KR20010022632A - Process for Producing LH-RH Derivatives - Google Patents

Abstract

The present invention is characterized by reacting the peptide fragment represented by the following general formula (1) and the peptide fragment represented by the following general formula (2) in the presence of chymotrypsin or chymotrypsin chymotrypsin. It provides a method for producing a LH-RH derivative represented by.

pGlu-His-Trp-OR ¹ (1)

H-Ser-Tyr-X-Leu-Arg-Pro-Y (2)

pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (3)

Where

R1 represents lower alkyl,

X represents an amino acid selected from the group consisting of D-type amino acids such as D-Leu, D-Ser (But), D-Trp, (2-naphthyl) -D-Ala and Gly,

Y represents Gly-NH ₂ , Azgly (azaglycine) -NH ₂ or NHR ² (R ² is lower alkyl).

Since it does not involve side reactions such as racemization and the like, the separation and purification of LH-RH derivatives is easy, and the yield is high, and since the unreacted fragments can be recovered and reused, it is very industrially useful.

Description

Process for Producing LH-RH Derivatives

<110> ITOHAM FOODS INC.

<120> Process for Producing LH-RH Derivatives

<130> FP0001 / H & A / JP

<160> 4

<170> KOPATIN 1.5

<210> 1

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Xaa denotes a pyroglutamic acid residue

<400> 1

Xaa His Trp Ser Tyr Gly Leu Arg Pro Gly

1 5 10

<210> 2

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<223> Xaa of the amino acid number 2 denotes Arg (NO2), and Xaa of the a

mino acid number 4 denotes Azgly (azaglycin) -NH2

<400> 2

Leu Xaa Pro Xaa

<210> 3

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<223> Xaa denotes Azgly-NH2

<400> 3

Leu Arg Pro Xaa

<210> 4

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Peptide

<400> 4

Ser Tyr Gly Leu Arg Pro Gly

1 5

Progesterone (LH) and ovarian stimulating hormone (FSH) are released from the anterior pituitary gland under the control of the luteinizing hormone releasing hormone LH-RH, which is produced in the hypothalamus. Since LH-RH and its derivatives have gonadotropin-releasing activity and inhibition of gonadotropy is recognized by continuous administration, for example, prophylactic and / or therapeutic drugs such as endometritis, central puberty, infertility, and prostate cancer It is applied as. Drugs already available include Busererin (Ref. 60-9519), Gosererin (Ref. 61-13480), Ryuprorelin (Ref. 53-14072), Naparerin ( Reference: Commando 63-56238.

In the method for producing the LH-RH and its derivatives, a chemical synthesis method by a liquid phase synthesis method in which a peptide fragment having a partial sequence corresponding to the polypeptide is formed by a liquid phase method or a coating method, and each fragment is coupled again in a liquid phase (see : Special Offices 56-47175, Special Offices 49-117468, Special Offices 57-29462, Special Offices 57-25540, Special Offices 63-17839, Special Offices 63-45398, Special Offices 48-40770, Special Offices 50-88069, Special Offices Point 49-41375, Point 49-41376, Point 48-99170, Point 52-20996, Point 49-35381, Point 52-8831, Point 57-61268, Point 53-14072, Point 57 -26506, Special Forces 60-22720, Special Forces 61-13480, and Special Engineers 3-71439.

However, in liquid phase synthesis, the solubility of the peptide changes slightly as the number of amino acid residues of the peptide increases, making it difficult to find a suitable solvent, thereby making it difficult to separate the target peptide from the unreacted or by-product. In particular, the fourth serine residue of LH-RH and its derivatives are easily racemized and thus remain as impurities and are not economically satisfactory since the treatment after the reaction is difficult and more economical, such as recovery of raw materials is impossible.

[Initiation of invention]

An object of the present invention is to provide a method for efficiently and inexpensively producing LH-RH derivatives.

As a result of intensive studies in order to solve the above problems, a method for producing an LH-RH derivative suitable for industrial production by an enzyme synthesis method was found and the present invention was completed.

The present invention relates to a method for efficiently preparing an LH-RH derivative which is a peptide useful as a medicament using an enzyme.

The present invention is characterized in that the peptide fragment represented by the following general formula (1) and the peptide fragment represented by the following general formula (2) are reacted in the presence of an enzyme selected from the group consisting of chymotrypsin or chymotrypsin-like enzyme. It is a manufacturing method of the LH-RH derivative represented by General formula (3).

pGlu-His-Trp-OR ¹ (1)

H-Ser-Tyr-X-Leu-Arg-Pro-Y (2)

pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (3)

Where

R ¹ represents lower alkyl,

X represents an amino acid selected from the group consisting of D-Leu, D-Ser (But), D-Trp, (2-naphthyl) -D-Ala, and Gly,

Y represents Gly-NH ₂ , Azgly (azaglycine) -NH ₂ or NHR ² (R ² is lower alkyl).

Here, R ¹ is a C1-3 alkyl group, R ^{2 It} is characterized in that the C1-3 alkyl group. In addition, the enzyme selected from the group consisting of the chymotrypsin or chymotrypsin-like enzyme is characterized in that the chymotrypsin.

In addition, the present invention provides a peptide fragment represented by the following general formula (4) and the peptide fragment represented by the following general formula (5) in the presence of an enzyme selected from the group consisting of chymotrypsin or chymotrypsin-like enzyme in the presence of water or buffer It is a manufacturing method of the LH-RH derivative represented by General formula (6) characterized by making it react with the solvent which mixed the solution and the organic solvent.

pGlu-His-Trp-OR ¹ (4)

H-Ser-Tyr-X-Leu-Arg-Pro-Y (5)

pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (6)

Where

R ¹ represents lower alkyl,

X represents an amino acid selected from the group consisting of D-Leu, D-Ser (But), D-Trp, (2-naphthyl) -D-Ala, and Gly,

Y represents Gly-NH ₂ , Azgly-NH ₂ or NHR ² (R ² is lower alkyl).

Wherein the solvent in which the water or the buffer solution and the organic solvent are mixed is a mixed solvent of water or the buffer solution and the water miscible organic solvent, or a mixed solvent saturated with an organic solvent that partially mixes the water or the buffer solution with water. It features.

In addition, the present invention provides an immobilized enzyme obtained by immobilizing an enzyme selected from the group consisting of a peptide fragment represented by the following general formula (7) and a peptide fragment represented by the following general formula (8): chymotrypsin or chymotrypsin-like enzyme. In the presence of water, a method for producing an LH-RH derivative represented by the formula (9), characterized in that the reaction is carried out in a mixed solvent of water or a buffer solution and an organic solvent.

pGlu-His-Trp-OR ¹ (7)

H-Ser-Tyr-X-Leu-Arg-Pro-Y (8)

pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (9)

Where

R ¹ represents lower alkyl,

X represents an amino acid selected from the group consisting of D-Leu, D-Ser (But), D-Trp, (2-naphthyl) -D-Ala, and Gly,

Y represents Gly-NH ₂ , Azgly-NH ₂ or NHR ² (R ² is lower alkyl).

In the present specification, LH-RH derivative is a general formula (10) (SEQ ID NO: 1)

pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂ (10)

It means that the 6th and 10th glycine of LH-RH represented by is substituted with other amino acids, special amino acids or modified amino acids. The amino acid at the position corresponding to the sixth glycine (hereinafter referred to as X) may be a D-type amino acid or a modified amino acid in which these are modified, and is not particularly limited. Specific D-type amino acids include D-type amino acids such as D-Leu, D-Trp, D-Ala, D-Phe, D-Val, and D-His, and as modified amino acids, D-Ser (But), (2-naphthyl) -D-Ala, etc. are mentioned. In addition, X may be an L-type amino acid. When X is the above D-type amino acid and modified amino acid, it is selected from the group consisting of D-Leu, D-Trp, D-Ala, D-Ser (But) and (2-naphthyl) -D-Ala In the case of L-type amino acids, glycine is preferable. The tenth glycine (hereinafter referred to as Y) is preferably Gly-NH ₂ , Azgly-NH ₂ or NHR ² (R ² is lower alkyl).

"Lower alkyl" means alkyl having 1 to 3 carbon atoms, and specific examples thereof include methyl, ethyl, propyl and isopropyl. R ¹ is preferably a methyl or ethyl group, and R ² is an ethyl or methyl group.

In addition, in this specification, when abbreviated about amino acid, a peptide, a protecting group, a solvent, and others, the regulation of International Pure and Applied Chemistry (IUPAC), the International Biochemical Union (IUB), or the tolerance in the said field | area. We shall follow preference. The example is shown below. However, when there exists an optical isomer with respect to an amino acid etc., unless otherwise indicated, it shall show L-form.

