1) Elemental analysis (as Css f4g+ 013 N++ 8) 0% 1% N% Calculated value 59°426.96 18.14 Measured value
59.29 7,17 12.8710) Ti
'(25-34); Z (OMe)-Arg(M
ts)-Lys (Z)-Lys (Z)-Leu
-G I n - As p (OBz l ) -Va
l -Hi 5-As n-Phe-NH2(101(
5) 5.7T71/7 2y-le JIOml, T
VA12, 40 me was added and stirred at 0°C for 1 hour. After TF'A was distilled off under reduced pressure and ether was added to the residue, the resulting precipitate was separated and dried to obtain a powder. To this was added 80 ml of DMF and 1.59 ml of Et3N to obtain a neutralized solution. C9'J 8. oa
oBDMF 20m1
The above neutralized solution was added to the AND solution obtained in the same manner as in the previous section 3) using a sampler, and the mixture was stirred at 4°C for 18 hours. Although the ninhydrino reaction of the reaction solution was negative, [9] 2. OOgDM
F l0m18.936
N-H(4!/DME' melti 1゜04m
1 Isoamyl nitrite 0.27ml 1E
t3N O, 86m1
The and solution prepared using the sample was added and stirred for 18 hours. The reaction solution was neutralized with several drops of acetic acid under cooling, and DMF was distilled off under reduced pressure. 3% aqueous acetic acid and ethyl acetate were added to the residue, and the resulting precipitate was separated and washed in the order of 8% aqueous acetic acid, 5% aqueous sodium bicarbonate, and water. DMSO-M e O)] was reprecipitated 33 times to obtain 10). Yield: 9.171 g (yield: 80.8%). Melting point; 226-230°C [α)''; -17.8° (C=1.0, I)M
SO) TLC; Rf2 20 40 Amino acid analysis; Leuo, 97 (1), Lys2
．． 02(1), Argl, 19(1), Aspl
, 96 (2) , Glue, 98 (1) , ValO
,97(11, pHe1.00 (1), HisO
079(1) 11) F (28-24) Hoc-Trp-Leu
-OH-1) CHA (l l ) Boc-Trp-ONplO, 65 g l) MF
Dissolved in 80 ml. To this, at 0°C, H-Leu-O
Ha. 9g in 10ml of water, I) MF 80ml1. The mixture was dissolved in 6.95 ml of Et3N and added, and the mixture was stirred at room temperature overnight. The solvent was distilled off under reduced pressure, and the residue was mixed with 5% aqueous sodium bicarbonate and ethyl acetate. Acidify the aqueous layer with citric acid,
Ethyl acetate was added and mixed. The organic layer was washed with 5% citric acid and water in that order, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The residue was dissolved in M e OH and I) CHA5
．． Add 4551. M e OH was distilled off under reduced pressure,
Ether was added to crystallize. Recrystallization from MeOH-ether gave [:IF). Yield: 10.15 g (yield: 97.0%). Melting point: 192-195°C [α), -22,9° (C-0°6, Meol-])
TLC; Rf2=0.67, Rf3=9.69
Elemental analysis [as C22H3105N3/Cl2H23N] 0% N% N% Calculated value 68.19 9.09 9.86 Measured value 68.07 9.14 9.2712
)F (22-24)Boc-Glu(OBzl)-T
rp-Leu-OH・DCIIA (12) [1
1) Add 10.0!9 to 50ml of ethyl acetate. The suspension was suspended in 50 ml of water, and 17.1 ml of IN hydrochloric acid was added at 0°C. After removing the precipitated salt, the organic layer was washed with brine and dried over anhydrous sodium sulfate. Ethyl acetate was distilled off under reduced pressure to obtain an oil. This includes 2% EDT 7-=-7-
Add 5.57 ml of water and 22.28 ml of TFA, and heat at 0°C.
The mixture was stirred for 1 hour under nitrogen gas. TTi'' A was distilled off under reduced pressure, washed three times with n-hexane, and dried. This was dissolved in 50 ml of T)MF, and Boc-Glu (OBz
l) 7.48jj of -0Su and 4.79ml of E13N were added, and the mixture was stirred at 4°C for 16 hours. DMF' was distilled off under reduced pressure, and the residue was dissolved in 5% aqueous ammonia and washed with ether. The aqueous layer was acidified with citric acid and the resulting oil was extracted with ethyl acetate. The ethyl acetate layer was washed with brine, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. Me the residue
Dissolve in OH, add 2.8 mg of DCI-(A,
e O)] was concentrated under reduced pressure and crystallized by adding ether. Recrystallized three times from M e OH-ether [12
] was obtained. Yield: 7.84 g (yield: 57.0%). Melting point; 149-150°C [α]”; -2L 10 (C=1.8, Me
OH)'I''L C; Rf 2ro 0.5B,
Rf3=0.71 amino acid analysis, TrpO,76(1
), Glut, 12 (su1,, e u 1 (1
) (4MIMS△, IIQ'c, P4 at i] Elemental analysis (as C34H4408N4 CI2 H23N) 0% 8% N% Calculated value 67.58 8.26 8.56 Measured value 67.28 8.28 8.6913 )F (
22-34), Boa-Glu (OBZI-)-
Trp-Leu-Arg(Mts)-Lys (
Z ) Lys (Z)-Leu-〇In-A
sp (OBzl)-Val-His-Asn-
Phe -Nf12 [13) [10] 7.0Of anisole 8.83 ml, -
T'FA I5.32 ml precipitate was collected in P1. After that, 5
% sodium bicarbonate solution and then water, and dried to obtain a powder. On the other hand, [12] DMF-HMPA-D in 4.3199
30ml of mixed liquid of M80(]:1:1)! , and 0.8579 N-methylformory were added and dissolved. DOC for this
l, 308? was added and stirred at 4°C for 18 hours. Since the reaction solution was positive in the ninhydrin reaction, 000
0218m9 was added and stirred for 18 hours. After the reaction, the mixture was neutralized with acetic acid under cooling, and the solvent was distilled off under reduced pressure. Add 3% acetic acid water and ethyl acetate to the residue, count the resulting precipitate, and add 3% aqueous acetic acid and ethyl acetate.
