JPS6342639B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides human parathyroid hormone (h-PTH)
[1-38] Regarding fragments. More specifically, the present invention provides the formula It relates to a peptide represented by or a salt thereof. The above-mentioned novel peptide [] has parathyroid hormone activity, and antibodies obtained using this as an antigen have immunological cross-reactive activity with h-PTH. Therefore, this peptide is also useful as a peptide for preparing h-PTH antibodies. The object compound [] of the present invention is constructed by condensing individual amino acids or lower peptides in the amino acid order represented by the formula [], and removing the protective group of the functional group of the side chain in the final step of the condensation reaction. It is obtained by 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 preparation of the raw materials for the compound of interest as well as all intermediates are those known in peptide synthesis and therefore can be carried out by known means such as hydrolysis, acid hydrolysis, reduction, aminolysis or hydrazinolysis. Therefore, a protecting group that can be easily removed is used. Such protecting groups are described in the literature and reference books in the field of peptide synthetic chemistry. For example, as a protecting group for an amino group,
Acyl groups such as formyl group, trifluoroacetyl group, phthaloyl group, p-toluenesulfonyl group, o-nitrophenylsulfenyl group, benzyloxycarbonyl group, o (or p)-bromobenzyloxycarbonyl group, o (or benzyloxycarbonyl groups such as p)-chlorobenzyloxycarbonyl group, p-nitrobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, trichloroethyloxycarbonyl group,
Aliphatic oxycarbonyl groups such as t-butyloxycarbonyl group, t-amyloxycarbonyl group, diisopropylmethyloxycarbonyl group, 2
-Phenyl-isopropoxycarbonyl group, 2-
Tolyl-isopropoxycarbonyl group, 2-p-
Examples include aralkyloxycarbonyl groups such as diphenyl-isopropoxycarbonyl groups. These amino groups can also be protected by forming enamines obtained by reacting with 1,3-diketones such as benzoylacetone and acetylacetone. Carboxyl groups are protected by amide formation, hydratide formation or esterification. That is, the amide group is a 3,4-dimethoxybenzyl group, a bis-
Substituted with (p-methoxyphenyl)methyl group, etc. The hydratide group is substituted with a benzyloxycarbonyl group, a trichloroethyloxycarbonyl group, a trifluoroacetyl group, a t-butyloxycarbonyl group, a trityl group, a 2-p-diphenyl-isopropoxycarbonyl group, and the like. Ester groups include methanol, ethanol,
Alkanols such as t-butanol and cyanomethyl alcohol, benzyl alcohol, p-bromobenzyl alcohol, p-chlorobenzyl alcohol, 2,6-dichlorobenzyl alcohol, p
-Aralkanols such as methoxybenzyl alcohol, p-nitrobenzyl alcohol, benzhydryl alcohol, benzoylmethyl alcohol, p-bromobenzoylmethyl alcohol, p-chlorobenzoylmethyl alcohol, 2,4,6
- Substituted with phenols such as trichlorophenol, 2,4,5-trichlorophenol, pentachlorophenol, p-nitrophenol, 2,4-dinitrophenol, thiophenols such as thiophenol, p-nitrothiophenol, etc. Ru. The hydroxyl group of serine can be protected, for example, by esterification or etherification. Groups suitable for this esterification include, for example, acetyl group, benzoyl group, benzyloxycarbonyl group, and ethyloxycarbonyl group. Further, examples of groups suitable for etherification include benzyl group, tetrahydropyranyl group, and t-butyl group. A 2,2,2-trifluoro-1-t-butyloxycarbonylaminoethyl group and a 2,2,2-trifluoro-1-benzylosacycarbonylamino group are also suitable for protecting these hydroxyl groups. However, it is not necessary to protect these hydroxyl groups. Groups used to protect the amino group in the guanidino group of arginine include, for example, a nitro group, a tosyl group, a benzyloxycarbonyl group, a mesitylene-2-sulfonyl group, but the guanidino group is not necessarily protected. There's no need. Examples of the group used to protect the imino group of histidine include benzyl group, trityl group, benzyloxycarbonyl group, tosyl group,
2,2,2-trifluoro-1-t-butyloxycarbonylaminoethyl group, 2,2,2-trifluoro-1-benzyloxycarbonylaminoethyl group, etc., but it is not necessary to protect this imino group. There isn't. The thiomethyl group of the methionine is preferably converted into a methyl sulfoxide group to prevent side reactions, but it is not necessarily necessary to protect it by oxidation. In the present invention, t-
Butyloxycarbonyl group, p-methoxybenzyloxycarbonyl group is used, benzyloxycarbonyl group is used to protect the side chain amino group, 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 amino group in the guanidino group. In the synthesis of the present target compound [ ], condensation of individual amino acids or lower peptides is carried out, for example, with an amino acid or peptide having a protected α-amino group and an activated terminal carboxyl group, and a free α
- reacting an amino acid or peptide with an amino group and 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 terminal carboxyl group; This can be carried out by reacting an amino acid or peptide having an α-amino 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-trichlorophenyl ester, 2,4,6-trichlorophenyl ester, pentachlorophenyl ester, N-hydroxysuccinimide ester, It can be activated by converting it into N-hydroxyphthalic acid imide ester or the like. Condensing agents such as carbodiimides, such as N,N'-dicyclohexyl-carbodiimide, N-ethyl-N'-3-dimethylaminopropyl-carbodiimide, N,N'-carbonyl-diimidazole or isoxolium salts, such as Woodward reagents, may also be used. It can be activated by using and reacting. Preferred condensation methods in the present invention are the azide method, active ester method and carbodiimide method. In each step of the 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, Biunsch method [Z.Naturforsch., 21b , 426 (1966). ]
or Geiger method [Chem.Ber., 103 , 788
(1970)] is suitable. The condensation order can be synthesized in any order as long as it is the amino acid order shown by the formula [], but C--
It is preferable to connect amino acids or peptides sequentially from the terminal side. For example, C--
It is preferable to condense the terminal fragment with a peptide fragment consisting of amino acids 25 to 28. This C-terminal fragment and the tetrapeptide 25-28
The azide method is suitable for the condensation. Before the resulting C-terminal fragment 25-38
Peptide fragments consisting of amino acids 22 to 24 are linked, and the Geiger method is suitably used. The obtained C-terminal fragment 22-38 is preceded by a peptide fragment consisting of amino acid sequences 19 to 21, a peptide fragment consisting of amino acids 16 to 18, a peptide fragment consisting of amino acids 13 to 15, 8
It is preferable to link a peptide fragment consisting of amino acid sequences 1 to 12, a peptide fragment consisting of amino acids 4 to 7, and a peptide fragment consisting of amino acids 1 to 3. These condensations are suitably carried out by the azide method. The above C-terminal fragment 25-38 has the remaining amino acid order of the C-terminal fragment 29-38.
It is preferable to link peptide fragments consisting of 25-28 by the azide method. C-terminal fragment 29-38 is hexapeptide fragment 33-38
It is preferable to link the dipeptide fragments 31-32 to the peptide by the azide method, and before that, link each amino acid to the remaining amino acids in the order by the active ester method. A protecting group for α-amino group in the above condensation reaction, such as t-butyloxycarbonyl group, p-
The methoxybenzyloxycarbonyl group is removed with trifluoroacetic acid. Protecting groups for α-carboxyl groups, such as methyl esters, can be decomposed with dilute sodium hydroxide solution or converted to protected hydratides such as hydratide or trichloroethoxycarbonyl hydratide, and benzyl ester groups can be decomposed with anhydrous hydrogen fluoride. It can be decomposed or converted to hydratide by hydrogenolysis. In this way, an octatriacontapeptide having a protected α-amino group, ξ-amino group, carboxyl group, side chain carboxyl group, guanidino group and/or hydroxyl group is obtained. These protecting groups are preferably removed in one step by acid decomposition, such as with anhydrous hydrogen fluoride or trifluoromethanesulfonic acid, to yield the target compound of formula []. When the methyl sulfoxide group of methionine is reduced to a thiomethyl group, it may be carried out using dithiothreitol, thioglycolic acid, mercaptoethanol, ethanedithiol, or the like. The peptide [] thus obtained can be separated and purified by known means for purifying peptides or proteins. For example, Cephadex G-25 (trade name, manufactured by Pharmacia, gelling agent, dextran gel), Cephadex G-25
-50 (trade name, manufactured by Pharmacia, gelling agent,
Dextran gel), gel filtration using a gel filtration agent such as Cephadex LH-20 (trade name, manufactured by Pharmacia, gel filtration agent, hydroxypropylated dextran gel), column chromatography using carboxymethyl cellulose, ion exchange resin, etc. This can be carried out by high performance liquid chromatography or the like. The peptide [ ] of the present invention can be obtained in the form of a base or a salt thereof depending on the conditions of the method. Examples of salts include inorganic acid salts and salts with organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, succinic acid, malic acid, tartaric acid, and citric acid. The present peptide [ ] 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. Endoclinol.92, Endoclinol.92,
692 (1973). Comparing the results, it was found that the cyclic AMP synthesis ability when using kidney cell membranes was
PTH (1-34) is 2300U/mg, whereas the h of the present invention
-PTH(1-38) has about 3 times the activity at 7000U/mg. Similarly, when bone cells are used, the cyclic AMP synthesis ability is determined by the known h-PTH (1-34).