Tyr: Tyrosine residue

Gly: Glycine residue

Azgly: Azaglycine residue

Glu: Glutamic acid residue

pGlu: Pyroglutamic acid residue

Ser: Serine residue

Arg: Arginine residue

Pro: Proline residue

Leu: Leucine residue

His: Histidine residue

Ala: Alanine residue

Trp: Tryptophan residue

Et: Ethyl

Boc: t-Butoxycarbonyl

Aoc: t-amyloxycarbonyl

Bz: Benzyl

Z: Benzyloxycarbonyl

Tos: Tosyl

OMe: Methyl ester

OBz: Benzyl ester

OSu: N-Hydroxysuccinimide ester

TFA: Trifluoroacetic acid

THF: Tetrahydrofuran

DMF: Dimethylformamide

DCC: Dicyclohexylcarbodimide

WSC: N-ethyl-N'-dimethylaminopropylcarbodiimide

HOSu: N-hydroxysucciniimide

HOBt: 1-hydroxybenzotriazole (1-Hydroxybenzotriazol)

MeOH: Methanol

EtOH: Ethanol

AcOH: Acetic acid

The peptide fragment represented by general formula (1) corresponds to amino acid residues 1 to 3 of amino acid sequence of the LH-RH derivative represented by general formula (3). In addition, the peptide fragment represented by General formula (2) corresponds to the 4th or less amino acid residue of the amino acid sequence of the LH-RH derivative represented by General formula (3).

The peptide fragment represented by the general formula (1) or the peptide fragment represented by the general formula (2) can be synthesized according to conventional means for known peptide synthesis, respectively. For example, "The Peptides" Vol. 1 (1966) [Schreder and Luhke, Academic Press, New York, USA], or "Peptide Synthesis" by Izumiya et al., Maruzen Corporation (1975) According to the method described in []], for example, the azido method, the acid chloride method, the acid anhydride method, the mixed acid anhydride method, the DCC method, the active ester method (p-nitrophenyl ester method, N-hydride) Oxysuccinimide ester method, cyanomethyl ester method, etc.), woodward reagent K, carbodiimidazole method, redox method, DCC-additive (HONB, HOBt, HOSu) method, coating method, etc. It can synthesize | combine by. By the synthesis of general peptides as described above, for example, by a so-called stepwise stretching method that condenses amino acids one by one according to the amino acid sequence to the C-terminal amino acids, or by dividing into several fragments to synthesize each fragment and It can manufacture by the fragment condensation method to couple.

In addition, in the synthetic reaction step of the peptide fragment represented by the general formula (1) or (2), the functional group not involved in the reaction is protected by a common protecting group, and the protecting group is removed after completion of the reaction. In addition, the functional groups involved in the reaction are usually activated. Each of these reaction methods is known, and the reagents used therein are also appropriately selected from known ones.

Examples of protecting groups for the amino group include benzyloxycarbonyl (Z), t-butyloxycarbonyl (Boc), t-amyloxycarbonyl (Aoc), isobornyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2- Chlorobenzyloxycarbonyl, adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, o-nitrophenylsulphenyl, diphenylphosphinothioyl and the like. Boc is generally used to protect the amino group, but when combining D-Ser (But), Z group can be used to remove only Z group without removing But (t-butyl) group.

Examples of protecting groups for carboxyl groups include alkyl esters (eg, methyl esters, ethyl esters, propyl esters, butyl esters, tert-butyl esters, etc.), benzyl esters, p-nitrobenzyl esters, methyl benzyl esters, p-chlorobenzyl esters, Benzhydryl ester, benzyloxycarbonyl hydrazide, tert-butyloxycarbonyl hydrazide, trityl hydrazide and the like. When hydrazide is used, it is preferable to use methyl ester or ethyl ester.

Activated carboxyl groups involved in peptide bonds include, for example, the corresponding acid chlorides, acid anhydrides or mixed acid anhydrides, azides, active esters (e.g., pentachlorophenol, p-nitrophenol, N-hydride). Hydroxysuccinimide, esters such as N-hydroxybenztriazole, N-hydroxy-5-novolene-2,3-dicarboxyimide), and the like. Among these, when condensing fragments, it is preferable to use the azide method with little racemization.

In addition, the peptide bond generation reaction may be carried out in the presence of a condensation agent, for example, a carbodiimide reagent such as dicyclohexylcarbodiimide, carbodiimidazole, tetraethylpyrophosphate or the like.

Conventionally, proteolytic enzymes have been mainly used for cleavage of peptide bonds, but it has long been known that they can also be involved in the reaction of generating peptide bonds, which is the reverse reaction. Enzyme synthesis of long-chain peptides is limited to having amino acids with limited substrate specificity in the structure or using enzymes with limited substrate specificity. Therefore, general enzymes such as trypsin and chymotrypsin are used to form peptides. Less work Therefore, the enzyme reaction is mainly used for relatively short-chain peptide bonds such as oligopeptides, but it takes time to examine the synthetic conditions. For this reason, in the production of peptides, chemical synthesis is generally used, and enzyme synthesis is attempted when there are many side reactions in chemical synthesis or when the reaction is difficult. Enzyme synthesis can solve the above problems in chemical synthesis depending on the conditions, and can be a very useful method by carrying out under mild conditions capable of mass production.

As a result of intensive examination, the present invention reacts the peptide fragment represented by formula (1) and the peptide fragment represented by formula (2) in the presence of chymotrypsin or chymotrypsin-like enzyme to bind both. It was successful to efficiently prepare the LH-RH derivative represented by the formula (3).

The chymotrypsin used in the present invention is an enzyme obtained from bovine pancreas, which is a kind of serine proteolytic enzyme registered with enzyme number EC.3.4.21.1 with the International Biochemical Association (I.U.B.) enzyme committee. Chymotrypsin is commercially available from SIGMA.

The chymotrypsin-like enzyme refers to proteolytic enzymes that recognize aromatics such as tyrosine and phenylalanine, and specific examples thereof include α-chymotrypsin.

It is preferable to use chymotrypsin or chymotrypsin-like enzyme because tryptophan, which is one of the recognition sites of chymotrypsin or chymotrypsin degrading enzyme, exists in the LH-RH derivative.

The reaction of chymotrypsin or chymotrypsin-like enzymes is typically carried out in a medium containing a buffer solution having a pH of about 5 to 10, preferably a pH of about 6 to 9, more preferably a pH of about 7.5 to 8.5.

A medium containing a buffer solution is a solvent which uses the buffer solution itself as a medium, a mixed solvent of various buffer solutions and water-miscible organic solvents described below, or an organic solvent which does not completely mix these buffer solutions with water. Say a solvent.

As a buffer solution, if pH value is in the said range, it will not specifically limit but various things can be used. For example, a tris hydrochloric acid buffer solution, a maxyl vane buffer solution, a phosphate buffer solution, an ammonium acetate buffer solution, an Atkins & pantin buffer solution, a veronal buffer solution, etc. are mentioned.

When such a buffer solution is used as the reaction medium, the buffer solution is usually used in admixture with a water miscible organic solvent. As the water-miscible organic solvent used here, for example, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dimethylimidazoleidinone (DMI), hexamethylphosphoryltriamide (HMPA), methanol ( MeOH), ethanol (EtOH), etc. are mentioned. Preferred are dimethylformamide, methanol and ethanol.

In addition, an organic solvent which is not completely miscible with water such as n-butanol (1-BuOH) and ethyl acetate (EtOAc) may be used. In the case of using an organic solvent which is not completely miscible with water, it is preferable to use butanol in order to facilitate the operation of partition chromatography in the post-treatment operation.

These organic solvents may be used independently, or may be used in combination of 2 or more type.

The chymotrypsin or chymotrypsin-like enzyme is not a solvent of water or a buffer solution alone, but is reacted in a solvent in which these and organic solvents are mixed. The yield of the desired peptide is significantly improved.

The mixing ratio of water-miscible organic solvent with water or buffer solution is generally preferably 50 vol% or less in terms of reactivity, but 80 vol% using immobilized chymotrypsin using an immobilizing enzyme such as celite. It is preferable that it is above. In addition, in the case of using an organic solvent that is not completely miscible with water, the use of saturated water of the organic solvent has the advantage that the reaction efficiency of the enzyme is high.

The above enzymatic reaction is usually carried out in the temperature range in which the chymotrypsin or chymotrypsin-like enzyme acts, generally in the range of about 0 to 50 ° C, preferably about 0 to 20 ° C. Reaction at about 10 ℃ has the effect of suppressing the hydrolysis reaction that occurs at the same time.