It was washed in the following order: acetic acid water, 5% sodium bicarbonate water, and water. DM F-
Reprecipitation was performed four times with M e OH to obtain [13]. Yield 5.013r (yield 58.2%) Melting point: 282°C (decomposition) [α': l -19.4° (0 = 0.8.DM80
)T L O; Rf2=0.43, Rf3=0
Amino acid analysis; Glu 1.99 (2), Leu
1.92 (2), Asp 1.96 (2), Va
n, 0.95 (]), phe 1.00 (1),
Lys 2.12 (2), Hiq O,
93(1), Arg O, 98(1) Elemental analysis [
Cj 123 H164N24027 S] 0%
8% N% Calculated value 60.47 6.77 13.76 Measured value 60.88 6.78 13.8814)
F (20-21); Z (OMe) -Arg (M
is) -Val -NHNHTroc [14:]Z
(OMe) -Van, -NHNH'l'roc
22.799 to Ani7-roux 0.74 meXT F
A 42.96m7! was added and stirred at 0°C for 1 hour. TFA was distilled off under reduced pressure, and the residue was washed three times with n-hexane and dried to obtain an oil. On the other hand, Z (OMe) -Arg(Mts) -0B-
OHA 30.02, ethyl acetate 150me, water 100me
ml, and 48.41 ml of IN hydrochloric acid was added at 0°C. After removing the precipitated salt, the organic layer was washed with brine and dried over anhydrous sodium sulfate. Ethyl acetate was distilled off under reduced pressure to obtain an oil. Dissolve this in THFlooml and make 81.3 ml of Et3N! of isobutyroxycarbonyl salt at -10°C.7.53 m/! was added and stirred for 5 minutes. D M F 50 of the above oily substance to this.
A solution adjusted to pH 7 with 6.78 ml of Et3N was added thereto, and the mixture was stirred at -10°C for 2 hours and at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, and the residue was mixed with ethyl acetate and 5% citric acid. The organic layer was washed with 5% citric acid, brine, 1% aqueous ammonia, and brine in this order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained oil was subjected to silica gel column chromatography [elution solvent 0HCA3-MeOI-] (20: 0.5
)). Crystallization from ethyl acetate-n-hexane gave [14]. Yield: 29.25 liters (yield: 7
417%) Melting point; 114-116°C [α]; -24,,6° (0=1.1, MeOH
) T L C; Rf2=0.62, Rf3=0.
46 elemental analysis CC32H4409H7S As CJ 3] 0% 8% N% Calculated value 47.50 5.48 12.12 Measured value 47.24 5.43 11.8815) F
(19-21) Boc-Glu (OBzl)
-Arg(Mts)-Val -NHNH〒roc
[15] [14') Anisole 3.89 in 7.309
15.57 ml of TFA was taken and dried to obtain a powder. This is DMF 50m1! Dissolve in Et3N
12.6-PI (adjusted to 7, Boc-Glu
(OEzl) -ON p 4.13 t and Et3N
12.6 ml was added and stirred at room temperature for 16 hours. The solvent was recrystallized under reduced pressure to obtain [15]. Yield 6.29
F (yield 7285%). Melting point; 159-160°C Ca]; -31,9° (0 = 1.1, MeOH) 11'
LO; Rf2 = 0.68, Rf3 = Q, 74 elemental analysis CC40N57011 N88 (as lJ3)
0% 8% N%] Calculated value 49.82 5.96 11.62 Measured value
49.85 5.97 11.6016) F (1
921);Boc-Glu(OBZI)-Arg
(Mts)-Val-NHNH2[16) [15'l 6.29 f was dissolved in 15 ml of acetic acid,
4.25 ml of zinc powder was added and stirred at room temperature for 8 hours. Acetic acid was distilled off under reduced pressure, and 5% aqueous sodium bicarbonate was added to the residue, and the resulting precipitate was collected. It was washed with a saturated aqueous EDTA solution and then with water. Recrystallization from MeOH-ether gave [16]. Yield 2.64f (yield 51.3%). Melting point: 186-187°C 0 [α] knee 5.6° (C = 0.7, DMF) T LO;
Rf2=0.58.1 (f3=Q, 55 amino acid analysis; GIul, 20 (1), Leu 1 (1), A
rg 1.07 (1) Elemental analysis [03? N5609 N88 °'/I(
20] 0% 8% N% Calculated value 55,69 7.20 14.04 Measured value 55.73 7.18 14.1717)F
(19-34); Boa-Glu (OB
zI)-Arg(Mts)-Val-Glu(O
Bzl)-T'rp-Leu-Phe-NH2[1
:17] [13] 4.80? (1,96mM) with anisole (containing 2% ED'I') 326me, T
FA 12.8 ml! was added and stirred at 0°C for 60 minutes. TFA was distilled off under reduced pressure, hexane was added to the residue, and the resulting precipitate was separated and dried to obtain a powder. DM for this
30 ml of F and 55 tnl of Et3N O were added to obtain a neutralized solution. [16] 2.322D M
F 10 m13.936N
-HCA/DMF solution 1.79me isoamyl nitrite 0.47 ml! E t 3 N
O,98meN-Methylmorpholine 0.78 me'' Using the reagent described in 1 above, 3)
The above neutralized solution was added to the azide solution obtained in the same manner as in the method described above, and the mixture was stirred at 4°C for 48 hours. After the reaction, the mixture was neutralized with acetic acid under cooling, and the solvent was distilled off under reduced pressure. Add 3% acetic acid water and ethyl acetate to the residue, collect the resulting precipitate, and add 3% acetic acid water,
It was washed in the order of 5% sodium bicarbonate solution and water. A crude product was obtained by reprecipitation with DM It'-ethyl acetate. This is OHCA
Column chromatography using 3-MeOf (=water (8:3:1) dissolved in 5 ml and charged onto silica gel (2,3X50c+n) and elution solvent CHC, l=3-MθOH-water (8:3:1) Purified by Rf2=
The elution fraction of 0.41 (excluding the fraction of Rf 2 = 0.51) was collected and concentrated under reduced pressure. Ethyl acetate was added to the residue to powder it to obtain [17]. Yield 4.079 (yield 6
7%). Melting point: 255°C (decomposition) [α') -1,0 (0=1.0.DMF') T T
, C; Rf2 = 0.41, Rf4 = 0.73 Amino acid analysis; Qlu a, C6 (3), Arg (2),
Val-1, 87 (2), Asp 2.0? (2),
Leu 2.03 (2), Phe 1.00 (1)
, Lys 2.16 (2), I(is 0.99 (
1) Elemental analysis C0155H2O3N3003482
] As y21120] 0% 8% N% Division value 59.54 6.80 18.4.4 Measured value 59.24 6.68 18.3718)F
(16-18); Boc-Asn-8er-
Met-OMe [18] Ohem, Pharm, BulJ, 27 (2), 4
Z (
OMe ) -Ser -Met -OMe18.65
12, anisole 9-78me, TFA 39.1
After adding 2 rne and stirring at 0°C for 60 minutes, TFA
was distilled off under reduced pressure. After washing the residue with hexane, ether was added and the resulting gummy precipitate was separated by decanting.