h-PTH (1-38) of the present invention for 3000U/mg
showed approximately 3 times the activity at 9000U/mg. As mentioned above, the h-PTH (1-38) of the present invention has much stronger biological activity than the known h-PTH (1-34). In addition, the abbreviations described in this specification have the following meanings. Phe; L-phenylalanine Tyr; L-tyrosine Asn; L-asparagine His; L-histidine Met; L-methionine Val; L-valine Asp; L-aspartic acid Gln; L-glutamine Leu; L-leucine Lys; L-lysine Arg; L-arginine Trp; L-tryptophan Glu; L-glutamic acid Ile; L-isoleucine Ser; L-serine Gly; glycine Z; benzyloxycarbonyl Boc; t-butoxycarbonyl Z (OMe); P-methoxy Benzyloxycarbonyl Mts; N ^G -mesitylene-2-sulfonyl OBzl; benzyl ester OMe; methyl ester OTCP; 2,4,5-trichlorophenyl ester ONp; P-nitrophenyl ester OSu; N-hydroxysuccinimide ester NHNHTroc; trichloroethyloxycarbonylhydrazide NHNH ₂ ; hydrazide TFMSA; trifluoromethanesulfonic acid MSA; methanesulfonic acid TFA; trifluoroacetic acid TosOH; P-toluenesulfonic acid HCl; hydrogen chloride Et ₃ N; triethylamine CHA; cyclohexylamine DCHA; dicyclohexyl Amine DCC; NN′-dicyclohexylcarbodiimide HOBT; 1-hydroxybenzotriazole EDTA; ethylenediaminetetraacetate DMF; dimethylformamide DMSO; dimethyl sulfoxide MeOH; methanol EtOH; ethanol BuOH; butanol CHCl ₃ ; chloroform THF; tetrahydrofuran ether; diethyl ether TLC ; Thin layer chromatography Next, the production examples of the present invention will be specifically explained with reference to Examples. The carriers and solvent systems for thin layer chromatography (TLC) and column chromatography used in these Examples, as well as the conditions for amino acid analysis, are as follows. <TLC and column chromatography> Support: Silica gel G Solvent system: 1 CHCl ₃ -MeOH (10:0.5) 2 CHCl ₃ -MeOH-water (8:3:1) 3 CHCl ₃ -MeOH-acetic acid (9:1) :0.5) 4 BuOH-pyridine-acetic acid-water (4:1:1:
2) 5 BuOH-pyridine-acetic acid-water (4:1:1:
1) <Amino acid analysis> Hydrolysis conditions: 1 Heat with 6N-hydrochloric acid at 110°C for 24 to 48 hours in a sealed tube. 2 Heat 4N-MSA in a sealed tube at 110°C for 24 to 48 hours. Example 1 h-PTH (1-38); H-Ser-Val-Ser-Glu
―Ile―Gln―Leu―Met―His―Asn―Leu―
Gly-Lys-His-Leu-Asn-Ser-Met-Glu
―Arg―Val―Glu―Trp―Leu―Arg―Lys―
Lys―Leu―Gln―Asp―Val―His―Asn―Phe
-Production of Val-Ala-Leu-Gly-OH Z-Ser-Val-Ser-Glu (OBzl)-Ile-Gln
―Leu―Met(O)―His―Asn―Leu―Gly―Lys
(Z)-His-Leu-Asn-Ser-Met(O)-Gln
(OBzl) - Arg (Mts) - Val - Glu (OBzl) - Trp
―Leu―Arg(Mts)―Lys(Z)―Leu―Gln―
Asp(OBzl)―Val―His―Asn―Phe―Val―Ala
-Leu-Gly-OBzl148.3mg (0.0254mM)-
Add 0.58ml (5.59mM) of cresol. to this
Add 5.59 ml of a TFA solution containing 2M-TFMSA and 2M thioanisole, stir at 0°C for 1 hour and at 25°C for 1 hour, then add ether and separate the resulting precipitate.
Dissolve the obtained powder in 10ml of water and add Amberlite.
Add 1.5g of CG-4B resin (acetate type) and stir for 30 minutes. After removing the resin, 2 ml of 1N ammonia water was added to the solution, stirred at 0°C for 30 minutes, acidified with acetic acid, and then freeze-dried. Dissolve this in 2ml of water, add 0.78g (5.08mM) of dithiothreitol, and heat at 60°C.
After stirring for 36 hours, add 1 ml of 1N acetic acid and inject into a Sephadex G-25 column (3 x 95 cm).
Elute with 1N acetic acid. Fractionate the eluate into 5 ml portions,
The active fractions (42nd to 56th bottles) are collected and lyophilized to obtain 62.8 mg of white powder. 60 mg of the obtained white powder
Dissolve in 0.01M ammonium acetate-8M urea aqueous solution, inject into a carboxymethylcellulose column (1.8 x 12cm), and add 300ml of 0.01M ammonium acetate-8M urea aqueous solution (PH5.1) and 0.1M ammonium acetate-8M urea aqueous solution (PH5). .1) Perform elution using a linear concentration gradient over 300 ml. 5 of the eluate
Fractionate in ml portions, collect active fractions (101st to 114th), dialyze, and apply to Sephadex G-25 column (3 x 95
cm) and freeze-dried to obtain 35 mg of white powder. Dissolve this in 30 ml of water, inject it into a carboxymethyl cellulose column (1.8 x 12 cm), and
Elution is performed by applying a linear concentration gradient between 150 ml and 0.2 M ammonium acetate aqueous solution. The eluate was fractionated into 5 ml portions, and the active fractions (37th to 45th) were collected, desalted using a Sephadex G-25 column (3 x 95 cm), and lyophilized to obtain 12.1 mg of white powder. This powder was packed in a Sephadex G-50 column (1.8 x 95 cm) filled with butanol-pyridine-acetic acid-0.6M ammonium acetate aqueous solution (5:3:0.2:10).
This is then eluted with as little as possible of the upper layer of butanol-pyridine-acetic acid-0.6M ammonium acetate aqueous solution (5:3:0.2:10). Fractionate the eluate into 3 ml portions and collect the active fraction (6th to 10th fraction)
Collect and concentrate under reduced pressure. Add this to Safedex G
Desalt with a -25 column (3 x 95 cm) and freeze-dry to obtain 6.0 mg of white powder. 1.125 mg of the obtained white powder was dissolved in 25 ml of water, injected into a Cosmosil 5C ₁₈ column (0.461 x 15 cm), and used as a solvent.
Perform preparative high-performance liquid chromatography using 0.1% acetic acid-acetonitrile (73:27) containing 0.1M phosphoric acid.
Desalt the product using a Sephadex G-25 column as an eluent using 1N acetic acid as an eluent, and freeze-dry to obtain 0.8 mg of a white powder. Fractionation was performed three times using high performance liquid chromatography to obtain 2.4 mg of the target product as a white powder. The above raw materials are produced as follows. P (1-38) Z-Ser-Val-Ser-Glu
(OBzl)-Ile-Gln-Leu-Met(O)-His-
Asn-Leu-Gly-Lys(Z)-His-Leu-Asn-
Ser―Met(O)―Glu(OBzl)―Arg(Mts)―
Val―Glu(OBzl)―Trp―Leu―Arg(Mts)―
Lys(Z)―Lys(Z)―Leu―Gln―Asp(OBzl)
―Val―His―Asn―Phe―Val―Ala―Leu―Gly
-Production of OBzl [44] P (37-38) Z (OMe) -Leu-Gly-OBzl [1] H-Gly-OBzl・TosOH42.1g in DMF200ml
After adjusting the pH to 7 with 18.3ml of Et ₃ N,
Add 54.6 g of (OMe)-Leu-ONp and 18.3 ml of Et ₃ N and stir at room temperature for 18 hours. After the reaction, DMF was distilled off under reduced pressure and the residue was dissolved in ethyl acetate. Wash with 5% citric acid, saline, 5% sodium bicarbonate, and saline in this order,
After drying over anhydrous sodium sulfate, concentrate under reduced pressure. Add ether to the residue to obtain crystals. Recrystallized from MeOH-ether, 42.3g of target product (yield 76%)
get. Melting point; 94-95℃ [α] ²⁰ _D ; -7.8゜ (C = 0.8, DMF) TLC; Rf ₁ = 0.53 Elemental analysis [as C ₂₄ H ₃₀ O ₆ N ₂ ] C% H% N% Calculated value 65.14 6.83 6.33 Measured value 65.40 6.67 6.51 P(36−38)Z(OMe)−Ala−Leu−Gly−
OBzl [2] [1] 5.02g, anisole 2.46ml, TFA 9.84ml
is added, and the residue is washed three times with n-hexane and dried. Dissolve this in 20ml of DMF and adjust the pH with 1.6ml of Et ₃ N.
After adjusting to 7, Z(OMe)-Ala-ONp4.27g,
Add 1.6 ml of Et ₃ N and stir at room temperature for 20 hours. After the reaction, DMF was distilled off under reduced pressure, and the residue was mixed with 5% citric acid,
Add ether and remove the resulting precipitate. This precipitate was washed with 5% citric acid, water, 5% sodium bicarbonate, and water in this order, and recrystallized from DMF-MeOH to obtain the desired product.
Obtain 3.20 g (81% yield). Melting point; 148-150℃ [α] ²⁰ _D ; -13.9゜ (C = 0.6, DMF) TLC; Rf ₁ = 0.38 Elemental analysis [as C ₂₇ H ₃₅ O ₇ N ₃ ] C% H% N% Calculated value 63.14 6.87 8.18 Measured value 62.87 7.13 7.95 P(34~35) Z(OMe)―Phe―Val―OMe
[3] Dissolve 25.15g of H-Val-OMe in 250ml of DMF,
After adjusting the pH to 7 with 21ml of Et ₃ N, Z(OMe)-Phe-
Dissolve 49.40g of OH in 250ml of THF and add. −10
Cool to ℃, add 37.14 g of DCC, and stir for 3 hours and then at room temperature for 15 hours. After the reaction, remove the precipitate and
DMF and THF are distilled off under reduced pressure, and the residue is mixed with ethyl acetate and 5% citric acid. The organic layer is washed with 5% citric acid, 5% aqueous sodium bicarbonate, and brine in this order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Add ether to the residue to obtain crystals. MeOH
-Recrystallize from ether to obtain 47.98 g (yield 72%) of the desired product. Melting point: 132-133℃ [α] ²⁰ _D : -8.7゜ (C = 1.0, DMF) TLC: Rf ₁ = 0.69 Elemental analysis [as C ₂₄ H ₃₀ O ₆ N ₂ ] C% H% N% Calculated value 65.14 6.83 6.33 Measured value 65.00 6.98 6.44 P(34−35)Z(OMe)―Phe―Val―NHNH ₂
[4] Dissolve 44.25 g of [3] in 400 ml of MeOH, add 25.04 ml of hydrazine hydrate, and leave at room temperature overnight. The precipitated crystals were collected with EtOH, and DMF-
Recrystallized from EtOH to yield 36.59 g of the target product (yield: 83
%). Melting point 206-208℃ [α] ²⁰ _D -8.0゜ (C = 0.8, DMF) TLC; Rf ₂ = 0.52, Rf ₃ = 0.60 Elemental analysis [as C ₂₃ H ₃₀ O ₅ N ₄ ] C% H% N% Calculated value 62.42 6.83 12.66 Measured value 62.16 6.78 12.57 P(34−38)Z(OMe)―Phe―Val―Ala―
Leu-Gly-OBzl [5] [2] 3.02g, anisole 1.91ml, TFA 7.46ml
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, dissolve 2.87 g of [4] in 8 ml of DMF, and add 3.26 g of 4.78N-HCl/DMF solution while cooling to -50℃.
ml, then 1.03 ml of isoamyl nitrite. −
After stirring at 20°C for 20 minutes, cool to -50°C again.