The amount of chymotrypsin or chymotrypsin-like enzyme is not particularly limited and can be appropriately adjusted according to the reaction conditions. For example, using about 50-100 mg of chymotrypsin with respect to 50 g of substrate can be used to synthesize the desired peptide in a range yield of about 75-85% in one hour.

Moreover, 1-5 mol, preferably 2-4 mol of peptide fragments represented by General formula (1) are normally used per mol of peptide fragments represented by General formula (2).

As the method of the present invention, chymotrypsin or chymotrypsin-like enzyme may be dissolved in water or a suitable buffer solution, or may be immobilized by a general method such as a carrier binding method, a crosslinking method, a comprehensive method, or other methods. May be used as an immobilizing enzyme. Examples of the carrier used in the carrier bonding method include cellulose, dextrin, derivatives of polysaccharides such as agarose, polyacrylamide gels, celite, porous glass, and the like. Examples of the crosslinking reagent used in the crosslinking method include glutaraldehyde, bisdiazobibenzidine, N, N-polymethylenebisiodacetamide, N, N-ethylenebismaleimide, and the like. As a raw material used by a comprehensive method, polyacrylamide gel, polyacryl alcohol gel, starch, konjac powder, nylon, polyurea, polystyrene, ethyl cellulose, collodione, cellulose nitrate, etc. are mentioned. However, the immobilization method is not limited to the method using these at all.

As a manufacturing method of this invention, it is following formula (11) as follows, for example.

pGlu-His-Trp-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt (11)

The LH-RH derivative represented by can be obtained.

That is, the peptide fragment represented by following formula (12) of predetermined amount

pGlu-His-Trp-OMe (12)

And a peptide fragment represented by the following formula (13) in a predetermined amount:

H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt (13)

In the presence of chymotrypsin having predetermined activity, the LH-RH derivative can be obtained by stirring at about 10 ° C. for about 1 hour in saturated water of n-butanol. The activity of chymotrypsin is preferably about 1,000 international units (U) / mg or more because it is easy to remove in a later step. The molar ratio of the peptide fragment represented by the formula (12) and the peptide fragment represented by the formula (13) used in this reaction is about 3: 1, and the yield is high. In addition, instead of dissolving in water or a buffer solution and adding the enzyme to the reaction solution as described above, for example, chymotrypsin may be fixed to agarose according to a conventional method and used as an immobilized enzyme. By using the immobilized enzyme, the reaction solution can be easily removed from the reaction solution by passing through a filter such as a glass filter after completion of the reaction, and can be reused.

As mentioned above, the said LH-RH derivative can be obtained and the obtained LH-RH derivative can be refine | purified as follows.

The LH-RH derivative prepared by the method of the present invention can be desalted and purified according to a conventional method. Here, desalting can be performed by various methods utilizing the difference in molecular size from salts, for example, gel filtration, ultrafiltration, dialysis and the like. Specifically, ultrafiltration using membranes made of cellulose acetate, gel filtration using Sephadex columns such as Sephadex LH-20, Sephadex-60, Sephadex G-25, ion exchange chromatography using DEAE-cellulose, and the like. Or the like. In the refining process, two types of liquids (eg, water-n-butanol, etc.) which are not mixed with each other are used, and the chromatographic chromatography using silica gel and the like as the stationary phase using the difference in the partition coefficient between these two liquid phases is used. High performance liquid chromatography (HPLC), such as chromatography or reversed phase chromatography using ODS-silica gel as the stationary phase, can be used.

For example, since the LH-RH derivative prepared by the method of the present invention is hydrophilic, it can be purified as follows using an organic solvent which is not completely miscible with water as described above. The reaction mixture reacted as described above is extracted with an organic solvent, water is added to the extract and partitioned, and solidified with a solvent such as ether. As an organic solvent used for extraction, n-butanol (n-BuOH) etc. are preferable. This is because these solvents have high polarity, and thus, extraction efficiency is high, and since they are not mixed with water, they are convenient for the distribution chromatography performed in the next step.

About 1/4 part of n-butanol (300 mL) is added to the reaction mixture (1,200 mL), and the chromatography of the reaction mixture with water is performed. Extraction with n-butanol may be appropriately repeated. The aqueous and organic layers are separated and concentrated respectively. At this time, since the peptide fragment of said (7) is collect | recovered from an aqueous layer, it can be used for reaction again.

The crude product obtained in the organic layer by partition chromatography is preferably solidified by diethyl ether, ethyl acetate, etc., in view of the fact that the LH-RH derivative is poorly soluble in these solvents. This solidified crude is then dissolved in a suitable buffer such as ammonium acetate and fractionated by column chromatography using a suitable stationary and mobile phase. When a weak cation exchange resin such as CM cellulose or a strong cation resin such as SP is used as the stationary phase, the interaction by the arginine residues contained in LH-RH can be obtained. As a mobile phase, when the buffer solution which melt | dissolved the solidified crude agent was used, when salt concentration is raised with a linear gradient, there exists an advantage that a yield is high. Specifically, 0.01 M ammonium acetate aqueous solution and 0.1 M ammonium acetate aqueous solution can be used. The obtained eluate can be obtained by appropriately selecting the stationary phase and the eluate and purifying by HPLC. In HPLC, ODS silica columns, such as TSK gel ODS-120T, can be used as a stationary phase, and use of ODS silica columns is optimal. In addition, 0.1% TFA-acetonitrile etc. can be used as a mobile phase. Herein, the content of acetonitrile is changed from 0.1% TFA-20% acetonitrile at the start of 1% per minute, and it is easy to separate the target LH-RH peptide by using a straight concentration tool containing 0.1% TFA-50% acetonitrile. Do. Purification is not limited to the above purification method, and in the case of using an immobilized enzyme, ion exchange can be performed without performing distribution chromatography.

The method of the present invention has the advantages as described below in comparison with the conventional known methods for producing LH-RH derivatives, and is very useful industrially.

A) Purification and separation is easy because it can be synthesized without enzymatic reactions such as racemization.

B) The yield is high and unreacted fragments are economically advantageous since recoverable materials are available.

The LH-RH derivative may be made into a salt, for example, acetate, hydrochloride, phosphate, or the like, which can be accepted as a medicine as necessary.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, this invention is not limited to these.

In the following examples, the obtained pure peptide was identified by the measurement of the retention time of high performance liquid chromatography (HPLC), the measurement of the photosensitivity, and the amino acid analysis. These measurements were performed by the following measuring methods and measurement conditions, unless otherwise indicated.

(1) High Performance Liquid Chromatography (HPLC)

LC-Module-1 (Japan Water Limited) was used as a detector for high performance liquid chromatography analysis.

(HPLC analysis condition)

Column: TSK gel ODS-120T (4.6 × 250 mm)

Eluent: 0.1% TFA-acetonitrile

(Linear gradient to change acetonitrile by 1% per minute from 20% to 50%)

Flow rate: 1mL / min

Detection wavelength: 220 nm

(2) optical intensity

DIC-370 (Japan Spectroscopy Industry Co., Ltd.) was used for the measurement of the linearity.

(Photometric measurement condition)

Rays: Na Lamp 589nm

Temperature: 20 ℃

Layer length: 100mm

Concentration: 5mg / mL

(3) amino acid analysis

The amino acid analysis was performed by hydrolyzing the obtained peptide in 110N hydrochloric acid (containing 0.1% phenol) at 110 ° C. for 20 hours using Hitachi Amino Acid Analyzer L-8500 (Hitachi Corporation).

Example 1 Preparation of pGlu-His-Trp-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt

(1-1) Preparation of Boc-Arg (Tos) -Pro-NHEt

214.3 g of Boc-Arg (Tos) -OH was dissolved in a mixed solvent of 150 mL of THF and 100 mL of DMF, and cooled to −20 ° C. with dry ice-ethanol. Next, 35 mL of N-methylmorpholine was dripped at this, 66 mL of isobutyl chloroformates were then dripped, and it stirred at -20 degreeC for 1 minute, and manufactured the corresponding mixed acid anhydride. The obtained reaction solution was mixed with a solution in which 71.1 g of H-Pro-NHEt was dissolved in 300 mL of THF, and stirred at 0 ° C. for 5 minutes and at room temperature for 30 minutes. Thereafter, the obtained reaction mixture was concentrated under reduced pressure, 1,200 mL (2 times) of ethyl acetate was added to the residue, the mixture was washed twice with 500 mL of water, and the ethyl acetate layer was concentrated under reduced pressure. The residue was treated with ether, solidified and dried. Thus, 221.18 g of Boc-Arg (Tos) -Pro-NHEt (yield 80.0%) was obtained.