Dry. To this, D MF 90 me, E t
After adding 3N 630W and stirring, Eoc-As
n-ONp 17.492, Et3N 6.9
3 ml was added and stirred for 16 hours. After the reaction, DMF was distilled off under reduced pressure, and 5% citric acid-containing saturated saline and ethyl acetate were added to the obtained oil and shaken. The ethyl acetate layer was washed with 5% citric acid solution, 5% sodium bicarbonate solution, and saturated saline (7
, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The residue was crystallized by treatment with ether and recrystallized with methanol-ether to obtain [18]. Yield 12.969 (yield 62.0%
) Melting point; 133-135°C [α)D -31.8° (C=0.9.DM]'i
”)TLC; Rf2 = 0.51,
Rf 3 = 0.24 elemental analysis CCl8H32N4
08 S and 1-]C% 11% N% Division value 46.54 6.94 12.06 Measured value 46,77 6.89 11.9819)F (
16-18); Boc-Aen-8er-Me
t -NHNI-12[191) [18:] 4.0Of was dissolved in MeOI] 50 tne, 2.15 mL of hydrazine hydrate was added thereto, and the mixture was left at room temperature for 18 hours. The precipitated crystals are E' t
After treatment with OI-1 and washing, it was washed with E t OI-1. Recrystallization from DMF-EtOIi yielded [19]. Yield 2.893r (yield 724%) Melting point: 212~
216℃ [α 5-26.40 (0=1.0, ])MTi
)'I' L C; Rf2 = 0.32, ]L
f3=0.10 amino acid analysis; Ser 0.88 (
1), Asp 1.00 (1), Met 0.40
(1) Elemental analysis C017H32N607 S] 0%
8% N% Calculated value 43.95 6.94 18.09 Measured value 43.86 7.02 1? , 8720) F (
16-34); Boc-Asn-Met-Gl
u(OBZI) -Arg, (Mts) -Val
, -Glu (OBZI) -Ichimon2 [I20] [:17] 500 m9 (0,161mM) with 0.35 ml of anisole (containing 2% EDT), TFA
After adding 1.40 ml and stirring at 0°C for 60 minutes,
'I'FA was distilled off under reduced pressure. Add hexane to the residue,
The resulting precipitate was separated and dried to obtain a powder. D to this
MF 5 me SEt 3N 45.2μ! was added to obtain a neutralized solution. [19] 150.5m9 D M F 0.20m13.
936 N, -He, #/DMF solution 0.20 m
l Isoamyl nitrite 517μ! Et3N
108.4μ! The above neutralized solution was added to the azide solution obtained using the above reagent in the same manner as in the method described in 3) above, and the mixture was stirred at 4°C for 48 hours. The reaction solution was neutralized with acetic acid under cooling, DMF was distilled off under reduced pressure, ether and 5% citric acid aqueous solution were added to the residue, and the resulting precipitate was collected. It was washed three times each with 5% citric acid solution, 5% sodium bicarbonate solution, and water, and reprecipitated five times with DMF-ethyl acetate to obtain [20]. Yield 468.8m9 (yield 88.9%) Melting point: 14
3-144℃ 5 [α'lID-2.70(C=0.8.DMF)']
'LO; Rf2 = 0.57, Rf, = 0.79 Amino acid analysis; Asp 3.18 (3), 8er 0
．． 90 (]), Met 0.70 (1), Glu
2.98 (3), Vall, 92 (2), Len
2.01 (2), Phe 1.00 (1), L
ye 2.14 (2), HisO196 (1), A
rg, 2.12 (2) Elemental analysis CC+6q H2z
s N3403983・4T (as 20) 0% 8
% N% Calculated value 57.24 6.79 18.59 Measured value
57.34 6.59 13.4721) F (1
4-15);Z (OMe) -Hls -Leu -
OMe[21] Z (OMe)-His-NHNH220,00
? 4.78N-HOA/DMF Book J 45.
21 me Isoamyl nitrite 9.58
m/! DMF 20
meNt:bN 40.
32meZ (OMe) -Hls -N1-INH2
to the azide solution obtained in the same manner as in the previous section 3),
D M Leu-OMe-I-IC79,08f
Dissolve in 50 ml and add Et3N? ,00m7! At PH
After adding the solution adjusted to No. 7, the mixture was stirred at 4° C. for 20 hours. DMF was distilled off under reduced pressure, and the residue was dissolved in ethyl acetate and 5%
I sprinkled it with baking soda and water. The organic layer was washed with 5% sodium bicarbonate solution and brine in that order, dried over anhydrous sulfuric acid, and concentrated under reduced pressure. The residue was crystallized by adding ether and diluted with MeOH.