Add 3.27 ml of Et ₃ N, and add a solution of the oil obtained above dissolved in 50 ml of DMF and adjusted to pH 7 with 0.82 ml of Et ₃ N. After stirring for 2 days at 0-7°C,
The reaction solution was concentrated under reduced pressure, and 5% citric acid and ether were added to the residue. The resulting precipitate is collected and washed with 5% citric acid, 5% aqueous sodium bicarbonate, and water in this order. DMF−
Recrystallized from MeOH to obtain 3.97 g of the target product (yield
87.7%). Melting point; 249-250℃ [α] ²⁰ _D ; -20.1゜ (C = 0.9, DMF) TLC; Rf ₁ = 0.26, Rf ₃ = 0.82 Amino acid analysis; Val 0.92 (1), Phe 1.02 (1),
Gly1.00(1), Ala1(1), Leu1.03(1) Elemental analysis [as C ₄₁ H ₅₃ O ₉ N ₅ ] C% H% N% Calculated value 64.80 7.03 9.22 Measured value 64.66 7.06 9.38 P(33 −38) Z(OMe)-Asn-Phe-Val-
Ala-Leu-Gly-OBzl [6] [5] 3.52g, anisole 1.51ml, TFA6.04ml
and stirred at 0°C for 1 hour. TFA is distilled off under reduced pressure, ether is added to the residue, and the resulting precipitate is collected. Powder obtained in DMSO10ml, DMF10
After adjusting the pH to 7 by adding 0.65 ml of Et ₃ N,
To this, HOBT0.1g, Z (OMe) -Asn-
Add 1.93 g of ONp and 0.65 ml of Et ₃ N and stir at room temperature for 2 days. Add 5% citric acid and ether to the reaction solution and collect the precipitate. 5% citric acid, water, 5%
Wash with baking soda and water in that order. Recrystallization from DMSO-MeOH yields 3.03 g (yield 74.9%) of the desired product. Melting point; 260-262°C [α] ²⁰ _D ; -20.1° (C = 1.1, DMSO) TLC; Rf ₂ = 0, Rf ₄ = 0 Amino acid analysis; Asp 0.99 (1), Gly 0.98 (1), Ala1
(1), Val0.96(1), Leu1.01(1), Phe0.95(1) Elemental analysis [as C ₄₅ H ₅₉ O ₁₁ N ₇ ] C% H% N% Calculated value 61.84 6.84 11.22 Measured value 61.82 6.82 11.41 P(31−38)Z(OMe)―Val―Asn―Phe―
Val―Ala―Leu―Gly―OBzl [7] [6] 2.73g, 1.69ml of anisole, 6.76ml of TFA
and stirred at 0°C for 1 hour. TFA is distilled off under reduced pressure, ether is added to the residue, and the resulting precipitate is collected. Z(OMe)-Val-His-NHNH ₂ 1.50g 4.78N-HCl/DMF 2.82ml Isoamyl nitrite 0.60ml DMF 5ml Et ₃ N 2.52ml Z(OMe)-Val-His-NHNH ₂ last time [5]
Dissolve the powder obtained above in 10 ml of DMSO and 10 ml of DMF in the azide solution obtained in the same manner as in the method of
Add a solution adjusted to pH 7 with 0.44 ml of Et ₃ N. 0
After stirring at ~7°C for 2 days, the reaction solution was concentrated under reduced pressure, and 5% citric acid and ethyl acetate were added to the residue. Collect the resulting precipitate and add 5% citric acid, water, 5% sodium bicarbonate solution,
Wash with water. Recrystallization from DMSO-MeOH yields 2.81 g (yield 81.2%) of the desired product. Melting point; 242-244°C [α] ²⁰ _D ; 21.8° (C = 1.0, DMSO) TLC; Rf ₂ = 0 Amino acid analysis; Val 1.93 (2), His 1.03 (1),
Asp1.01(1), Gly0.98(1), Ala1(1), Leu1.00(1),
Phe0.98(1) Elemental analysis [as C ₅₆ H ₇₅ O ₁₃ N ₁₁・H ₂ O] C% H% N% Calculated value 59.61 6.88 13.66 Measured value 59.55 6.96 13.59 P (30-38) Z (OMe) - Asp(OBzl)―Val―
His―Asn―Phe―Val―Ala―Leu―Gly―
OBzl [8] [7] 2.81g, anisole 1.64ml, TFA 6.56ml
and stirred at 0°C for 1 hour. TFA is distilled off under reduced pressure, ether is added to the residue, and the resulting precipitate is collected. Add the obtained powder to DMSO10ml, DMF10
ml, add 0.71ml of Et ₃ N, adjust the pH to 7,
To this, Z(OMe)-Asp(OBzl)-ONp1.54g,
Add 0.43 ml of Et ₃ N and stir at room temperature for 2 days. The reaction solution was concentrated under reduced pressure, and 5% citric acid and ethyl acetate were added to the residue to collect the precipitate. Wash in the following order: 5% citric acid, 5% sodium bicarbonate solution, and water. Recrystallization from DMSO-MeOH gives 2.55 g (yield 76.5%) of the desired product. Melting point: Decomposes above 250°C [α] ²⁰ _D : -16.4° (C = 1.0, DMSO) TLC: Rf ₂ = 0, Rf ₃ = 0 Amino acid analysis: Asp2.05(2), Gly0.98(1) ,Ala1
(1), Val2.01(2), Leu1.01(1), Phe0.99(1), His0.87
(1) Elemental analysis [as C ₆₇ H ₈₆ O ₁₆ N ₁₂・3H ₂ O] C% H% N% Calculated value 58.76 6.77 12.27 Measured value 58.84 6.68 11.87 P(29-38)Z(OMe)-Gln-Asp (OBzl) -
Val―His―Asn―Phe―Val―Ala―Leu―Gly
-OBzl

[9] [8] Add 1.26 ml of anisole and 5.04 ml of TFA to 2.55, and stir at 0°C for 1 hour. TFA is distilled off under reduced pressure, ether is added to the residue, and the resulting precipitate is collected. The obtained powder was dissolved in 6 ml of DMSO and 6 ml of DMF, and 0.54 ml of Et ₃ N was added to adjust the pH to 7.
Add 1.17 g of (OMe)-Gln-ONp and 0.38 ml of Et ₃ N and stir at room temperature for 2 days. The reaction solution was concentrated under reduced pressure, and 5% citric acid and ethyl acetate were added to the residue to collect the precipitate. Wash in the following order: 5% citric acid, water, 5% sodium bicarbonate solution, and water. Recrystallization from DMSO-MeOH yields 2.76 g (yield 98.6%) of the desired product. Melting point; 248-249°C [α] ²⁰ _D ; -24.6° (C = 1.1, DMSO) TLC; Rf ₂ = 0, Rf ₃ = 0 Amino acid analysis; Glu1.05(1), Asp1.95(2),
Gly0.99(1), Ala(1), Val1.98(2), Leu1.00(1),
Phe0.99(1), His0.89(1) Elemental analysis [as C ₇₂ H ₉₄ O ₁₈ N ₁₄・1/2H ₂ O] C% H% N% Calculated value 59.53 6.59 13.50 Measured value 59.58 6.71 13.20 P( 27-28) Z (OMe) - Lys (Z) - Leu -
OMe [10] Dissolve 2.91g of H-Leu-OMe in 50ml of DMF,
After cooling to 0℃ and adjusting the pH to 7 with 2.24ml of Et ₃ N, Z
Add a solution of 10 g of (OMe)-Lys(Z)-OTCP dissolved in 50 ml of THF and 2.24 ml of Et ₃ N, and stir 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 is washed with 5% citric acid, brine, 5% sodium bicarbonate, and brine in this order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Add ether to the residue to obtain crystals.
MeOH-7.33g of target product recrystallized from ether
(yield 80.1%). Melting point: 107-108℃ [α] ²⁰ _D : -21.2゜ (C = 0.9, MeOH) TLC: Rf ₂ = 0.73, Rf ₃ = 0.63 Elemental analysis [as C ₃₀ H ₄₁ O ₃ N ₈ ] C% H% N% Calculated value 63.03 7.23 7.35 Measured value 63.18 7.23 7.26 P (26-28) Z (OMe) - Lys (Z) - Lys (Z)
-Leu-OMe [11] [10] 5.72g, 2.17ml of anisole, 8.68ml of TFA
and stirred at 0°C for 1 hour. TFA is distilled off under reduced pressure, and the residue is washed three times with n-hexane and dried. Dissolve this in 20ml of DMF and adjust the pH with 1.4ml of Et ₃ N.