The melting point of Boc-Arg (Tos) -Pro-NHEt, photoluminescence, Rf of TLC and elemental analysis values are shown below.

m.p. : 100 to 102 ° C

[α] _D : -30.3 (c = 1.0, MeOH)

Rf: 0.69 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental analysis value (as C ₂₅ H ₄₀ N ₆ O ₆ SH ₂ O)

Theoretical C: 52.61% H: 7.42% N: 14.73%

Found: C: 52.85% H: 7.30% N: 14.41%

(1-2) Preparation of Z-Leu-Arg (Tos) -Pro-NHEt

110.54 g of Boc-Arg (Tos) -Pro-NHEt prepared in (1-1) was dissolved in 150 mL of dichloromethane (DCM). TFA150mL was added to this under ice cooling, and it stirred at room temperature for 30 minutes. The obtained reaction liquid was concentrated under reduced pressure, 1,500 mL of ether was added to the residue, solidified, and dried. Thus, H-Arg (Tos) -Pro-NHEtTFA was obtained.

The electric deprotector H-Arg (Tos) -Pro-NHEt.TFA was dissolved in a mixed solvent of 80 mL of DMF and 200 mL of THF, and neutralized with N-methylmorpholine while cooling. Subsequently, a solution obtained by dissolving 53.06 g of Z-Leu-OH and 23.02 g of HOSu in 200 mL of THF and 36.4 mL of WSC were added, followed by stirring at room temperature overnight at 0 ° C. for 5 minutes. After confirming with ninhydrin, the reaction mixture was concentrated under reduced pressure.

1,500 mL of ethyl acetate was added to the residue, and the mixture was washed twice with 500 mL of water and twice with 500 mL of saturated saline. Subsequently, the ethyl acetate layer was concentrated under reduced pressure, and the residue was treated with ether to solidify and dry. Thus, 119.5 g (yield 85.1%) of Z-Leu-Arg (Tos) -Pro-NHEt was obtained.

The melting point of Z-Leu-Arg (Tos) -Pro-NHEt, photoluminescence, Rf of TLC and elemental analysis values are shown below.

m.p. : 99-103 ℃

[α] _D : -40.6 (c = 1.0, MeOH)

Rf: 0.75 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₃₄ H ₄₉ N ₇ O ₇ S)

Theoretical C: 58.35% H: 7.06% N: 14.01%

Found C: 58.40% H: 7.26% N: 13.78%

(1-3) Preparation of Z-D-Ser (But) -Leu-Arg-Pro-NHEt

To 20.99 g of Z-Leu-Arg (Tos) -Pro-NHEt prepared in (1-2) was added 32 mL of anisole, followed by addition of about 200 mL of HF while cooling to -70 ° C with dry ice-ethanol. It stirred at 0 degreeC for 1 hour. Thereafter, the residue was concentrated under reduced pressure and the residue was treated with ether. The precipitated product was filtered off and dried in vacuo over sodium hydroxide to give H-Leu-Arg-Pro-NHEt.HF.

8.86 g of Z-D-Ser (But) -OH was dissolved in 100 mL of THF. This was cooled to -20 DEG C with dry ice-ethanol, 3.3 mL of N-methylmorpholine was added dropwise, and then 3.96 mL of isobutylchloroformate was added dropwise, followed by stirring at -20 DEG C for 1 minute to give a corresponding mixed acid. Anhydrides were prepared. The obtained reaction solution was mixed with a 200 mL solution of H-Leu-Arg-Pro-NHEt.HF (neutralized with N-methylmorpholine), and stirred at 0 ° C for 5 minutes and at room temperature for 30 minutes. Thereafter, the mixture was concentrated under reduced pressure, 500 mL of water was added to the residue, and extracted with 200 mL of n-butanol five times. The extract was washed five times with water, and then the n-butanol layer was concentrated under reduced pressure and the residue was treated with ether to solidify. Thus, 22.89 g (yield 90.5%) of Z-D-Ser (But) -Leu-Arg-Pro-NHEt was obtained.

The melting point of Z-D-Ser (But) -Leu-Arg-Pro-NHEt, photoluminescence, Rf of TLC and elemental analysis values are shown below.

m.p. : 121 ~ 123 ℃

[α] _D : -49.0 (c = 1.0, MeOH)

Rf: 0.51 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₃₄ H ₅₆ N ₈ O ₇ )

Theoretical C: 59.28% H: 8.19% N: 16.27%

Found C: 59.01% H: 8.15% N: 16.19%

(1-4) Preparation of Z-Ser-Tyr-OMe

23.9 g of Z-Leu-OH and 23.2 g of H-Tyr-OMe were dissolved in 50 mL of DMF, and 12.7 g of HOSu and 20 mL of WSC were added, followed by stirring at 0 ° C. for 5 minutes and at room temperature overnight. After confirming with ninhydrin, the reaction mixture was concentrated under reduced pressure. 500 mL of ethyl acetate was added to the residue, followed by washing twice with 200 mL of 1N hydrochloric acid, twice with 200 mL of saturated aqueous sodium bicarbonate, and twice with brine. The ethyl acetate layer was concentrated under reduced pressure, and the residue was treated with hexane to solidify and dried. Thus, 38.44 g (yield 92.3%) of Z-Ser-Tyr-OMe was obtained.

The melting point of Z-Ser-Tyr-OMe, photoluminescence, Rf of TLC and elemental analysis are shown below.

m.p. : 49-53 ° C (decomposition)

[α] _D : +2.3 (c = 1.0, MeOH)

Rf: 0.89 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₂₁ H ₂₄ N ₂ O ₇ )

Theoretical C: 60.57% H: 5.81% N: 6.73%

Measured value C: 60.88% H: 5.88% N: 6.99%

(1-5) Preparation of Z-Ser-Tyr-NHNH ₂

38.0 g of Z-Ser-Tyr-OMe prepared in (1-4) was dissolved in 200 mL of methanol, 50 g of hydrazine was added, and the mixture was allowed to stand at room temperature overnight. Thereafter, the mixture was concentrated under reduced pressure and washed with methanol. In this way by the _{Z-Ser-Tyr-NHNH 2} 34.2g ( yield 90.0%) it was obtained.

The melting point of Z-Ser-Tyr-NHNH _2, the photoluminescence, the Rf of TLC and the elemental analysis values are shown below.

m.p. : 194-197 ℃

[α] _D : +9.9 (c = 1.0, DMF)

Rf: 0.64 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₂₀ H ₂₄ N ₄ O ₆ )

Theoretical C: 57.69% H: 5.81% N: 13.45%

Found C: 57.85% H: 5.83% N: 13.49%

(1-6) Preparation of Z-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt

28.0 g of Z-D-Ser (But) -Leu-Arg-Pro-NHEt prepared in (1-3) was dissolved in 300 mL of methanol, and hydrogen was added in the presence of 10% palladium / 2.5 g of carbon. After stirring for 7 hours at room temperature, the catalyst was removed, concentrated under reduced pressure, and the residue was treated with ether. Thus, H-D-Ser (But) -Leu-Arg-Pro-NHEt was obtained.

20.1 g of Z-Ser-Tyr-NHNH ₂ prepared in (1-5) was dissolved in 150 mL of DMF, and cooled to −20 ° C. with dry ice-ethanol. 43.5 mL of 4N HCl-dioxane and 6.5 mL of isoamyl nitride were added to the above solution to obtain an azide compound. Furthermore, 24.4 mL of triethylamine was added and neutralized. The mixture was transferred to a 150 mL solution of DMF containing HD-Ser (But) -Leu-Arg-Pro-NHEt, stirred at -20 ° C for 2 hours, and at 4 ° C for 17 hours, and then concentrated. 500 mL of water was added to the residue, and extracted with 200 mL of n-butanol five times. After washing five times with water, the n-butanol layer was concentrated under reduced pressure and the residue was treated with ether to solidify. Thus, 33.2 g of Z-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt (yield 87.5%) was obtained.