- recrystallized from ether [2 fables] 16.325' (
A yield of 78.1% was obtained. Melting point, 110-111℃ 20 - [α], , 24.1° (0 = 1.1,
MeOH)TLO; Rf2=0.65, Rf3
=Q, 17 elemental analysis as C022H2O06N4]0
% 8% N% Calculated value 59.18 6.77 12.55 Measured value
58,93 6.70 12.3222) F (1
3-15);Boc-Lys(Z)-His-L
eu-OMe [22] [21]5. 2.49 ml of anisole in Hf! ,
9.96 ml of 'I' F A was added and stirred at 0°C for 1 hour. After 'I'FA was distilled off under reduced pressure and ether was added to the residue, the resulting precipitate was separated and dried. The obtained powder was dissolved in DMF20 me and E t 3N 3.
After adding 19 ml and adjusting the pH to 7, Boc-Lys
(Z)-0Su was added and stirred at room temperature for 20 hours. DMF was distilled off under reduced pressure, and the residue was dissolved in ethyl acetate and 5%
I sprinkled it with baking soda and water. The organic layer was washed with 5% sodium bicarbonate solution and brine in that order, and dried over anhydrous sodium sulfate. Ethyl acetate was distilled off under reduced pressure, the residue was crystallized by adding ether, and recrystallized from MeOH-ether to give C22] 5.4
29 (yield 787%) was obtained. Melting point 1144-147℃ 20, - [: a ] n, 3 & 6° (C=0
．． 7, MeOT()TLO; Rf2 = 0.65 Elemental analysis C032114808N6] C%
8% N% Calculated value 59.61 7.50 18.04 Measured value
59.61? , 45 18.0323) F
(13-15);Boc-Lys(Z)-T(i
s -Leu-NUN) (2[23] [22+ 2.65 y was dissolved in 20 ml of MeOH, 1.03 ml of hydine hydrate! was added, and the mixture was left at room temperature for 2 days. M e OH was distilled off under reduced pressure, water was added to the residue, and the resulting precipitate was filtered. Recrystallized from MeOH-ethyl acetate [23] 2.10 t<yield 79.3%
) I got it. Melting point, 167-169°C Ca; -21,4° (C = 1.0. DMF)
TLC; Rf2 = 0.48 Amino acid analysis; Leu 1 (1), Lye 0.
97 (1), His O, 91 (1) Elemental analysis [031H4807N8 トL TE]C%
8% N% Reduction value 57.74 7.50 17.38 Measured value
57.58 7.52 17.3824)F
(13-34); Boc-T,ys (Z)
-Hi8-Leu -Asn -8er -Met -
G], u (OBZI)-Arg(Mts)-Val
-Glu (OBzl)-Trp -Leu -A
rg (Mts)-Lys (Z) Lye
(Z)-Leu-Gln-Asp (OBZI)
-Val -Hi, s -Asn -Phe-NI(
2 [24) [201435m9 (0,127mM) with anisole (containing 2% EDT) 0.41 m7! ,'I'F
After adding 1.64 ml of A and stirring at 0°C for 90 minutes, 'I'FA was distilled off under reduced pressure. Hexano was added to the residue, the resulting oil was separated by decanting, and ether was added. .Count the resulting sediment,
A powder was obtained by drying. DMF5 me, E t3 for this
35μ of N was added to obtain a neutralized solution. [23) 163.8m9D
M F 3 m13.93
6N-BOA/DMF solution 232μ! Nitrite inoa-41μ chisel 2 and Et3N 85μ! The above neutralized solution was added to the azide solution obtained using the above reagent in the same manner as in the method described in 3) above, and the mixture was stirred at 4°C for 60 hours. The reaction solution was neutralized with acetic acid under cooling, and DMF was distilled off under reduced pressure. 3% aqueous acetic acid and ether were added to the residue under cooling, and the resulting precipitate was washed in the order of 3% aqueous acetic acid, 5% aqueous sodium bicarbonate, and water. Reprecipitate with DMF-ethyl acetate 5 times [24]
I got it. Yield 482.3 mti (yield 96.1%)
Melting point, 118-120°C [α]D -27° (0 = 1.1.DMF) TLO;
Rfg = 0.38, Rfa = 0.75 amino acid analysis;
Lys 3.12 (3), Lys 8.18 (:
Q Hls2.1s (2), Asp &10 (3)
, S e r O, 79 (1), Glu 2.99 (
3), Val 1.96 (2), Met 0.48
(1), Phe 1.00 (1), Arg 1.99
(2) Elemental analysis C0193H264N4004483 ・6H
20] C% 8% N% Calculated value 57.20 6.86 13.83 Measured value 57.07 6.58 13.672! I)
F (11-12);z (OMe)-Leu-Gl
y -OMe [25] )] -Gly -OMe-HCZ 12.56 t was dissolved in DMTi'〆5゜, and P was dissolved in Et3N14m/
After adjusting to H7, Z (OMe) -Leu -ON
14 ml of p41.64v1Et3N was added and stirred at room temperature for 18 hours. DMF' was distilled off under reduced pressure, and the residue was dissolved in ethyl acetate. After crystallization by adding 5% quether, recrystallization from ethyl acetate-ether gave [2
5] 28.98 g (yield 79.1%) was obtained. Melting point near 8-79℃ [ff]”: ; 13.1° (C=0.7, DM
]+')TLO;Rf2=o,'7i As elemental analysis C018H2606N2]C%'H
% N% Division value 59.00 7.15 7.65 Measured value
59.17 7.16 7.5926) F (10
-12);Z (OMe) -Asn-Leu-Gly
-OMe [26) [25] Anisole 1 & 26 ml in 22.369,
TFA5&04me was added and stirred at 0°C for 1 hour. T'FA was distilled off under reduced pressure, the residue was washed three times with n-hexane, and then dried 1.1. =. DMF 100 this
ml, dissolved, Bt3N8.54'm7! At PH7
After adjusting to Z (OMe) -Asn -0NF2
5.46 t, Et3 N 8. Add '54 me,
Stirred at room temperature for 20 hours. DMF was distilled off under reduced pressure to obtain a residue of 5% 22.42 f (yield 76.5%). r; Melting point: 196-197°C [α temperature 2; -15,40 (0=0.8, DMF) T L
C; Rf2 = 0.56, Rf3 = 0.