After adjustment to 7, Z (OMe) - Lys (Z) - OTCP6.24
g, 1.4 ml of Et ₃ N was added, and the mixture was stirred at room temperature for 20 hours. After the reaction, DMF is distilled off under reduced pressure, 5% citric acid and ether are added to the residue, and the resulting precipitate is collected. This precipitate is washed in the following order: 5% citric acid, water, 5% sodium bicarbonate solution, and water. Recrystallization from hot MeOH-ether gives 6.71 g (yield 80.5%) of the desired product. Melting point; 153-154℃ [α] ²⁰ _D ; -15.8゜ (C = 1.1, DMF) TLC; Rf ₁ = 0.41 Elemental analysis [as C ₄₄ H ₅₉ O ₁₁ N ₅ ] C% H% N% Calculated value 63.37 7.13 8.40 Measured value 63.54 7.21 8.38 P(25−28)Z(OMe)―Arg(Mts)―Lys
(Z)-Lys(Z)-Leu-OMe [12] [11] 6.00g, anisole 2.33ml, TFA 9.33ml
and stirred at 0°C for 1 hour. After TFA is distilled off under reduced pressure and ether is added to the residue, the resulting precipitate is collected and dried to obtain a powder. On the other hand, Z(OMe)-Arg(Mts)-OH・
6.71g of CHA with 20ml of ethyl acetate and 8.64ml of 1N hydrochloric acid
Mix with ml. After washing the organic layer with 20ml of water,
Concentrate under reduced pressure. Dissolve this residue in 20ml of THF,
Add 1.45 ml of Et ₃ N, add 1.36 ml of isobutyroxycarbonyl chloride at -10°C, and stir for 5 minutes. Dissolve the above powder in 20ml of DMF and add 1.00ml of Et ₃ N to this.
Add the solution adjusted to pH 7 and stir at 0°C for 4 hours. The reaction solution was concentrated under reduced pressure, and the residue was mixed with 5% citric acid and
Add ether and remove the resulting precipitate. Wash in the following order: 5% citric acid, 5% sodium bicarbonate solution, and water. DMF―
Recrystallization from ethyl acetate yielded 5.78 g of the target product (yield
68.5%). Melting point: 151-152℃ [α] ²⁰ _D : -8.9゜ (C = 0.7, DMF) TLC: Rf ₂ = 0.76, Rf ₃ = 0.75 Elemental analysis [as C ₅₉ H ₈₁ O ₁₄ N ₉ S] C% H % N% Calculated value 60.44 6.96 10.75 Measured value 60.48 6.97 10.65 P(25−28)Z(OMe)―Arg(Mts)―Lys
Dissolve 5.78 g of (Z)-Lys(Z)-Leu-NHNH ₂ [13] [12] in 50 ml of MeOH, add 1.23 ml of hydrazine hydrate, and let stand overnight at room temperature. Take the precipitated crystals and wash them with EtOH.
5.25g of target product recrystallized from MeOH-EtOH
(yield 91.3%). Melting point: 178-180℃ [α] ²⁰ _D : -10.1゜ (C = 0.7, DMF) TLC: Rf ₂ = 0.48 Amino acid analysis: Leu1(1), Lys1.94(2), Arg1.01
(1) Elemental analysis [as C ₅₈ H ₈₁ O ₁₃ N ₁₁ S] C% H% N% Calculated value 59.42 6.96 13.14 Measured value 59.29 7.17 12.87 P (23-24) Boc-Trp-Leu-OH・DCHA
[14] Dissolve 10.65 g of Boc-Trp-ONp in 30 ml of DMF. Add 3.9 g of H-Leu-OH to this at 0°C and add 10 g of water.
ml, dissolved in 30 ml of DMF and 6.95 ml of Et ₃ N, and stirred overnight at room temperature. The solvent was distilled off under reduced pressure,
The residue was mixed with 5% sodium bicarbonate solution and ethyl acetate. Acidify the aqueous layer with citric acid, add ethyl acetate, and mix. The organic layer is washed successively with 5% citric acid and water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Dissolve the residue in MeOH and add 5.45 g of DCHA.
Add. MeOH was distilled off under reduced pressure, and ether was added for crystallization. Recrystallization from MeOH-ether yields 10.15 g (yield 97.0%) of the desired product. Melting point: 192-195℃ [α] ²⁰ _D : -22.9゜ (C = 0.6, MeOH) TLC; Rf ₂ = 0.67, Rf ₃ = 0.69 Elemental analysis [C ₂₂ H ₃₁ O ₅ N ₃・C ₁₂ H ₂₃ N ] C% H% N% Calculated value 68.19 9.09 9.36 Measured value 68.07 9.14 9.27 P(22−24)Boc−Glu(OBzl)―Trp―Leu―
Suspend 10.0 g of OH・DCHA [15] [14] in 50 ml of ethyl acetate and 50 ml of water, and add 17.1 ml of 1N hydrochloric acid at 0°C. After removing the precipitated salt, the organic layer is washed with brine and dried over anhydrous sodium sulfate. Ethyl acetate was distilled off under reduced pressure to obtain an oil. Add 5.57 ml of anisole containing 2% EDT and 22.28 ml of TFA to this, and
Stir for 1 hour under nitrogen gas. TFA is distilled off under reduced pressure, washed three times with n-hexane, and then dried.
Dissolve this in 50ml of DMF, Boc-Glu(OBzl)-
Add 7.43 g of OSu and 4.79 ml of Et ₃ N, and stir 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 is acidified with citric acid and the resulting oil is extracted with ethyl acetate. The ethyl acetate layer is washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Dissolve the residue in MeOH, add 2.8 ml of DCHA, concentrate MeOH under reduced pressure, and add ether to crystallize.
MeOH-Recrystallized three times from ether to obtain the desired product
Obtain 7.84 g (yield 57.0%). Melting point: 149-150℃ [α] ²⁰ _D : -23.1゜ (C=1.3, MeOH) TLC: Rf ₂ = 0.53, Rf ₃ = 0.71 Amino acid analysis ² ; Trp0.76(1), Glu1.12(1) , Leu1
(1) Elemental analysis [as C ₃₄ H ₄₄ O ₈ N ₄・C ₁₂ H ₂₃ N] C% H% N% Calculated value 67.53 8.26 8.56 Measured value 67.23 8.28 8.69 P(20-21)Z(OMe)-Arg (Mts) -Val-
NHNHTroc [16] Add 10.74 ml of anisole and 42.96 ml of TFA to 22.79 g of Z(OMe)-Val-NHNHTroc and stir for 1 hour at 0°C. 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)-OH・
Suspend 30.0 g of CHA in 150 ml of ethyl acetate and 100 ml of water, and add 48.41 ml of 1N hydrochloric acid at 0°C. After removing the precipitated salt, the organic layer is washed with brine and dried over anhydrous sodium sulfate. Ethyl acetate was distilled off under reduced pressure to obtain an oil. Dissolve this in 100 ml of THF, add 81.3 ml of Et ₃ N, add 7.53 ml of isobutyroxycarbonyl chloride at -10°C, and stir for 5 minutes. Dissolve the above oil in 50ml of DMF,
Add a solution adjusted to pH 7 with 6.78 ml of Et ₃ N, and store at -10°C.
Stir for 2 hours at room temperature and 16 hours at room temperature. The reaction solution was concentrated under reduced pressure, and the residue was mixed with ethyl acetate and 5% citric acid. The organic layer is 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 purified by silica gel column chromatography [eluent: CHCl ₃ -MeOH (20:0.5)]. Crystallization from ethyl acetate-n-hexane yields 29.25 g (yield 74.7%) of the desired product. Melting point: 114-116°C [α] ²⁰ _D : -24.6° (C = 1.1, MeOH) TLC: Rf ₂ = 0.62, Rf ₃ = 0.46 Elemental analysis [as C ₃₂ H ₄₄ O ₉ N ₇ SCl ₃ ] C% H% N% Calculated value 47.50 5.48 12.12 Measured value 47.24 5.43 11.88 P (19-21) Boc―Glu(OBzl)―Arg(Mts)―
Val―NHNHTroc [17] [16] 7.30g and anisole 3.89ml,
Add 15.57 ml of TFA and stir at 0°C for 1 hour. TFA
After distilling off under reduced pressure and adding ether to the residue, the resulting precipitate is collected and dried to obtain a powder. Dissolve this in 50ml of DMF, adjust the pH to 7 with 12.6ml of Et ₃ N, and add 4.13g of Boc-Glu(OBzl)-ONp.
Add 12.6 ml of Et ₃ N and stir at room temperature for 16 hours. The solvent was distilled off under reduced pressure, 5% citric acid and ether were added to the residue, and the resulting precipitate was collected. Wash in the following order: 5% citric acid, water, 5% sodium bicarbonate, water, and MeOH
-Recrystallized from ether to yield 6.29g of the desired product (yield
72.5%). Melting point: 159-160℃ [α] ²⁰ _D : -31.9゜ (C = 1.1, MeOH) TLC: Rf ₂ = 0.68, Rf ₃ = 0.74 Elemental analysis [as C ₄₀ H ₅₇ O ₁₁ N ₈ SCl ₃ ] C% H% N% Calculated value 49.82 5.96 11.62 Measured value 49.85 5.97 11.60 P(19-21) Boc―Glu(OBzl)―Arg(Mts)―
Dissolve 6.29 g of Val-NHNH ₂ [18] [17] in 15 ml of acetic acid, add 4.25 g of zinc powder
g 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 to collect the resulting precipitate. Wash with saturated EDTA aqueous solution and then with water. MeOH-Recrystallized from ether to obtain the desired product
Obtain 2.64 g (yield 51.3%). Melting point; 186-187℃ [α] ²⁰ _D ; -5.6゜ (C = 0.7, DMF) TLC; Rf ₂ = 0.58, Rf ₃ = 0.55 Amino acid analysis ¹ ; Glu1.20(1), Leu(1), Arg1 .07
(1) Elemental analysis [as C ₃₇ H ₅₆ O ₉ N ₈ S・1/2H ₂ O] C% H% N% Calculated value 55.69 7.20 14.04 Measured value 55.73 7.18 14.17 P(17−18)Z(OMe)− Ser−Met(O)−OMe
[19] Add 5.71 ml of anisole and 22.84 ml of TFA to 9.02 g of Z(OMe)-Met(O)-OMe and stir 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. Z(OMe)―Ser―NHNH ₂ 8.95ml 5.55N―HCl/DMF 13.66ml Isoamyl nitrite 5.05ml DMF 20ml Et ₃ N 15.92ml Z(OMe)―Ser―NHNH ₂ in the same manner as the previous method [5] In the azide solution obtained above, the oil obtained above was dissolved in 40 ml of DMF and mixed with 3.68 ml of Et ₃ N.