The melting point of Z-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt, photoluminescence, Rf of TLC and elemental analysis are shown below.

m.p. : 115 ~ 118 ℃

[α] _D : -27.6 (c = 1.0, DMF)

Rf: 0.36 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₄₆ H ₇₀ N ₁₀ O ₁₁ )

Theoretical C: 58.83% H: 7.51% N: 14.91%

Found C: 58.88% H: 7.55% N: 14.90%

(1-7) Preparation of Z-pGlu-His-OH

52.6 g of Z-pGlu-OH was dissolved in 400 mL of THF. The solution was cooled to -20 DEG C with dry ice-ethanol, 22.0 mL of N-methylmorpholine and 26.4 mL of isobutylchloroformate were added dropwise, followed by stirring at -20 DEG C for 1 minute to give the corresponding mixed acid anhydride. Was prepared. HOSu 25.3g was added to the obtained reaction mixture, and stirred for 30 minutes. Then, the solution which melt | dissolved 62.9 g of H-His-OH * HCl and 28.0 mL of triethylamine (TEA) in 400 mL of water was added to this. It stirred for 1 hour at 0 degreeC and overnight at room temperature, and concentrated under reduced pressure. The remaining aqueous solution was washed with ethyl acetate and the aqueous layer was extracted with n-butanol. After washing several times with water, the n-butanol layer was concentrated under reduced pressure, solidified with ether, dissolved in water and adjusted to pH 5. The aqueous solution was poured into a column (5.0 × 20 cm) filled with HP-20, washed with water until the Pauli reaction of the effluent reached ±, and then eluted with methanol. The eluted methanol was concentrated under reduced pressure and the residue was treated with ether. Subsequently, it was recrystallized from methanol ether. Thus, 59.5 g (yield 74.3%) of Z-pGlu-His-OH were obtained.

The melting point of Z-pGlu-His-OH, photoluminescence, Rf of TLC and elemental analysis are shown below.

m.p. : 146-149 ° C (decomposition)

[α] _D : -0.91 (c = 1.0, DMF)

Rf: 0.14 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₁₉ H ₂₀ N ₄ O ₆ )

Theoretical C: 57.00% H: 5.03% N: 13.99%

Found C: 57.23% H: 5.05% N: 14.05%

(1-8) Preparation of Z-pGlu-His-Trp-OMe

40.0 g of Z-pGlu-His-OH, 19.6 g of H-Trp-OMe-HCl and 12.7 g of HOSu prepared in (1-7) were dissolved in 100 mL of DMF. Subsequently, after neutralizing with N-methylmorpholine while cooling this solution, 2.0 mL of WSC was added, and it stirred at 0 degreeC for 5 minutes, and overnight at room temperature. After confirming with ninhydrin, the reaction mixture was concentrated under reduced pressure, 200 mL of ethyl acetate-n-butanol (1: 1) was added to the residue, and the organic layer was washed three times with 100 mL of water. The organic layer was concentrated under reduced pressure and the residue was treated with ether to solidify. Thus, 48.5 g (yield 92.3%) of Z-pGlu-His-Trp-OMe was obtained.

The melting point of Z-pGlu-His-Trp-OMe, photoluminescence, Rf of TLC and elemental analysis values are shown below.

m.p. : 112 to 115 ° C (decomposition)

[a] _D : -1.5 (c = 1.0, DMF)

Rf: 0.3 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₃₁ H ₃₂ N ₆ O ₇ S)

Theoretical C: 61.99% H: 5.37% N: 13.99%

Measured value C: 61.54% H: 5.33% N: 13.80%

(1-9) Preparation of pGlu-His-Trp-OMe

40.0 g of Z-pGlu-His-Trp-OMe prepared in (1-8) was dissolved in 50 mL of DMF, and hydrogen was added in the presence of 500 mg of 10% palladium / carbon. After stirring for 7 hours at room temperature, the catalyst was removed, concentrated under reduced pressure, and the residue was solidified by treating with ether. Thus, 28.5 g (yield 91.8%) of pGlu-His-Trp-OMe were obtained.

The melting point of pGlu-His-Trp-OMe, photoluminescence, Rf of TLC and elemental analysis are shown below.

m.p. : 132-137 degreeC (analysis)

[α] _D : +4.1 (c = 1.0, DMF)

Rf: 0.18 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₂₃ H ₂₆ N ₆ O ₅ )

Theoretical C: 59.22% H: 5.62% N: 18.02%

Found C: 59.01% H: 5.55% N: 17.89%

(1-10) Preparation of H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt

93.0 g of Z-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt prepared in (1-6) was dissolved in 800 mL of methanol, and hydrogen was added in the presence of 5.0 g of 10% palladium / carbon. Added. After stirring for 7 hours at room temperature, the catalyst was removed, concentrated under reduced pressure, and the residue was solidified by treating with ether. Thus, 74.0 g of H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt (yield 90.3%) was obtained.

Melting points, optical fluorescence, Rf of TLC and elemental analysis of H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt are shown below.

m.p. : 124-128 degreeC (decomposition)

[α] _D : +10.1 (c = 1.0, DMF)

Rf: 0.16 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₃₈ H ₆₄ N ₁₀ O ₉ )

Theoretical C: 56.70% H: 8.01% N: 17.40%

Found C: 55.55% H: 7.97% N: 17.18%

(1-11) Preparation of pGlu-His-Trp-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt

PGlu-His-Trp-OMe 30g prepared in (1-9) and H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt 22g prepared in (1-10) were n It was dissolved in 1,300 mL of butanol saturated water. The pH was adjusted to 7.8 using 1 M hydrochloric acid or ammonia water, and a solution of 78 mg of 1,000 U / mg chymotrypsin in 2.5 mL of n-butanol saturated water was added thereto, followed by stirring at 10 ° C for 1 hour. 300 mL of n-butanol was added to the reaction mixture, and the synthesized peptide was partitioned into water and n-butanol, and separated into a lower layer (aqueous layer) and an upper layer (n-butanol layer). The same partition chromatography was repeated three more times by adding n-butanol (200 mL) to the lower layer. The upper layer was washed five times with 100 mL of water, and the organic and aqueous layers were each concentrated to solidify each with ether. H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt recovered from the aqueous layer was used for the reaction. The organic layer (n-butanol) was concentrated and powdered by adding ether to the residue.

The powder obtained in the organic layer was dissolved in an aqueous 0.01 M ammonium acetate solution and applied to a column (4 × 30 cm) filled with CM-cellulose. 500 mL of 0.01 M aqueous ammonium acetate solution (pH 4.4) to 500 mL of 0.1 M aqueous ammonium acetate solution (pH 4.4) was used for linear farming equipment dissolution (60 mL / hour), and the eluate was fractionated in 10 mL portions. The eluate was analyzed by high performance liquid chromatography, and the desired fractions were lyophilized. Thus, 22.4 g of pGlu-His-Trp-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt was obtained (yield 74.9%).

The photoluminescence, elemental analysis and amino acid analysis of pGlu-His-Trp-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt are shown below.

[α] _D : -46.0 (c = 1.0, H ₂ O)

Elemental Analysis Values (as C ₆₀ H ₈₆ N ₁₆ O ₁₃ CH ₃ COOH ₂ H ₂ O)

Theoretical C: 55.76% H: 7.09% N: 16.78%

Measured value C: 55.60% H: 7.10% N: 16.79%

Amino acid composition;

Ser 2.05 (2), Glu 1.08 (1), Pro 0.99 (1), Leu 1.09 (1), Tyr 0.92 (1), His 0.93 (1), Arg 1.06 (1)

(1-12) Synthesis of pGlu-His-Trp-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt by Immobilized Enzyme

(1) Synthesis of pGlu-His-Trp-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt

2.00 g of pGlu-His-Trp-OMe prepared in (1-9) and H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt prepared in (1-10) above Was dissolved in 200 mL of water and adjusted to pH 7.8 with 1 M hydrochloric acid or ammonia water. To this solution was suspended by adding 2.5 g of immobilized chymotrypsin (agarose carrier) at 2000 U / g and confirming that the pH was 7.8, and stirred at 10 ° C for 1 hour. The reaction solution was filtered through a glass filter to immobilize chymotrypsin chymotrypsin.

The filtrate was concentrated and applied to a column packed with CM-cellulose (4 × 30 cm). 500 mL of 0.01 M aqueous ammonium acetate solution (pH 4.4) to 500 mL of 0.1 M aqueous ammonium acetate solution (pH 4.4) were evaporated (60 mL / hour), and the elution was fractionated by 10 mL. The eluate was analyzed by HPLC and the desired fractions were lyophilized. Thus, 1.45 g of pGlu-His-Trp-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt was obtained (yield 82.4%).

Further, the melting point, the optical intensity, the Rf value of TLC and the elemental analysis value of the obtained compound were pGlu-His-Trp-Ser-Tyr-D-Ser (But) -Leu- prepared in (1-11) of Example 1. It is consistent with that of Arg-Pro-NHEt.