33 elemental analysis [022H320s N4 ToL, in]
C% 8% N% Calculated value 54.99 6.71 11.66 Measured value
55,14 6.86 11.6027) F (9
-12) ;Z (OMe) -His -Asn-
Leu-Gly-OMe [27)[26':l
Anisole 9.52 me on 14.03 f, T
38.08 ml of FA was added and stirred at 0°C for 1 hour. T
After FA was distilled off under reduced pressure and ether was added to the residue, the resulting precipitate was separated and dried to obtain a powder. Z (OMe) -Hls -NHNH211,67f
5.55N-HOA/DMF solution 22.70ml
Isoamyl nitrite 5.59 mlD
MF 20 meEt
3N 22.54+++7!
A solution obtained by dissolving the powder obtained above in DMF'70 solution and adjusting the pH to 7 with 4.09 ml of Et3N was added to the azide solution obtained using the above reagent in the same manner as in the previous section 3). . The mixture was stirred at 4°C for 20 hours. DMF was distilled off under reduced pressure, and the residue was washed with 5% sodium bicarbonate water and ether with water in that order, and recrystallized from DMF-MeOH to obtain C27]15.1.
3f (yield 88.9%) was obtained. Melting point: 226-227℃ [α grandson 2 knee 11.1° (C=0.8, DMF) TLC
; Rf2=0.37 Elemental analysis [as C2s H3209N4] 0%
8% N% Calculated value 54,45 6.36 15.88 Measured value
54,31 6.38 15.7328) F
(8-12); Boc -Met -T(is -As
n −' LeuGly OM e [28] [27] 5.089 Near = 7-L4. OmeST
FA 12.0+++/ was added and stirred at 0°C for 1 hour. After TFA was distilled off under reduced pressure and ether was added, the resulting precipitate was separated and dried to obtain a powder. DMF for this
After adding 50 ml and 2.28 ml of Et3N and stirring, Boc-Met-08112,859
, Et3N 1.14 me and stirred for 24 hours. The reaction solution was neutralized with acetic acid under cooling, and DMT+' was distilled off under reduced pressure. The residue was dissolved in butanol, washed four times with water, and then concentrated under reduced pressure. Ether was added to the residue, and the resulting gel-like powder was separated and recrystallized twice from Me OTl -Fr t OH to obtain [28]. Yield 2572 (yield 45.7%). Melting point: 122-125°C Crt]D-26,70 (c=0.7, DMF)
TLC; Rfg, -0,59 29) F (812); Boc -Met -H
ls -Aen-Leu-01,y -NHNH2[
29] [28] 8.279 was converted into DMF-MeOH (4:
1) It was dissolved in 30 ml, 1.20 me of hydrazine hydrate was added thereto, and the mixture was left at room temperature for 18 hours. The solvent was distilled off under reduced pressure, and the residue was treated with E t OT-1.
．． After the crystals (heated), M e OHE t
Recrystallization twice from OH gave [29]. Yield: 2.989 (909% yield). Melting point: 184-186°C [α] -33.1° (C=0.9, DMF)
T'LO; Rfg = 0.31 amino acid analysis;
Asp 0.95 (1), G], y 1.01 (
1), Me t 0.92 (] ), T, eu 1.
00 (1), His O, 95 (2) Elemental analysis CC
28H48NIOOs As S〕0% 8% N
% Calculated value 49.11 7.07 20.46 Measured value
49.04 7.14 20.2330) F (
8-34): 13oc-Met-Hls-A
sn-Leu-Gly-Lys (Z,) -Hls
-Leu-Asn-8er-Met -Glo (OB
ZI) Arg (Mts) -Val -Glu
(OBZI)-Trp-Leu-Arg (Mt
s)-Lys (Z)-L12 (Z)-Leu
-Gln -Asp(OBzl) -Val, -His
-Aen -Phe -Nl2 [30] [24
] 0.46 ml of anisole (containing 2% ED') in 4482.3m9 (0,122mM), T F
After adding 1.84 ml of A and stirring at 0°C for 90 minutes, TFA was distilled off under reduced pressure. After washing the residue with hexane, ether was added, and the resulting precipitate was filtered and dried. DMF to the obtained powder
Add 5 ml, Et3N 51.0 pi,
A neutralized solution was obtained. [29] 167.1myD
MF 3 ml & 9
36N-HC/TIIMF solution 2283μ! Isoamyl nitrite 390μkEt3N
The above neutralized solution was added to the azide solution obtained in the same manner as in the previous section 3) using the above reagent of 163.3 μm, and the mixture was stirred at 4° C. for 48 hours. The reaction solution was neutralized with acetic acid under cooling, and DMF was distilled off under reduced pressure. Add 5% citric acid water and ether to the residue, count the resulting precipitate, and add 5% citric acid water and ether.
% citric acid solution, 5% sodium bicarbonate solution, and water in this order. D.M.I.
? The crude product [3o] was obtained by reprecipitation with '-ethyl acetate five times. This was dissolved in 10 ml of DMF and eluted with Sephaded DMF'. Each fraction was divided into 9 ml, and the 43rd to 55th fractions were collected and dried under reduced pressure. The residue was treated with ethyl acetate to powder [3
0] was obtained. Yield: 360.9 m9 (67% yield). Melting point: 135-137°C [α']D-41.70(C20.6.DMIi')T
L C; Rfg = 0.34, Rf4 =
0.69 amino acid analysis; A day p 4.27 (4),
Gly 1.23 (1), Met 1.61 (
2), Leu 4.36 (4), H
ls 8.06 (3), Ser O, 81 (
1), G1. u 8.03 (3), Val 2.0
0 (2), Phe 1.00 (1), Lys 3.