Add solution adjusted to pH 7. Stir for 2 days at 4°C. DMF was distilled off under reduced pressure and the residue was converted into n-BuOH
The solution was washed with 5% citric acid, brine, and 5% sodium bicarbonate in this order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained oil was subjected to silica gel column chromatography [eluent: CHCl ₃ -MeOH]
(10:05)]. Crystallization from MeOH-ethyl acetate yields 7.22 g (yield 63.8%) of the desired product. Melting point: 109-110℃ [α] ²⁰ _D : -36.4゜ (C = 1.0, DMF) TLC: Rf ₂ = 0.6, Rf ₃ = 0.25 Elemental analysis: [as C ₁₈ H ₂₆ O ₈ N ₂ S] C% H% N% Calculated value 50.22 6.09 6.51 Measured value 50.31 6.05 6.43 P (16-18) Boc―Asn―Ser―Met(0)―
OMe [20] [19] 7.20g, anisole 3.63ml, TFA14.54
ml and stirred at 0°C for 1 hour. After TFA is distilled off under reduced pressure and ether is added to the residue, the resulting precipitate is collected and dried. The obtained powder is DMF25
After adjusting the pH to 7 with 2.34 ml of Et ₃ N,
Add Boc―Asn―O Np5.91g, Et ₃ N2.34ml,
Stir at room temperature for 18 hours. DMF was distilled off under reduced pressure,
After adding ethyl acetate to the residue, the precipitated crystals are collected and washed five times with ethyl acetate. Recrystallization from MeOH-ethyl acetate yielded 6.44 g of the target product (yield
80.1%). Melting point: 181-182℃ [α] ²⁰ _D ; -49.3゜ (C = 0.8, DMF) TLC; Rf ₂ = 0.46, Rf ₃ = 0.06 Elemental analysis [as C ₁₈ H ₃₂ O ₉ N ₄ S] C% H % N% Calculated value 44.99 6.71 11.66 Measured value 45.10 6.83 11.45 P(16−18)Boc−Asn−Ser−Met(0)—
Dissolve 5.25 g of NHNH ₂ [21] [20] in 30 ml of MeOH, add 2.73 ml of hydrazine hydrate, and let stand overnight at room temperature. Take the precipitated crystals and wash them with EtOH.
Recrystallized from DMSO-MeOH to obtain 4.73g of target product
(yield 90.4%). Melting point 218-219℃ [α] ²⁰ _D −27.0゜ (C=1.0, DMSO) TLC; Rf ₂ = 0.06, Rf ₄ = 0.49 Amino acid analysis ¹ ; Asp1(1), Ser0.88(1), Met+
Met(0)0.75(1) Elemental analysis [as C ₁₇ H ₃₂ O ₈ N ₆ S・1/2H ₂ O] C% H% N% Calculated value 41.71 6.79 17.17 Measured value 41.64 6.59 17.07 P (14-15) Z(OMe)-His-Leu-OMe [22] Z(OMe)-His-NHNH ₂ 20.00g 4.78N-HCl/DMF 45.21ml Isoamyl nitrite 9.58ml DMF 20ml Ht ₃ N 40.32ml Z(OMe)-His -H-Leu was added to the azide solution obtained by adding NHNH ₂ in the same manner as in the previous method [5].
-Dissolve 9.08g of OMe・Hcl in 50ml of DMF,
After adding a solution adjusted to pH 7 with 7.00 ml of Et ₃ N,
Stir at 4°C for 20 hours. DMF was distilled off under reduced pressure,
The residue is triturated with ethyl acetate and 5% aqueous sodium bicarbonate. The organic layer is washed with 5% multistory water and brine in that order, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was crystallized by adding ether and MeOH
-Recrystallize from ether to obtain 16.32 g (yield 73.1%) of the desired product. Melting point: 110-111℃ [α] ²⁰ _D : -24.1゜ (C = 1.1, MeOH) TLC: Rf ₂ = 0.65, Rf ₃ = 0.17 Elemental analysis [as C ₂₂ H ₃₀ O ₆ N ₄ ] C% H% N% Calculated value 59.18 6.77 12.55 Measured value 58.93 6.70 12.32 P(13−15)Boc―Lys(Z)―His―Leu―
OMe [23] [22] 5.11g, anisole 2.49ml, TFA 9.96ml
and stirred at 0°C for 1 hour. After TFA is distilled off under reduced pressure and ether is added to the residue, the resulting precipitate is collected and dried. The obtained powder was added to DMF20
ml, add 3.19ml of Et ₃ N and adjust the pH to 7,
Add Boc-Lys(Z)-OSu and stir at room temperature for 20 hours. DMF was distilled off under reduced pressure, and the residue was mixed with ethyl acetate and 5% aqueous sodium bicarbonate. The organic layer is 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 obtain 5.42 g of the target product (yield 73.7
%). Melting point; 144-147℃ [α] ²⁰ _D ; -33.6゜ (C = 0.7, MeOH) TLC; Rf ₂ = 0.65 Elemental analysis [as C ₃₂ H ₄₈ O ₈ N ₆ ] C% H% N% Calculated value 59.61 7.50 13.04 Measured value 59.61 7.45 13.03 P(13−15)Boc―Lys(Z)―His―Leu―
Dissolve 2.65 g of NHNH ₂ [24] [23] in 20 ml of MeOH, add 1.03 ml of hydrazine hydrate, and let stand at room temperature for 2 days.
After removing MeOH under reduced pressure and adding water to the residue,
Collect the resulting precipitate. Recrystallization from MeOH-ethyl acetate yields 2.10 g (yield 79.3%) of the desired product. Melting point; 167-169℃ [α] ²⁰ _D ; -21.4゜ (C = 1.0, DMF) TLC; Rf ₂ = 0.48 Amino acid analysis ¹ ; Leu1(1), Lys0.97(1), His0.91
(1) Elemental analysis [as C ₃₁ H ₄₈ O ₇ N ₈ ] C% H% N% Calculated value 57.74 7.50 17.38 Measured value 57.58 7.52 17.38 P(11-12)Z(OMe)-Leu-Gly-OMe[25 ] H-Gly-O Me・HCl12.56g in DMF/50ml
After adjusting the pH to 7 with 14 ml of Et ₃ N,
Add 41.64 g of (OMe)-Leu-ON and 14 ml of Et ₃ N, and stir at room temperature for 18 hours. DMF was distilled off under reduced pressure and the residue was dissolved in ethyl acetate. Wash with 5% citric acid, 5% sodium bicarbonate solution, and brine in this order, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. The residue was crystallized by adding ether, and then recrystallized from ethyl acetate-ether to obtain 28.98 g of the desired product (yield 79.1%).
get. Melting point; 78-79℃ [α] ²⁵ _D ; 13.1゜ (C = 0.7, DMF) TLC; Rf ₂ = 0.71 Elemental analysis [as C ₁₈ H ₂₆ O ₆ N ₂ ] C% H% N% Calculated value 59.00 7.15 7.65 Measured value 59.17 7.16 7.59 P(10−12)Z(OMe)―Asn―Leu―Gly―
OMe〔26〕〔25〕22.36g and anisole 13.26ml,
Add 53.04 ml of TFA and stir at 0°C for 1 hour. TFA
is distilled off under reduced pressure, and the residue is washed three times with n-hexane and dried. Dissolve this in 100ml of DMF,
After adjusting the pH to 7 with 8.54ml of Et ₃ N, Z (OMe) -
Add 25.46 g of Asn-ONp and 8.54 ml of Et ₃ N and stir at room temperature.
Stir for 20 hours. DMF is distilled off under reduced pressure, 5% citric acid and ethyl acetate are added to the residue, and the resulting precipitate is collected. The product is washed with 5% citric acid, 5% aqueous sodium bicarbonate, and water in this order, and recrystallized from DMF-ethyl acetate to obtain 22.42 g (yield: 76.5%) of the desired product. Melting point: 196-197℃ [α] ²² _D : -15.4゜ (C = 0.8, DMF) TLC: Rf ₂ = 0.56, Rf ₃ = 0.33 Elemental analysis [as C ₂₂ H ₃₂ O ₈ N ₄ ] C% H% N% Calculated value 54.99 6.71 11.66 Measured value 55.14 6.86 11.60 P(9-12)Z(OMe)―His―Asn―Leu―
Gly-OMe [27] [26] 14.03g, anisole 9.52ml, TFA38.08
ml and stirred at 0°C for 1 hour. After TFA is distilled off under reduced pressure and ether is added to the residue, the resulting precipitate is collected and dried to obtain a powder. Z(OMe)-His-NHNH ₂ 11.67g 5.55N-HCl/DMF 22.70ml Isoamyl nitrite 5.59ml DMF 20ml Et ₃ N 22.54ml Z(OMe)-His-NHNH ₂ in the same manner as the previous method [5] In the azide solution obtained above, dissolve the powder obtained above in 70 ml of DMF, and adjust the pH to 7 with 4.09 ml of Et ₃ N.