(2) Examination of substrate specificity

Under the above enzyme reaction conditions, the peptides shown in Table 1 were reacted as substrates, and peptides generated 2 hours after the start of the reaction were analyzed by HPLC, and yields were calculated from the area of each peak.

Substrate used yield(%) pGlu-His-Trp-OMe + H-Ser-Tyr-D-Ser (tBu) -Leu-Arg-Pro-NHEtpGlu-His-D-Trp-OMe + H-Ser-Tyr-D-Ser (tBu)- Leu-Arg-Pro-NHEtpGlu-D-His-Trp-OMe + H-Ser-Tyr-D-Ser (tBu) -Leu-Arg-Pro-NHEtpGlu-His-Trp-OMe + HD-Ser-Tyr-D -Ser (tBu) -Leu-Arg-Pro-NHEt 78.80.07.80.0

As shown in Table 1, when tryptophan, an enzyme recognition site, uses a D-type substrate, the yield was 0% and the reaction did not proceed at all. In addition, when the histidine adjacent to the N-terminal side of the enzyme was used as a D-type substrate, the yield was low as 7.8%, but it was confirmed that there was a product. It was also confirmed that the yield was 0% even when serine, which is an enzyme binding site, using a D-type substrate, and the reaction did not proceed.

In the above, it was confirmed that racemization of serine and histidine is unlikely to occur due to substrate specificity of the enzyme, and thus a product having high optical purity can be obtained.

Example 2

In the present Example, ZD-Ser (But) -Leu-Arg-Pro-NHEt prepared according to (1-1) to (1-3) of Example 1 is represented by the following (2-1) to (2- Except that prepared according to 3), pGlu-His-Trp-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt was prepared in the same manner as in Example 1.

(2-1) Preparation of Z-Leu-Arg-NHNH ₂

172.5 g of Z-Leu-OH and 178.9 g of H-Arg-OEt.2HCl were dissolved in 800 mL of a mixed solution of DMF / THF (1/1), and neutralized by addition of 143 mL of N-ethylmorpholine at 0 ° C. Then, DCD 147.5 mL was added and stirred at 0 ° C. for 5 minutes and at room temperature for 15 hours. The reaction solution was concentrated under reduced pressure, 1,000 mL of water was added to the obtained residue, the mixture was washed twice with 500 mL of ethyl acetate, and the aqueous layer was extracted three times with 1,000 mL of n-butanol. Z-Leu-Arg-OEt was obtained by concentrating the obtained n-butanol layer under reduced pressure.

Z-Leu-Arg-OEt was dissolved in 500 mL of methanol, 150 mL of NH ₂ NH ₂ · H ₂ O was added under ice-cooling, and the mixture was stirred at room temperature for 18 hours. 500 mL of water was added to the residue obtained by concentrating a reaction liquid under reduced pressure, and it extracted 5 times with 1,000 mL of n-butanol. The extract was washed four times with 500 mL of water and then concentrated under reduced pressure. The obtained residue was treated with ether to solidify and dry. In this way a 209.7g (yield: 74.1%) Z-Leu-Arg -NHNH 2 was obtained.

The melting point of Z-Leu-Arg-NHNH _2, the photoluminescence, the Rf value of TLC and the elemental analysis value are shown below.

m.p. : 105 ~ 107 ℃

[α] _D : -30.6 (c = 1.0, MeOH)

Rf: 0.60 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₂₀ H ₃₃ N ₇ O ₄ )

Theoretical C: 55.61% H: 7.64% N: 22.51%

Found C: 55.84% H: 7.42% N: 22.41%

(2-2) Preparation of Z-Leu-Arg-Pro-NHEt

130.8 g of Z-Leu-Arg-NHNH ₂ prepared in (2-1) was dissolved in 500 mL of DMF, and cooled to -20 ° C with dry ice-ethanol. 225 mL of 4N HCl-dioxane and 40.5 mL of isoamyl nitrate were added to the solution to obtain an azide compound. Furthermore, 126 mL of triethylamine was added and neutralized. The mixture was transferred to a 200 mL solution of DMF containing 42.6 g of H-Pro-NHEt, stirred at -20 ° C for 2 hours, and at 4 ° C for 16 hours, and then concentrated. After adding 800 mL of water to the residue and washing twice with 400 mL of ethyl acetate, the aqueous layer was extracted three times with 800 mL of n-butanol and washed with 400 mL of water again. The obtained n-butanol layer was concentrated under reduced pressure, and the obtained residue was treated with ether to solidify and dried. In this manner, 154.5 g (yield 94.3%) of Z-Leu-Arg-Pro-NHEt was obtained.

The melting point of Z-Leu-Arg-Pro-NHEt, photoluminescence, Rf of TLC, and elemental analysis values are shown below.

m.p. 125 to 126 ° C

[α] _D : -66.6 (c = 1.0, MeOH)

Rf: 0.45 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₂₇ H ₄₃ N ₇ O ₅ )

Theoretical C: 59.43% H: 7.94% N: 17.97%

Found C: 59.31% H: 7.78% N: 18.13%

(2-3) Preparation of Z-D-Ser (But) -Leu-Arg-Pro-NHEt

154.5 g of Z-Leu-Arg-NHEt prepared in (2-2) was dissolved in 1,500 mL of methanol, and hydrogen was added in the presence of 14 g of 10% palladium / carbon. After stirring at room temperature for 16 hours, the catalyst was removed and concentrated under reduced pressure to obtain H-Leu-Arg-Pro-NHEt.

82.3 g of ZD-Ser (But) -OH was dissolved in 200 mL of THF, cooled to −20 ° C. with dry ice-ethanol, 30.7 mL of N-methylmorpholine was added dropwise, and then 36.3 mL of isobutylchloroformate was added dropwise. Then, the mixture was stirred for 1 minute at -20 ° C to prepare a corresponding mixed acid anhydride. The obtained reaction solution was mixed with a 700 mL solution of DMF containing H-Leu-Arg-Pro-NHEt and stirred at 0 ° C. for 5 minutes and at room temperature for 30 minutes. Thereafter, the mixture was concentrated under reduced pressure, 1,000 mL of water was added to the residue, the mixture was washed twice with 500 mL of ethyl acetate, and the aqueous layer was extracted twice with 1,000 mL of an ethyl acetate / n-butanol (2/1) mixed solution. The extract was washed 5 times with 500 mL of water, and the residue obtained by concentration under reduced pressure was treated with ether to solidify and dried. Thus, 178.3 g (yield 91.2%) of D-Ser- (But) -Leu-Arg-Pro-NHEt was obtained.

In addition, melting | fusing point of the obtained compound, luminous intensity, Rf of TLC, and elemental analysis value were the same as that of D-Ser (But) -Leu-Arg-Pro-NHEt manufactured by (1-3) of Example 1.

Example 3: Preparation of pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt

(3-1) Preparation of Boc-D-Leu-Leu-Arg (Tos) -Pro-NHEt

20.99 g of Z-Leu-Arg (Tos) -Pro-NHEt prepared in (1-2) of Example 1 was dissolved in 200 mL of methanol, and hydrogen was added in the presence of 10 g of palladium / 1.5 g of carbon. After stirring for 7 hours at room temperature, the catalyst was removed, concentrated under reduced pressure, and the residue was treated with ether to solidify. Thus, H-Leu-Arg (Tos) -Pro-NHEt was obtained.

H-Leu-Arg (Tos) -Pro-NHEt was dissolved in 80 mL DMF and 200 mL THF and neutralized with N-methylmorpholine while cooling the solution. Subsequently, a solution obtained by dissolving 8.86 g of Boc-D-Leu-OH and 23.02 g of HOSu in 200 mL of THF and 36.4 mL of WSC were added thereto, and the mixture was stirred at 0 ° C. for 5 minutes at room temperature overnight. After confirming with ninhydrin, the reaction mixture was concentrated under reduced pressure. 500 mL of water was added to the residue, and extracted with 200 mL of n-butanol five times. After washing five times with water, the n-butanol layer was concentrated under reduced pressure and the residue was treated with ether to solidify. Thus, 21.5 g of Boc-D-Leu-Leu-Arg (Tos) -Pro-NHEt (yield 92.1%) was obtained.