15 (3), Arg 2.00 (2) Elemental analysis C0
21) 1300 N4805OS+・5II20] 0% 8% N% Division value 56,55 6.81 14.66 Measured value 56,32 6.66 14. ．． 7731) F
(6-7);Z (OMe) -Gln -Leu -
NHNHTroc [31] Z (OMe) -Leu -NHNHTroc 20
．． 009 with 8.96 ml of anisole, TFA
35.84 me was added and stirred at 0°C for 1 hour. 'I
"FA was distilled under reduced pressure with F, and the residue was washed with n-hexane and dried. This was dissolved in DMF 120?ne, 11.52 ml of Et3N, Z (OMe
) -01,n-ONp 5% sodium bicarbonate solution and then water, recrystallized from DMF-ethyl acetate to obtain [31'320
．． 7Or (yield 20%) is obtained. Melting point: 188-190℃ [63 points 0; -17,10 (0=1.0, DM:
F' ) TL C; Rf4 =0.65, Rf3=0
．． 44 elemental analysis as C023H3208H5Cp3]
0% ) 1% N% Calculated value 45.07 5.26 11.43 Measured value 45.52 5.41 11.5232) F
(5-7)Z (OMe) -11e -Gin 7L
11.00 ml of anisole and 44.0 ml of TFA were added to eu-NT-INB[31] 20.709, and the mixture was stirred at 0°C for 1 hour. T'F'A was distilled off under reduced pressure, and the residue was washed three times with n-hexane and then dried. Dissolve this in 200 ml of DMF, and add Et3N948
me, Z (OMe) -Tle -ONp 1
4.099 was added and stirred at room temperature for 18 hours. DM]i
' was distilled off under reduced pressure and crystallized to 17.43r (yield 79
, 1%). Melting point: 205-207°C 0 [α]; -15.3° (0=0.9, DMF)T
I, (j; Rf2=0.62, Rf3=0.3
Two-element analysis C029H4309H6 (as J3) 0%
8% N% Calculated value 47,97 5.97 11.58 Measured value
48,16 6.17 11.6633) li'
(4-7)'Boc -Gln (OBZI) -11
e -Gln -Le+i -NT-IN)iTroc
[33') [32] 15. Of anisole 8.
Add 99 ml, TFA 35.69-, and θ
The mixture was stirred at ℃ for 1 hour. After 'I'FA was distilled off under reduced pressure and ether was added to the residue, the resulting precipitate was separated and dried. Dissolve this in 50 ml of DMF and add Et3N
5.80 me % Boc -01,u (OBZI
)-08u8.999 was added and stirred at room temperature for 20 hours. 15.89 after recrystallization from DM'FM e OH
r (yield 87.1%) was obtained. Melting point, 223-225°C [α] too”; -26.3° (C=0.6, DMSO)T
LO; Rfg=0.59, Rf3=0.49 Elemental analysis C
0378560il H7 (as J3) 0% 8%
N% Calculated value 50.43 6.41 11.13 Measured value 50,33 6.52 11.6334) F (
4-7); Boc-Glu (OBZI)-11e
-GlnLeu -NHNH2 [34] [33] 9. Dissolve (1?) in 40 ml of MT', add 6.81 me of acetic acid and 6.679 of zinc powder,
Stirred at room temperature for 18 hours. After removing the zinc dust, the p solution was concentrated under reduced pressure, and a saturated E]) 'I' A solution was added to the residue, and then sodium bicarbonate was added to adjust the concentration to T) H7, forming a precipitate. The precipitate was washed successively with saturated ED'l'A solution and water, recrystallized twice from DMF-MeOH (3
4] 5.31? (Yield 78.7%) was obtained. Melting point: Decomposes above 260°C 8 [α] Knee 22.3° (0 = 0.6, DMSO
) T T, C; Rf2=0.84 Amino acid analysisゝ; G], u 2.06 (2), I]
, e 1.01 (1) Leu 1 (1) Elemental analysis as C034H5509Nq] 0% 8%
N% Calculated value 57.85 7.85 13.89 Measured value 57.76 8.00 13.6435)F(4
-34) ;Boc-G+, u(OT3z])-Tle
-Gln -1Jeu -Met -His -As
n -Leu -G'1y-Lys (Z)-His-
Leu-Aen-8er-Met-G], u (O
BZI)-Arg (Mts)-V+a1- Glu
(OBzl) -Trp -Leu -Arg, (
Mts) -Lys(Z)-LyEl(Z)-1
-+eu -G], n -A31) (OBZl,)
-Val -His -Asn -Phe -NH2[
35] [30) 357.1. mg (0,0794m
M) Anisole 035m1! XT' F A 1.4
After adding me and stirring at 0°C for 90 minutes, TEA was distilled off under reduced pressure. Hexane was added to the residue, the resulting oil was separated by a decanting method, ether was added, and the resulting precipitate was separated and dried to obtain a powder. DMF 5 to this
A neutralized solution was obtained by adding ml 1Et3N 44.31'A. [34] 112.1mgT
) MF 1 company 3.93
6 N-BOA/DMF' acid solution 96.8μ! Isoamyl nitrite 25.3 μm Et 3N
79.7μ! The above neutralized solution was added to the azide solution obtained using the above reagent in the same manner as in the method described in 3) above, and the mixture was stirred at 4°C for 48 hours. The reaction solution was neutralized with acetic acid under cooling, and DMF was distilled off under reduced pressure. 5 for residue
% citric acid solution and ether were added, and one portion of the resulting precipitate was washed in the order of 5% citric acid solution, 5% sodium bicarbonate solution, and water. D
Reprecipitation was performed twice with MF-ethyl acetate to obtain crude [35]. This was dissolved in 5 ml of DMF and loaded onto a Sephadex-L H-60 column (8.3X 140ct
Charge n) and DME? It was eluted. Each fraction is 9m7! The 54th to 61st fractions were collected and dried under reduced pressure. The residue was triturated with ethyl acetate to obtain [35]. Yield: 240.3 mg (yield: 59.7%) Melting point: 138-141°C [α] -4.0° (C=0.5.DMF) T L
O; Rf2=0.43, Rf4=0.77 Amino acid analysis; Glu 4.93 (5), Ile 1.0
7 (1), Leu 5.405), Asp 4.24
(4), Ser 0.83 (1), Glyl, 22
(1), Val2.04 (2), Met 1.75
(2), Phe 1.00 (1), T,ys 8.0
0 (3), His 2.92 (3), Argl,
94 (2) Elemental analysis CC245I343 N5305
As 78a/7H20]0% T-1% N% Calculated value 56.62 a, 92 14.28 Measured value 56.47 6.91 14.2936) F
(2-3);Z (OMe) -Val -Ser
-OMe C36]H-Ser -OMe 15.56
Dissolve f in I) M F 80 me, Et3N
The pH was adjusted to 7 with 14.0 ml. Z in this solution
(OMe) -Val-O1128,13t T' H
After adding the solution dissolved in F 140 me, -10℃
D 0024. ．． 762 was added and stirred for 3 hours and then at room temperature for 15 hours. After removing the precipitate, the solvent was distilled off under reduced pressure, and the precipitated crystals were washed with EtOH. Recrystallization from THF-EtOH gave [36] 22.199 (yield 58.0
%) was obtained. Melting point: 160-161°C 20 . [α], 9.9° (0=0.9, DM'F) TLC;
Rfa=0.63, Rf3=0.64 elemental analysis COIF
(As l-I2607 N2) C% 14%
N% Calculated value 56.53 6,85 7.33 Measured value
56.70 6.96 7.3637) F(13)
;Z-8er-N'al-8er-OMe[37)C3
6] 4.06 ml of anisole in 7.159, T
F A16.24 me was added and stirred at 0°C for 1 hour. TTi'A was distilled off under reduced pressure, and the residue was washed three times with n-hexane and dried to obtain an oil. DMF this
Dissolve in 35 ml, E t 3 N 2.62 m
l! was added to obtain a neutralized solution. Z -Ser -NHNH25,68?5.55 N
HOA/DMF Seat f<, 9.70 me Isoamyl nitrite 3.58+n/! DMF
10 m1Et3N
11.31 ml" (1) without using the reagent described in the previous section 3.