Add the adjusted solution. Stir at 4°C for 20 hours. DMF was distilled off under reduced pressure, and the residue was mixed with 5% sodium bicarbonate solution and
After adding ether, remove the resulting precipitate. 5
% sodium bicarbonate solution and then water, and recrystallized from DMF-MeOH to obtain 15.13 g (yield: 83.9%) of the desired product. Melting point; 226-227℃ [α] ²² _D ; -11.1゜ (C = 0.8, DMF) TLC; Rf ₂ = 0.37 Elemental analysis [as C ₂₈ H ₃₂ O ₉ N ₄ ] C% H% N% Calculated value 54.45 6.36 15.88 Measured value 54.31 6.38 15.73 P(8-12)Boc-Met(0)-His-Asn-Leu
-Gly-OMe [28] [27] 10.00g, anisole 5.28ml, TFA21.12
ml and stirred at 0°C for 1 hour. After distilling off TFA under reduced pressure and adding ether, the resulting precipitate is collected and dried to obtain a powder. On the other hand, add 5.16 g of Boc-Met (0)-OH to 30 ml of THF.
Add 3.26 ml of Et ₃ N, add 3.06 ml of isobutyroxycarbonyl chloride at -10°C, and stir for 5 minutes. Dissolve the above powder in 50ml of DMF,
Add a solution adjusted to pH 7 with 4.57 ml of Et ₃ N, and cool at 0°C.
Stir for 4 hours. Filter the reaction solution and concentrate under reduced pressure. After adding EtoH to the residue, collect the resulting precipitate. Recrystallize three times from DMF-EtoH to obtain 6.39 g (yield 56.3%) of the desired product. Melting point; 218-219℃ [α] ²² _D ; -11.4゜ (C = 0.9, DMF) TLC; Rf ₂ = 0.22 Elemental analysis [as C ₂₉ H ₄₈ O ₁₀ N ₈ S] C% H% N% Calculated value 49.70 6.90 15.99 Measured value 49.74 6.80 15.74 P(8-12)Boc-Met(0)-His-Asn-Leu
Dissolve 5.21 g of -Gly-NHNH ₂ [29] [28] in 30 ml of DMF and 20 ml of MeOH, add 1.86 ml of hydrazine hydrate, and stir at room temperature for 20 hours. After evaporating the solvent under reduced pressure and adding EtOH, the resulting precipitate was collected. Recrystallization from DMF-MeOH yields 4.39 g (yield 84.2%) of the desired product. Melting point: 197-198℃ [α] ¹⁸ _D : -19.7゜ (C = 1.0, DMSO) TLC: Rf ₂ = 0.13 Amino acid analysis: Asp1.02(1), Gly1(1), Met+
Met (0) 0.82 (1), Leu 1.03 (1), His 0.98 (1) Elemental analysis [as C ₂₈ H ₄₈ O ₉ N ₁₀ S] C% H% N% Calculated value 47.98 6.90 19.99 Measured value 47.72 6.84 19.70 P(6-7)Z(OMe)-Gln-Leu-
NHNHTroc [30] Add 8.96 ml of anisole and 35.84 ml of TFA to 20.00 g of Z(OMe)-Leu-NHNHTroc and stir for 1 hour at 0°C. TFA is distilled off under reduced pressure, and the residue is washed with n-hexane and dried. Dissolve this in 120 ml of DMF, add 11.52 ml of Et ₃ N and Z(OMe)-Gln-ONp, and stir at room temperature for 18 hours. DMF was distilled off under reduced pressure, 5% citric acid and ethyl acetate were added to the residue, and the resulting precipitate was collected. 5% citric acid, 5
% sodium bicarbonate solution and then water, and recrystallized from DMF-ethyl acetate to obtain 20.70 g (yield: 82.0%) of the target product.
get. Melting point: 188-190℃ [α] ²⁰ _D : -17.1゜ (C = 1.0, DMF) TLC: Rf ₂ = 0.65, Rf ₃ = 0.44 Elemental analysis [as C ₂₃ H ₃₂ O ₈ N ₅ Cl ₃ ] C% H% N% Calculated value 45.07 5.26 11.43 Measured value 45.52 5.41 11.52 P(5-7)Z(OMe)―Ile―Gln―Leu―
NHNHTroc [31] [30] 20.70g, anisole 11.00ml, TFA44.0
ml and stirred at 0°C for 1 hour. After distilling off TFA under reduced pressure and washing the residue three times with n-hexane,
dry. Dissolve this in 200ml of DMF,
Add 9.48 ml of Et ₃ N and 14.09 g of Z(OMe)-Ile-ONp, and stir at room temperature for 18 hours. DMF was distilled off under reduced pressure, 5% citric acid and ethyl acetate were added to the residue, and the resulting precipitate was collected. 5% citric acid, 5
% sodium bicarbonate solution and then water, and recrystallized from DMF-ethyl acetate to obtain 17.43 g of the target product (yield 79.1%).
get. Melting point: 205-207℃ [α] ²⁰ _D : -15.3゜ (C = 0.9, DMF) TLC: Rf ₂ = 0.62, Rf ₃ = 0.32 Elemental analysis [as C ₂₉ H ₄₃ O ₉ N ₆ Cl ₃ ] C% H% N% Calculated value 47.97 5.97 11.58 Measured value 48.16 6.17 11.66 P(4-7)Boc−Glu(OBzl)−Ile−Gln−Leu
-NHNHTroc [32] [31] 15.0g, anisole 8.99ml, TFA35.69
ml and stirred at 0°C for 1 hour. After TFA is distilled off under reduced pressure and ether is added to the residue, the resulting precipitate is collected and dried. Dissolve this in DMF50ml, _Et3N5.80ml , Boc-Glu(OBzl)-OSu8.99
g and stirred at room temperature for 20 hours. DMF was distilled off under reduced pressure, 5% citric acid and ether were added to the residue, and the resulting precipitate was collected. 5% citric acid,
Wash with 5% sodium bicarbonate solution and then water. DMF―MeOH
15.89g of target product (yield 87.1%) was recrystallized from
get. Melting point: 223-225℃ [α] ¹⁸ _D ; -26.3゜ (C = 0.6, DMSO) TLC; Rf ₂ = 0.59, Rf ₃ = 0.49 Elemental analysis [as C ₃₇ H ₅₆ O ₁₁ N ₇ Cl ₃ ] C% H% N% Calculated value 50.43 6.41 11.13 Measured value 50.33 6.52 11.63 P(4-7) Boc―Glu(OBzl)―Ile―Gln―
Dissolve 9.0 g of Leu-NHNH ₂ [33] [32] in 40 ml of DMF, 6.81 ml of acetic acid,
Add 6.67 g of zinc powder and stir at room temperature for 18 hours. After removing the zinc dust, concentrate the liquid under reduced pressure to obtain a saturated residue.
After adding the EDTA solution, add baking soda to adjust the pH to 7 to form a precipitate. saturate this precipitate
Washed with EDTA solution and water in that order, and recrystallized twice from DMF-MeOH to obtain 5.31 g of the target product (yield 73.7%).
get. Melting point: Decomposes above 260°C [α] ¹⁸ _D : -22.3° (C = 0.6, DMSO) TLC: Rf ₂ = 0.48 Amino acid analysis ¹ : Glu2.06(2), Ile1.01(1), Leu1(1) ) Elemental analysis [as C ₃₄ H ₅₅ O ₉ N ₇ ] C% H% N% Calculated value 57.85 7.85 13.89 Measured value 57.76 8.00 13.64 P(2-3)Z(OMe)-Val-Ser-OMe[34] H ―Ser―Dissolve 15.56g of OMe in 80ml of DMF,
Adjust the pH to 7 with 14.0ml of Et ₃ N. Z in this solution
After adding a solution of 28.13 g of (OMe)-Val-OH dissolved in 140 ml of THF, 24.76 g of DCC was added at -10°C 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 collected with EtOH. Recrystallization from THF-EtOH yields 22.19 g (yield 58.0%) of the desired product. Melting point; 160-161℃ [α] ²⁰ _D ; 9.9゜ (C = 0.9, DMF) TLC; Rf ₂ = 0.63, Rf ₃ = 0.64 Elemental analysis [as C ₁₈ H ₂₆ O ₇ N ₂ ] C% H% N % Calculated value 56.53 6.85 7.33 Measured value 56.70 6.96 7.36 P (1-3) Z-Ser-Val-Ser-OMe [35] [34] 7.15g, anisole 4.06ml, TFA16.24
ml and stirred at 0°C for 1 hour. After distilling off TFA under reduced pressure and washing the residue three times with n-hexane,
Dry to obtain an oil. Z-Ser-NHNH ₂ 5.68g 5.55N-HCl/DMF 9.70ml Isoamyl nitrite 3.58ml DMF 10ml Et ₃ N 11.31ml Azide solution obtained from Z-Ser-NHNH ₂ in the same manner as in the previous method [5] Then, a solution of the oil obtained above in 35 ml of DMF and adjusted to pH 7 with 2.62 ml of Et ₃ N is added. Stir for 2 days at 4°C. After removing the precipitate and concentrating the liquid under reduced pressure, the residue
Add EtOH. Remove the resulting precipitate and place it in DMF.
Recrystallized from MeOH to yield 4.94 g of the target product (yield 60.1
%). Melting point; 211-213℃ [α] ²⁰ _D ; 6.4゜ (C = 0.9, DMF) TLC; Rf ₂ = 0.66 Elemental analysis [as C ₂₀ H ₂₉ O ₈ N ₃ ] C% H% N% Calculated value 54.66 6.65 9.56 Measured value 54.75 6.52 9.45 P(1-3)Z―Ser―Val―Ser―NHNH ₂
[36] [35] Dissolve 3.27 g in 20 ml of DMF and 10 ml of MeOH, add 1.86 ml of hydrazine hydrate, and leave overnight. The precipitated crystals were collected with MeOH, washed with EtOH, and then recrystallized from DMSO-MeOH to obtain the desired product.