The melting point of Boc-D-Leu-Leu-Arg (Tos) -Pro-NHEt, the photoluminescence, the Rf of TLC and the elemental analysis values are shown below.

m.p. : 119 ~ 121 ℃

[α] _D : -45.2 (c = 1.0, MeOH)

Rf: 0.50 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₃₀ H ₅₆ N ₈ O ₆ )

Theoretical C: 57.67% H: 9.03% N: 17.93%

Found C: 57.43% H: 8.89% N: 17.80%

(3-2) Preparation of Z-Ser-Tyr-D-Leu-Leu-Arg (Tos) -Pro-NHEt

25.8 g of Boc-D-Leu-Leu-Arg (Tos) -Pro-NHEt prepared in (3-1) was dissolved in 10 mL of DCM. 10 mL of TFA was added under ice cooling, and the mixture was stirred at room temperature for 30 minutes. Then, the mixture was concentrated under reduced pressure, and 200 mL of ether was added to the residue to solidify and dry. Thus, H-D-Leu-Leu-Arg (Tos) -Pro-NHEt TFA was obtained.

16.6 g of Z-Ser-Tyr-NHNH ₂ was dissolved in 100 mL of DMF and cooled to −20 ° C. with dry ice-ethanol. 29.8 mL of 4N HCl-dioxane and 5.3 mL of isoamyl nitrate were added to the solution to obtain an azide compound. In addition, 16.7 mL of triethylamine was added to the mixture, which was neutralized, and then transferred to a 200 mL solution of DMF containing HD-Leu-Leu-Arg (Tos) -Pro-NHEtTFA, followed by 2 hours at -20 ° C and 17 hours at 4 ° C. After stirring, it was concentrated. 500 mL of water was added to the residue, and extracted with 200 mL of n-butanol five times. After washing five times with water, the n-butanol layer was concentrated under reduced pressure and the residue was treated with ether to solidify. Thus, 25.0 g of Z-Ser-Tyr-D-Leu-Leu-Arg (Tos) -Pro-NHEt (yield 83.2%) was obtained.

The melting point of Z-Ser-Tyr-D-Leu-Leu-Arg (Tos) -Pro-NHEt, the photoluminescence, Rf of TLC, and elemental analysis values are shown below.

m.p. : 114 to 118 ° C (decomposition)

[α] _D : -30.1 (c = 1.0, DMF)

Rf: 0.32 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₄₅ H ₆₈ N ₁₀ O ₁₀ )

Theoretical C: 59.45% H: 7.54% N: 15.41%

Measured value C: 59.33% H: 7.52% N: 15.32%

(3-3) Preparation of H-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt

32 mL of anisole was added to 20.4 g of Z-Ser-Tyr-D-Leu-Leu-Arg (Tos) -Pro-NHEt prepared in (3-2) above, and cooled to -70 ° C with dry ice-ethanol. . HF 200mL was added here and it stirred at 0 degreeC for 1 hour. Thereafter, the mixture was concentrated under reduced pressure, the residue was treated with ether, and the precipitated product was filtered and dried in vacuo over sodium hydroxide. Thus, 16.0 g (yield 92.4%) of H-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt was obtained.

The melting point of H-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt, the photoluminescence, Rf of TLC, and elemental analysis values are shown below.

m.p. : 121 ~ 123 ℃

[α] _D : +11.0 (c = 1.0, DMF)

Rf: 0.20 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₃₇ H ₆₂ N ₁₀ O ₈ )

Theoretical C: 57.35% H: 8.06% N: 18.07%

Found C: 57.21% H: 8.01% N: 18.10%

(3-4) Preparation of pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt

Instead of H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt, H-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt 1.30 prepared above (3-3) Except for using g, 1.33 g of pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt was obtained by the same method as in (1-11) of Example 1 (yield 76.4 %).

The photoluminescence, elemental analysis and amino acid analysis of pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt are shown below.

[a] _D : -32.0 (c = 0.52, 5% AcOH)

Elemental Analysis Values (as C ₅₉ H ₈₄ N ₁₆ O ₁₂ )

Theoretical C: 58.59% H: 7.00% N: 18.53%

Found: C: 58.40% H: 7.10% N: 18.31%

Amino acid composition

Ser 1.05 (1), Glu 1.08 (1), Pro 0.99 (1), Leu 2.09 (2), Tyr 0.92 (1), His 0.93 (1), Arg 1.06 (1)

Example 4 Preparation of pGlu-His-Trp-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-Azgly-NH ₂

(4-1) Preparation of Z-Pro-Azgly-NH ₂

24.9 g of Z-Pro-OH, 11.2 g of semicarbazide hydrochloride and 14.5 g of triethylamine were dissolved in 200 mL of DMF. It cooled to 0 degreeC, the DCC20.6g was added, and it stirred at 4 degreeC for 16 hours. Thereafter, dicyclohexylurea (DCU) was removed from the reaction mixture by filtration and concentrated under reduced pressure. 500 mL of ethyl acetate was added to the residue, and the mixture was washed twice with 200 mL of water. Concentrated under reduced pressure and the residue was treated with ether to solidify. Thus, 16.7 g (yield 54.3%) of Z-Pro-Azgly-NH ₂ were obtained.

The melting point of Z-Pro-Azgly-NH _2, the photoluminescence, the Rf of TLC and the elemental analysis values are shown below.

m.p. : 189 ~ 190 ℃

[α] _D : -43.6 (c = 1.4, DMF)

Rf: 0.62 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₁₄ H ₁₈ N ₄ O ₄ )

Theoretical C: 54.89% H: 5.95% N: 18.29%

Measured value C: 54.41% H: 7.74% N: 15.60%

(4-2) Preparation of Boc-Arg (NO ₂ ) -Pro-Azgly-NH ₂

16.5 g of Z-Pro-Azgly-NH ₂ prepared in (4-1) was dissolved in 500 mL of DMF, and hydrogen was added in the presence of 1.1 g of 10% palladium / carbon. After stirring for 7 hours at room temperature, the catalyst was removed and concentrated under reduced pressure. 1,500 mL of ether was added to the residue, solidified and dried. Thus, H-Pro-Azgly-NH ₂ · TFA was obtained.

13.5 g of Boc-Arg (NO ₂ ) -OH was dissolved in 100 mL of THF, cooled to −20 ° C. with dry ice-ethanol, and 3.3 mL of N-methylmorpholine was added dropwise, followed by 3.96 mL of isobutylchloroformate. After dropping, the mixture was stirred for 1 minute at -20 ° C to prepare a corresponding mixed acid anhydride. The obtained reaction solution was mixed with 200 mL of a DMF solution (neutralized with N-methylmorpholine) of H-Pro-Azgly-NH ₂ · TFA, stirred at 0 ° C. for 5 minutes, at room temperature for 30 minutes, and then concentrated under reduced pressure. 500 mL of water was added to the residue, and extracted with 200 mL of n-butanol five times. After washing five times with water, the n-butanol layer was concentrated under reduced pressure and the residue was treated with ether to solidify. In this manner, the _{Boc-Arg (NO 2) -Pro} -Azgly-NH 2 16.7g ( yield 88.6%) was obtained.

Melting points, optical fluorescence, Rf of TLC and elemental analysis of Boc-Arg (NO ₂ ) -Pro-Azgly-NH ₂ are shown below.

m.p. : 135 to 137 ° C (decomposition)

[a] _D : +35.3 (c = 1.0, DMF)

Rf: 0.49 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₁₇ H ₃₁ N ₉ O ₇ )

Theoretical C: 43.12% H: 6.60% N: 26.62%

Measured value C: 54.41% H: 7.74% N: 15.60%

(4-3) Preparation of Z-Leu-Arg (NO ₂ ) -Pro-Azgly-NH ₂ (SEQ ID NO: 2)

110.54 g of Boc-Arg- (NO ₂ ) -Pro-NHEt prepared in (4-2) was dissolved in 150 mL of DCM. 150 mL of TFA was added to this solution under ice cooling, and it stirred at room temperature for 30 minutes. Subsequently, the reaction mixture was concentrated under reduced pressure, 1,500 mL of ether was added to the residue, solidified and dried. Thus, H-Arg (NO 2) -Pro-NHEt TFA was obtained.

H-Arg (NO ₂ ) -Pro-NHEt.TFA was dissolved in a mixed solvent of 80 mL of DMF and 200 mL of THF, and the solution was neutralized with N-methylmorpholine while cooling. Subsequently, a solution obtained by dissolving 53.06 g (0.2 mole) of Z-Leu-OH and 23.02 g (0.22 mole) of HOSu in THF 200 mL and 36.4 mL of WSC were added thereto, and the mixture was stirred at 0 ° C. for 5 minutes at room temperature overnight. . After confirming with ninhydrin, the reaction mixture was concentrated under reduced pressure. 1,500 mL of ethyl acetate was added to the residue, and washed twice with 500 mL of water and twice with 500 mL of saturated saline. The ethyl acetate layer was then concentrated under reduced pressure, and the residue was treated with ether to solidify and dry. Thus, 27.52 g (yield 98.6%) of Z-Leu-Arg (NO ₂ ) -Pro-Azgly-NH ₂ (SEQ ID NO: 2) was obtained.