) The above neutralized solution was added to the azide solution obtained in the same manner as in the method described above, and the mixture was stirred at 4°C for 48 hours. After removing the precipitate and concentrating the filtrate under reduced pressure, EtOH was added to the residue. The resulting precipitate was collected and recrystallized from I) MP-MθOH [
37] 4.94r (yield 601%) was obtained. Melting point 1211-213°C [α]; 6.4° (C-0,9,DMF) T L C
; Rf2 = 0.66 Elemental analysis CC20I (as 290s N3) 0%
11% N% Estimated value 54. ．． 66 6,65 9.56 measured value
54.75 6,52 9.45 was dissolved, 1.86 ml of hydrazine hydrate was added, and the mixture was left overnight. The precipitated crystals were collected with MeO) and washed with E t OH,
Recrystallize from DMS O-MeOH [38] 2
．． 65y (yield 81.3%) was obtained. Melting point; 240°C [α'); -8.4° (C=0.6, DMSO)T
LC; Rf2=0.44 Amino acid analysis; Ser 1.83 (2), Vall
(1) Elemental analysis CC10N2902 as N5〕0
% N% N% Calculated value 51.92 6.65 15.94 Measured value
51.63 6.65 15.9839) F (1
-34); Z-8er-Val-8er-Glu
(OBzl) -11e -01n -Leu -Me
t -Hls -Asn-Leu-Gly-Lys (
Z) -Hls-Leu -Asn -Ser -Me
t -Glu (OBzI) -Arg (Mts)-
Val -Glu + (OBzl) -'rrp-L
eu-Arg(Mts)-Lys(Z)-Lys
(Z) -Leu-Gln -ASI) (OB
ZI) -Van -Ir1s -Asn -
Phe-N)J2[39] [38] 240.3 m9 (0,0474mM) with anisole (containing 2% ED'l'), TFA 0
．． After adding 84 ml and stirring at 0°C for 90 minutes, T
FA was distilled off under reduced pressure. The residue was washed with hexane, ether was added, and the resulting precipitate was dried several times to obtain a powder.
26.4μ! was added to obtain a neutralized solution. (:38) 41. tmy])
M P 1 m13.936
N-11CA/DMF solution 57.8μ! Inoaamyl nitrite 15.1μ! Et3N
47.6μ! ” Using the reagent described in 1 above, 3)
To the azide solution obtained in the same manner as in the above method, the neutralizing solution described in section 1 was added, and the mixture was stirred at 4°C for 48 hours. The reaction solution was neutralized with acetic acid under cooling, and ]) MP was distilled off under reduced pressure. 3% in residue
Add acetic acid water and ether, collect the resulting precipitate, and reduce to 3%
It was washed in the following order: acetic acid water, 5% sodium bicarbonate water, and water. Reprecipitation was performed five times with DMF-ethyl acetate to obtain crude (39) 199.8
I got m9. This was dissolved in DM
0 cIn) and eluted with a mixture of DMF and water (95:5). Each fraction was separated into 10me fractions. Each fraction was tracked at 280 nm, and the 75th to 89th fractions were collected and dried under reduced pressure. The residue was treated with ethyl acetate to obtain 141.3 m9 of powder. DMIi'5 m
Column chromatography was performed under the same conditions as above. The 76th to 88th fractions were collected and dried under reduced pressure. The residue was purified by treatment with ethyl acetate [3
9] was obtained. Yield 111.3m9 Melting point: 145-148
℃ [α] −3,5° (0=0.3.DMF) T L
O; Rfg = 0.60, Rf4 = 0.81 Amino acid analysis; Ser 2.78 (3), Val a, i
s (3), Asp 4.21 (4), G], u,
5.10 (5), Gly 1.08 (1), Met
1.49 (2), 11e 1.07 (1), Le
u 5.18 (5), Phe1.00 (1), Ly
s2.91 (3), Hls2.45 (3), Ar
g 1.91 (2) Amino acid analysis [4M-M5A, 110°C, 48 hours];
Glu 5.13 (5), Ile 1.02 (
1), Leu 5.36 (5), Asp 4.4
0 (4), Ser 0.94 (3)
, Gly 1.22 (1), Vanl
, 90 (3), Met 1.78 (2), Phe
1.00 (1), Trp 0.59 (1), Lys
2.96 (3), His 2.92 (3)
, Argl, 91(2) Elemental analysis C0259N360
N56062 As S4/7H20〕0% N%
N% Autopsy value 56,51 6.85 14.24 Measured value
56.55 6.81 13.9840) h -P
T T-] (1-34) N1-12[39] 2
00 my (0,0372mM) m-cresol 0.78m7! then IM-TFMSA and IM
Add 7.44 m/ml of thioanisole-containing TFA solution and
After stirring for 2 hours at °C, hexane was added and the resulting oil was separated by decanting. Add ether to powder,
It was quickly removed and dried. Cool the resulting powder under sewage water for 10 minutes.
me, and add Amberlite CG-4B resin (
(acetate form) was added and stirred for 30 minutes. After removing the resin, the pH of the p-liquid was adjusted to 10 with 5N ammonia water under water cooling.