Obtain 2.65 g (yield 81.3%). Melting point; 240℃ [α] ²² _D ; −3.4° (C = 0.6, DMSO) TLC; Rf ₂ = 0.44 Amino acid analysis ¹ ; Ser1.83(2), Val1(1) Elemental analysis [C ₁₉ H ₂₉ O ₂ As N ₅ ] C% H% N% Calculated value 51.92 6.65 15.94 Measured value 51.63 6.65 15.98 P (25-38) Z (OMe) - Arg (Mts) - Lys
(Z)―Lys(Z)―Leu―Gln―Asp(OBzl)―
Val―His―Asn―Phe―Val―Ala―Leu―Gly
―OBzl〔37〕

[9] 2.76g with 1.65ml of anisole and 6.60ml of TFA
and stir at -10℃ for 60 minutes. After the reaction is complete, TFA is distilled off under reduced pressure, ether is added to the residue, and the resulting powder is separated and dried under reduced pressure. [13] 2.69g 4.78NHCl/DMF 1.16ml Isoamyl nitrite 0.38ml DMF 10ml Et ₃ N 0.31ml [13] was prepared in the same manner as in the previous [5], and the obtained dry powder was added to the obtained azide solution. DMF,
Add a solution of 0.54 ml of Et ₃ N dissolved in 5 ml of DMSO each and stir at 4° C. for 48 hours. A few drops of acetic acid is added to the reaction solution in which a precipitate has been deposited to neutralize it. The solvent was distilled off under reduced pressure, and ethyl acetate and 5% citric acid water were added to the residue to obtain a powder. After suspending and washing this powder in the order of 5% citric acid solution, 5% sodium bicarbonate solution, and water,
Recrystallize with DMSO-MeOH to obtain 3.50 g. Melting point: decomposes above 260℃ [α] ²⁰ _D ; -15.9゜(C=0.6, DMSO) TLC;
Rf ₄₅ = 0 Elemental analysis [as C ₁₂₁ H ₁₆₃ O ₂₈ N ₂₃ S1.5H ₂ O] C% H% N% Calculated value 59.39 6.84 13.17 Measured value 59.29 6.83 13.23 Amino acid analysis ¹ ; Leu1.92(2), Lys2 .00(2),
Arg1.04(1), Asp2.03(2), Glu1.11(1), Gly0.99(1),
Ala1.00(1), Val1.82(2), Phe0.95(1), His0.90(1), P(22−38) Boc―Glu(OBzl)―Trp―Leu―
Arg(Mts)-Lys(Z)-Lys(Z)-Leu-Gln
-Asp(OBzl)-Val-His-Asn-Phe-Val
-Ala-Leu-Gly-OBzl [38] [37] 3.50g with 1.58ml of anisole and 6.32ml of TFA
and stir at 0°C for 60 minutes. After the reaction is completed, TFA is distilled off under reduced pressure, and the residue is washed with hexane to obtain an oily substance. Add ether and 5% sodium bicarbonate solution to this to obtain a powder. This powder is suspended and washed in the order of 5% sodium bicarbonate solution and water, and then dried under reduced pressure. [15] 2.33g, 10ml of ethyl acetate and 10ml of 0.2N hydrochloric acid
Add ml and mix. The organic layer is separated, washed with water, dried over anhydrous sodium sulfate, the desiccant is removed, and ethyl acetate is distilled off. To the resulting residue, 10 ml each of DMSO and DMF are added and dissolved. This is combined with the previously obtained dry powder.
After adding and dissolving 0.54 g of HOBT, 0.72 g of DCC was added and stirred at 4°C for 72 hours. The reaction solution in which a precipitate was deposited was concentrated under reduced pressure, and 5% citric acid water and ether were added to the resulting oily residue to obtain a powder. This powder was suspended and washed in the order of 5% citric acid solution, 5% sodium bicarbonate solution, and water, then dissolved in DMSO and left overnight. After removing insoluble matter, recrystallization is performed with DMSO-MeOH to obtain 2.85 g (yield 68.5%). Melting point: Decomposes above 260°C [α] ²⁰ _D : -19.8° (C = 0.8, DMSO) TLC: Rf ₂₃ = 0 Amino acid analysis ² : Glu1.98(2), Leu2.84(3),
Trp0.74(1), Asp1.97(2), Gly1.02(1), Ala1.00(1),
Val1.67(2), Phe0.93(1), Lys2.00(2), His0.78(1),
Arg1.02 Elemental analysis [as C ₁₄₆ H ₁₉₇ O ₃₂ N ₂₇ S・7H ₂ O] C% H% N% Calculated value 58.43 6.83 12.61 Measured value 58.44 7.09 12.58 P(19−38) Boc―Glu(OBzl)― Arg (Mts) -
Val―Glu(OBzl)―Trp―Leu―Arg(Mts)―
Lys(Z)―Lys(Z)―Leu―Gln―Asp(OBzl)
―Val―His―Asn―Phe―Val―Ala―Leu―
Gly-OBzl [39] [38] Anisole 2.13ml containing 2% EDT in 2.81g
Add 8.52 ml of TFA and stir at -10°C for 60 minutes. TFA is distilled off under reduced pressure, and hexane is added to the residue to obtain an oil. Ether is added to this and the resulting powder is dried under reduced pressure. [18] 1.54g 5.55NHCl/DMF 0.85ml Isoamyl nitrite 0.31ml Et ₃ N 0.66ml DMF 3.0ml Et ₃ N 0.27ml Add [18] to the obtained azide solution in the same manner as in the previous [5] method. , the dry powder obtained earlier
Dissolved in DMF, DMSO each 10ml solution, Et ₃ NO, 27
ml solution and stir at 4°C for 48 hours.
Add several drops of acetic acid to the reaction solution to neutralize it, and then concentrate under reduced pressure. 5% citric acid water and ethyl acetate are added to the obtained residue to obtain a powder. After suspending and washing this powder in the order of 5% citric acid solution, 5% sodium bicarbonate solution, and water,
Recrystallize with DMSO-MeOH to obtain 3.01 g. Melting point; 165-168°C [α] ²⁰ _D ; 25.3° (C = 0.4, DMF) TLC; Rf ₂ = 0 Amino acid analysis; Glu2.97(3), Val2.79(3),
Arg1.89(2), Asp1.94(2), Gly1.00(1), Ala1.00(1),
Leu2.96(3), Phe1.00(1), Lys1.92(2), His1.02(1),
Trp0 (not measured) (1) Elemental analysis [as C ₁₇₈ H ₂₄₁ O ₃₉ N ₃₃ S ₂・11H ₂ O] C% H% N% Calculated value 57.32 7.11 12.40 Measured value 56.98 6.65 12.92 P (16-38) Boc―Asn―Ser―Met(O)―Glu
(OBzl) - Arg (Mts) - Val - Glu (OBzl) -
Trp－Leu－Arg(Mts)－Lys(Z)－Lys(Z)
―Leu―Gln―Asp(OBzl)―Val―His―Asn
-Phe-Val-Ala-Leu-Gly-OBzl [40] [39] 2.95g contains 2% EDT anisole 2.27ml
Add 9.08 ml of TFA and stir at -10°C for 60 minutes under a nitrogen stream. TFA is distilled off under reduced pressure, and hexane is added to the residue to obtain an oily substance. Ether is added to this, and the resulting powder is dried under reduced pressure. [21] 0.80g 5.55NHCl/DMF 0.72ml Isoamyl nitrite 0.27ml Et ₃ N 0.56ml DMF 3.00ml Et ₃ N 0.28ml Add [21] to the solution obtained in the same manner as in the previous [5] method. The dry powder obtained in DMF,
A solution of 5 ml each of DMSO and 0.23 ml of Et ₃ N was added thereto, and the mixture was stirred at 4° C. for 48 hours. The reaction solution was neutralized with acetic acid, and then concentrated under reduced pressure. Ether and 5% citric acid water were added to the resulting oily residue to obtain a powder. This powder is suspended and washed in the following order: 5% citric acid solution, 5% sodium bicarbonate solution, and water, and then recrystallized from DMSO-ethyl acetate. Divide this into three times, dissolve it in DMSO, and add it to a Sephadex LH-60 column (6x
150 cm, DMSO) and elute with DMSO.
The eluate was fractionated into 15 ml portions, and the main eluate portion (95 bottles ~
(116 bottles) are collected, DMSO is distilled off, and ethyl acetate is added to crystallize. Process the other two parts in the same way to obtain a total of 2.40 g. Melting point; 160-161°C [α] ²⁰ _D ; -9.9° (C = 0.8, DMF) TLC; Rf ₂ = 0.45 Amino acid analysis ² ; Asp2.91(3), Ser0.81(1),
Met0.11, Met(0)0.61(1), Glu3.12(3), Gly
(3), Gly1.01(1), Ala1(1), Val2.85(3), Leu3.02
(3), Phe0.99(1), Lys2.01(2), His0.93(1),
Arg2.03(2) Elemental analysis [as C ₁₉₀ H ₂₆₁ O ₄₅ N ₃₇ S ₃・10H ₂ O] C% H% N% Calculated value 56.21 6.98 12.77 Measured value 56.18 6.65 13.27 P (13−38) Boc−Lys (Z)-His-Leu-Asn
-Ser-Met(O)-Glu(OBzl)-Arg(Mts)
―Val―Glu(OBzl)―Trp―Leu―Arg(Mts)
―Lys(Z)―Lys(Z)―Leu―Gln―Asp
(OBzl)-Val-His-Asn-Phe-Val-Ala
-Leu-Gly-OBzl [41] [40] 2.37g contains 2% EDT anisole 1.33ml
Add 5.32 ml of TFA and stir at -10°C for 60 minutes. TFA is distilled off under reduced pressure, hexane is added to the residue, ether is added to the obtained oily substance, and the obtained precipitate is dried. [24] 0.79g 5.55NHCl/DMF 0.79ml Isoamyl nitrite 0.19ml Et ₃ N 0.61ml DMF 2.0ml Et ₃ N 0.20ml Add [24] to the obtained azide solution in the same manner as in the previous [5] method. , the dry powder obtained earlier
A solution prepared by dissolving in 15 ml of DMF and adding 0.17 ml of Et ₃ N is added, and the mixture is stirred at 4° C. for 24 hours. After neutralizing the reaction solution with acetic acid, it is concentrated under reduced pressure. 5% to the resulting oily residue
Add citric acid water and ether to obtain a viscous precipitate. This is suspended and washed three times with 5% sodium bicarbonate solution and then with water to obtain a powder. This was reprecipitated five times with DMF-ethyl acetate to obtain 2.22 g. Melting point; 149-151℃ [α] ²⁰ _D ; 1.3゜ (C = 0.8, DMF) TLC; Rf ₂ = 0.59 Amino acid analysis ¹ ; Leu4.18(4), His1.87(2),
Lys3.48(3), Asp2.91(3), Ser0.79(1), Glu3.11
(3), Gly1.08(1), Ala1(1), Val2.91(3), Met0.82
(1), Phe0.96(1), Trp0 (not measured) (1), Arg1.61
(2) Elemental analysis [as C ₂₁₆ H ₂₉₇ O ₅₀ N ₄₃ S ₃・6H ₂ O] C% H% N% Calculated value 57.65 6.92 13.38 Measured value 57.35 6.86 13.69 P(8-38) Boc-Met(O) ―His―Asn―Leu
―Gly―Lys(Z)―His―Leu―Asn―Ser―
Met(O)-Glu(OBzl)-Arg(Mts)-Val-
Glu(OBzl)-Trp-Leu-Arg(Mts)-Lys
(Z)―Lys(Z)―Leu―Gln―Asp(OBzl)―
Val―His―Asn―Phe―Val―Ala―Leu―Gly
-OBzl [42] [41] 1.57g contains 2% EDT anisole 1.17ml
Add 4.68 ml of TFA and stir at -10°C for 60 minutes. Hexane was added to the residue after TFA was distilled off under reduced pressure.