．． Adjusted to 0. Acetic acid (Ehrlich reaction) was dissolved again in 5 ml of IN acetic acid water, and Sephadex G-50 was added.
column (2,8x143 cm) and I
Gel filtration was performed with N acetic acid water. The eluate was fractionated into 10 ml portions and tracked at 280 nm. The 36th to 57th fractions F-1 and the 58th to 72nd fractions F-[ were freeze-dried to obtain powder F-131.8m9 (yield 18.3%).
) and powder F-11140, 1m9 (yield 80.8
%) was obtained. p-[140+++9 was dissolved in 5 ml of 0.01M ammonium acetate aqueous solution (PI(5,1)) containing 8M urea, and this was dissolved in advance in 0.01M ammonium acetate aqueous solution (PI(5,1)).
A column (24 x 5 cm) of CM-cellulose equilibrated with PH5,1) was charged. Wash with 40 ml of 0.01M ammonium acetate aqueous solution (PH5,1), and add 0.01M ammonium acetate aqueous solution (PH5,1).
)300m/and 0.3M ammonium acetate aqueous solution (P
I] 5.1) Fractionate between 300 ml and trace each fraction at 280 nm.35th to 60th fraction F-[-1,61st to 85th fraction F-11-2,86~ 115th fraction F
-1-3 and 116th to 150th fractions F-
11-4 was obtained. Each fraction was collected and lyophilized,
To remove ammonium hydroxide, water was added and freeze-dried three times to obtain 7.5 mti of F-If-1 powder (yield 5.
4%), 20.5 m9 of F-11-2 powder (yield 1
4.5%), F-17-3 powder 41.5 mg (yield 2
9.6%) and F-11-4 powder (31.0 m9 (yield 22.1%)) were obtained. and gel filtration with IN aqueous acetic acid. 1 eluate
Fractionation was performed by ime, each fraction was tracked at 280 nm, and the 39th to 49th fractions were collected and freeze-dried to obtain 40 rng of powder. The above powder was dissolved in 5 ml of water, and 53 ml of dithiothretol was added thereto and reduced at 30°C for 48 hours. The reaction solution was transferred to a Sephadex G-25 column (1.8 x 140 t
yn) and eluted with IN aqueous acetic acid. The eluate was fractionated into 1 ome portions, and each fraction was divided into 280
The 27th to 38th fractions were charged to a collection column (2,4X 94 tx) and eluted with the above layer solution. The eluate was fractionated into 5.3 me fractions, and each fraction was colored using the Folin-Lowry method.
Tracking was performed by measuring absorbance at 50 nm. 35-40
th fraction I'-II-31, 41st to 49th fractions F-II-32 (TLO; Rf5=0.
48) and the 50th to 75th fractions F −n
-33 was obtained. The F-[-32 fractions were collected, concentrated under reduced pressure, and then freeze-dried four times to obtain h-P T I
-](1-34)NH2 was obtained. Yield 22.8 my'
I'LC, R5 = 0.48 [α') -55,2° (0 = 0.2, OIN acetic acid water) Amino acid analysis; Asp 4.00 (4), S
er 2.34 (3), Olu 4.80 (5),
G171.11 (1), Val 3.18 (3), M
et 1.25 (2), lle 1.23 (
]), Leu 5.20 (5), Phe 1.0
0 (1), Lys 2.87 (3), Hls 2
．． 38 (3),; Asp 4.11 (4),
Ser 2.97 (3), G], u 4B6
(5), Gly 1.07 (1), Val 3.1
6 (3), Met 1.55 (2), 11e
1.01 (1), Leu 5.12 (5), Phe
1.00 (1), Trp 0.64 (1), L
ys 3.2 (1 (3), I (is 2.76 (3)
), Arg 2.00 (2) Amino acid analysis [Sigma, leucine aminopeptidase Lot, No. L-6007, 38°C, 48 hours]
, Asp O, 92 (1), Asn 2.88 (3
), Ser 2.91 (3), Glu 2.93 (
3), Gln 1.91 (2), Gly 1.03
(1), Va'13.11 (3), Met 1.84
(2), Ile O, 94 (1), Leu 495
(5), Phe 1.00 (1), Trp 0.78
(1), Lys 8.25 (3), His 2.5
8 (3), Arg 1.99 (2) Elemental analysis [C
l81 N292 N56050 S2・9cH3CO
As OH・15B20] 0% T-3% N% Calculated value 48°50 7.32 15.92 Measured value 48.55 7.65 15.79 High performance liquid chromatography column; p Bondapak 018 (0,25"
×1') Buffer solution, 0.1 M IJ containing 0.1% acetic acid. Linear concentration gradient flow rate of 0 to 50:50; 1 m/! /min Detection: 280nm Result: 7.76 min 1 spot detection disc, etc. focused electrophoresis (8M urea gel, PH3~
10. Length 0.5 x 6cm, 1mA, 200
V); has only one band at a position 0.75 crn from P H10,0. PTI (active, rat kidney P'T'H receptor assay results are 5100 U/my, h-
PTI] (1-34) (manufactured by Toyo Jozo Co., Ltd., 3300u/
75-428-

Claims (1)

[Claims] (1) In producing h-PTH (1-84) amide salts represented by formula %), the carboxyl group of the C-terminal phenylalanyl group is converted to an amide group, and the formula [I ] individual protected amino acids and/or protected peptides are condensed with the amino acid order of The group was removed by acid decomposition, and the obtained 11-P'
A method for producing highly active h-PTI ((1-84·) amide, which comprises separating and purifying rH(1-84) amide by column chromatography using a gel filtration agent and an adsorbent.