Obtain an oily substance. Ether is added to this and the resulting powder is dried under reduced pressure. [29] 0.50ml 5.55NHCl/DMF 0.46ml Isoamyl nitrite 0.12ml Et ₃ N 0.36ml DMF 5.0ml Et ₃ N 0.12ml Add [29] to the obtained azide solution in the same manner as in the previous [5] method. , the dry powder obtained earlier
Add a solution of 0.15 ml of Et ₃ N dissolved in 15 ml of DMF, and stir at 4° C. for 48 hours. The same amount of the azide solution obtained from [29] was further added to this reaction solution,
Stir for 18 hours. This reaction solution was neutralized with acetic acid and concentrated under reduced pressure. To the resulting oily residue were added 3% aqueous acetic acid and ether to form a powder. After suspending and washing this powder in the order of 3% acetic acid, water, 5% sodium bicarbonate, and water,
Re-precipitate three times with DMF-ethyl acetate, apply to a silica gel column (2 x 14 cm), elute with solvent system (2), collect the fraction with Rf ₂ = 0.46, concentrate,
Add ethyl acetate to obtain 872 mg of precipitate. Melting point; 158-160℃ [α] ¹⁸ _D ; -20.8゜ (C = 0.2, DMSO) TLC; Rf ₂ = 0.46 Amino acid analysis ¹ ; Asp3.87(4), Gly1.94(2), Met
+Met(0)1.27(2), Leu4.67(5), His2.16(3),
Ser0.62(1), Glu3.16(3), Ala1(1), Val3.08(3),
Phe0.97(1), Lys2.84(3), Arg1.91(2), Trp0 (not measured)(1) Elemental analysis [as C ₂₃₉ H ₃₃₃ O ₃₇ N ₅₁ S ₄・3H ₂ O] C % H% N% Calculated value 57.24 6.81 14.25 Measured value 57.07 6.96 14.41 P(4-38) Boc―Glu(OBzl)―Ile―Gln―
Leu-Met(O)-His-Asn-Leu-Gly-Lys
(Z)-His-Leu-Asn-Ser-Met(O)-
Glu (OBzl) - Arg (Mts) - Val - Glu (OBzl)
―Trp―Leu―Arg(Mts)―Lys(Z)―Lys
(Z)-Leu-Gln-Asp(OBzl)-Val-His-
Asn―Phe―Val―Ala―Leu―Gly―OBzl [43] [42] Anisole 0.48ml containing 2% EDT in 0.54g
Add 1.92 ml of TFA and stir at -10°C for 60 minutes. After the reaction is complete, TFA is distilled off under reduced pressure, and hexane is added to the residue to obtain an oily substance. Ether is added to this and the obtained powder is dried under reduced pressure. [33] 0.31g 5.55NHCl/DMF 0.19ml Isoamyl nitrite 0.07ml Et ₃ N 0.15ml DMF 1.0ml Et ₃ N 74μl [33] Add the azide solution obtained using the same method as in the previous [5] to 0.31g. obtain. The previously obtained dry powder was dissolved in 3 ml of DMF, and 61 μl of Et ₃ N was added. The resulting solution was added to the azide solution and stirred at 4° C. for 20 hours.
Furthermore, using 0.31 g of [33], add the azide solution obtained in the same manner, and stir at 4°C for 20 hours. The reaction solution was neutralized with acetic acid and concentrated under reduced pressure to give an oily residue.
% acetic acid water and ether are added and cooled to obtain a powder. This was suspended and washed in the order of 3% aqueous acetic acid, 5% aqueous sodium bicarbonate, and water, and then reprecipitated twice with DMF-ethyl acetate. Half of the obtained precipitate was added to the cefdex.
Inject into LH-60 column (3.4 x 140 cm, DMF) and elute with DMF. Fractionate the eluate into 8 ml portions,
Collect the main eluted portion (58 to 70 bottles), concentrate, and add ethyl acetate to obtain a precipitate. The remaining half of the precipitate was treated in the same manner to obtain a total of 440 mg. Melting point; 155-157°C [α] ²⁰ _D ; -30.4° (C = 0.1, DMF) TLC; Rf ₂ = 0.52 Amino acid analysis 1; Glu5.61(5), Ile1.22(1),
Leu6.00(6), Asp3.60(4), Ser0.72(1), Gly1.91(2),
Ala1(1), Val2.92(3), Met+Met(0)1.22(2),
Phe0.99(1), Trp0 (not measured) (0), Lys3.00(3),
His2.84(3), Arg2.02(2) Elemental analysis [as C ₂₆₈ H ₃₇₆ O ₆₄ N ₅₆ S ₄・6H ₂ O] C% H% N% Calculated value 57.04 6.93 13.90 Measured value 56.79 7.19 14.39 P( 1-38) Z-Ser-Val-Ser-Glu (OBzl)
―Ile―Gln―Leu―Met(O)―His―Asn―
Leu-Gly-Lys(Z)-His-Leu-Asn-Ser
-Met(O)-Glu(OBzl)-Arg(Mts)-Val
-Glu(OBzl)-Trp-Leu-Arg(Mts)-Lys
(Z)―Lys(Z)―Leu―Gln―Asp(OBzl)―
Val―His―Asn―Phe―Val―Ala―Leu―Gly
-OBzl〔44〕〔43〕420mg containing 2% EDT anisole 0.41ml
Add 1.64 ml of TFA and stir at -10°C for 60 minutes. After the reaction is complete, TFA is distilled off under reduced pressure, and hexane is added to the residue to obtain an oily substance. Ether is added to this, and the resulting powder is dried under reduced pressure. [36] 133mg 5.55NHCl/DMF 130μl Isoamyl nitrite 48.5μl Et ₃ N 102μl DMF 1ml Et ₃ N 51μl [36] 133mg in the same manner as in the previous [5] method,
Obtain the azide solution. The dry powder obtained earlier is
A solution obtained by dissolving in 5 ml of DMF and adding 42.5 μl of Et ₃ N is added to the azide solution and stirred at 4° C. for 18 hours. Further, using 133 mg of [36], add the azide solution obtained in the same manner to the reaction solution, and stir at 4°C for 36 hours. The reaction solution was neutralized with acetic acid and concentrated under reduced pressure. To the resulting oily residue were added 5% aqueous acetic acid and ether to obtain a viscous precipitate. This was suspended and washed in the order of 5% acetic acid water, 5% sodium bicarbonate water, and water, and the resulting powder was diluted with DMF-ethyl acetate.
Recrystallize twice to obtain 460 mg. Dissolve half of this in DMF and add it to a Sephadex LH-60 column (3x
140cm, DMF) and elute with DMF. The eluate was fractionated into 8 ml portions, and the main eluate portion (47 to 58
), concentrate, and add ethyl acetate to obtain a powder. Process the remaining half in the same way, totaling 375
Get mg. Further, this powder was divided into six parts, one part was dissolved in DMF containing 5% water, injected into a Sefa Seal S-200 column (2 x 144 cm) prepared with the same solvent, and eluted with the same solvent. The eluate is fractionated into 5 ml portions, the main eluate portions (71 to 81 bottles) are collected, concentrated, and ethyl acetate is added to obtain a powder. 5 left
Two parts were treated in the same way to obtain a total of 208.3 mg. Melting point; 159-160°C [α] ¹⁸ _D ; -17.9° (C = 0.2, DMSO) TLC; Rf ₂ = 0.59 Amino acid analysis; Asp3.84(4), Ser3.18(3),
Glu5.38(5), Gly2.22(2), Ala1(1), Val4.61(4),
Met + Met (0) 1.21 (2), Ile 1.11 (1), Leu 5.76 (6),
Phe1.07(1), Trp0 (not measured)(1), Lys3.05(3),
His2.73(3), Arg1.91(2) Elemental analysis [as C ₂₈₂ H ₃₉₃ O ₆₉ N ₅₉ S ₄・7H ₂ O] C% H% N% Calculated value 56.75 6.87 13.85 Measured value 56.55 6.54 13.72

Claims (1)

[Claims] 1 Formula H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met
―His―Asn―Leu―Gly―Lys―His―Leu―
Asn―Ser―Met―Glu―Arg―Val―Glu―Trp
―Leu―Arg―Lys―Lys―Leu―Gln―Asp―
Val―His―Asn―Phe―Val―Ala―Leu―Gly
-Peptide or its salt represented by OH.