The melting point of Z-Leu-Arg (NO ₂ ) -Pro-Azgly-NH _2, the photoluminescence, the Rf of TLC and the elemental analysis values are shown below.

m.p. : 88 ~ 90 ℃

[a] _D : -30.2 (c = 1.5, DMF)

Rf: 0.57 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₂₆ H ₄₀ N ₁₀ O ₈ )

Theoretical C: 50.31% H: 6.50% N: 22.57%

Measured value C: 50.24% H: 6.41% N: 22.45%

(4-4) Preparation of ZD-Ser (But) -Leu-Arg-Pro-Azgly-NH ₂

16.5 g of Z-Leu-Arg (NO ₂ ) -Pro-Azgly-NH ₂ (SEQ ID NO: 2) prepared in (4-3) was dissolved in 500 mL of DMF, and hydrogen was present in the presence of 1.1 g of 10% palladium / carbon. Was added. After stirring for 7 hours at room temperature, the catalyst was removed and concentrated under reduced pressure. 1,500 mL of ether was added to the residue, solidified and dried. Thus, H-Leu-Arg-Pro-Azgly-NH ₂ (SEQ ID NO: 3) was obtained.

8.86 g of ZD-Ser (But) -OH was dissolved in 100 mL of THF, cooled to −20 ° C. with dry ice-ethanol, and 3.3 mL of N-methylmorpholine was added dropwise, followed by dropwise addition of 3.96 mL of isobutylchloroformate. After that, the mixture was stirred for 1 minute at -20 ° C to prepare a corresponding mixed acid anhydride. The reaction solution was mixed with a 200 mL solution of DMF containing H-Leu-Arg-Pro-Azgly-NH ₂ (neutralized with N-methylmorpholine), stirred at 0 ° C. for 5 minutes, and at room temperature for 30 minutes, and then decompressed. Concentrated. 500 mL of water was added to the residue, and extracted with 200 mL of n-butanol five times. After washing five times with water, the n-butanol layer was concentrated under reduced pressure and the residue was treated with ether to solidify. Thus, 16.9 g of ZD-Ser (But) -Leu-Arg-Pro-Azgly-NH ₂ (yield 78.3%) was obtained.

The melting point of ZD-Ser (But) -Leu-Arg-Pro-Azgly-NH _2, the photoluminescence, Rf of TLC, and elemental analysis values are shown below.

m.p. : 115 ~ 118 ℃

[α] _D : -45.8 (c = 1.0, DMF)

Rf: 0.51 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₃₃ H ₅₄ N ₁₀ O ₈ )

Theoretical C: 55.14% H: 7.57% N: 19.48%

Found C: 55.01% H: 7.42% N: 19.33%

(4-5) Preparation of Z-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-Azgly-NH ₂

26.7 g of ZD-Ser (But) -Leu-Arg-Pro-Azgly-NH ₂ prepared in (4-4) was dissolved in 500 mL of methanol, and hydrogen was added in the presence of 1.9 g of 10% palladium / carbon. After stirring for 7 hours at room temperature, the catalyst was removed and concentrated under reduced pressure. The residue was treated with ether and the precipitated product was filtered and dried in vacuo over sodium hydroxide. Thus, HD-Ser (But) -Leu-Arg-Pro-Azgly-NH ₂ was obtained.

18.5 g of Z-Ser-Tyr-NHNH ₂ prepared in (1-5) of Example 1 was dissolved in 150 mL of DMF, and cooled to −20 ° C. with dry ice-ethanol. 33.4 mL of 4N HCl-dioxane and 6.0 mL of isoamyl nitrate were added to the solution to obtain an azide compound. In addition, 18.7 mL of triethylamine was added to the mixture, which was neutralized, and transferred to a 150 mL solution of DMF containing HD-Ser (But) -Leu-Arg-Pro-Azgly-NH ₂ . After stirring for time, it was concentrated. 500 mL of water was added to the residue, and extracted with 200 mL of n-butanol five times. After washing five times with water, the n-butanol layer was concentrated under reduced pressure and the residue was treated with ether to solidify. Thus, 28.3 g (yield 78.6%) of Z-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-Azgly-NH ₂ was obtained.

The melting point, photoluminescence, Rf of TLC and elemental analysis of Z-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-Azgly-NH ₂ are shown below.

m.p. : 110 to 113 ° C (decomposition)

[α] _D : -28.2 (c = 1.0, DMF)

Rf: 0.36 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₄₅ H ₆₈ N ₁₂ O ₁₂ )

Theoretical C: 55.77% H: 7.07% N: 17.34%

Found C: 55.64% H: 7.01% N: 17.29%

(4-6) Preparation of H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-Azgly-NH ₂

25.4 g of Z-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-Azgly-NH ₂ prepared in (4-5) above was dissolved in 500 mL of methanol and 1.3 g of 10% palladium / carbon was present. Hydrogen was added under. After stirring for 7 hours at room temperature, the catalyst was removed and concentrated under reduced pressure. The residue was treated with ether to solidify. Thus, 20.7 g (yield 94.6%) of H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-Azgly-NH ₂ was obtained.

The melting point, linearity, Rf of TLC, and elemental analysis of H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-Azgly-NH ₂ are shown below.

m.p. : 120-122 degreeC

[a] _D : +11.2 (c = 1.0, DMF)

Rf: 0.31 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₃₇ H ₆₂ N ₁₂ O ₁₀ )

Theoretical C: 53.22% H: 7.48% N: 20.13%

Found C: 53.193% H: 7.43% N: 20.01%

(4-7) Preparation of pGlu-His-Trp-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-Azgly-NH ₂

Instead of H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt, H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro prepared as (4-6) above PGlu-His-Trp-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro by the same method as (1-11) of Example 1, except that 1.30 g of -Azgly-NH ₂ was used. 1.29 g (Yield 75.9%) of -Azgly-NH ₂ was obtained.

The melting point of the pGlu-His-Trp-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-Azgly-NH _2, the photoluminescence, the Rf of the TLC and the elemental analysis values are shown below.

[α] _D : -52.4 (c = 1.0, DMF)

Elemental Analysis Values (as C ₅₉ H ₈₄ N ₁₈ O ₁₄ )

Theoretical C: 55.82% H: 6.67% N: 19.86%

Measured value C: 55.70% H: 6.55% N: 19.72%

Amino acid composition

Ser 2.02 (2), Glu 1.07 (1), Pro 1.00 (1), Leu 1.03 (1), Tyr 0.92 (1), His 0.94 (1), Arg 1.02 (1)

Example 5: Preparation of pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH ₂

(5-1) Preparation of H-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH ₂

Solid phase synthesis was performed using a peptide synthesizer 9600 manufactured by Milligen Bioresearch as a solid phase synthesis apparatus.

First, 694 mg of p-methylbenzhydrylamine (MBHA) resin (Peptide Researcher, 0.72 mmol / g amino group) was added to a reaction vessel for peptide high phase synthesis, DCM 8 mL (4 times, 1 min each), DCM containing 60% TFA 8 mL of solution (20 minutes), 4 mL of DCM (3 times, 15 seconds each), 3 mL of DMF solution containing 1 mL of DIEA (2 times, 1 minute each) 8 mL of DMF (6 times, 40 seconds each) in the argon gas stream Agitation was performed. In addition, filtration was performed for each treatment.

On the other hand, ₂ mmol of Boc-Gly-OH reacting with the 10th amino acid residue of the amino acid sequence of pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH ₂ was dissolved in 4 mL of DCM. . This solution was added to the amino acid activation vessel, and 3 mL of DCC (0.5M-DCM solution) and 4 mL of HOBt (0.5M-DCM solution) were added to react for 30 minutes. The reaction solution was filtered and transferred to a concentration vessel. 3 mL of DMF was added thereto, DCM was distilled off under an argon gas stream, and then 3 mL of DMF was added to the reaction vessel for peptide solid phase synthesis, followed by reaction for 30 minutes. Then washed with 8 mL of DCM (6 times, 20 seconds each). Further, 2 mmol of Boc-Gly-OH was dissolved in 4 mL of DCM, and then subjected to a so-called double coupling method in which the same operation was repeated in an amino acid activation reaction vessel, followed by filtration to obtain a Boc-Gly-MBHA resin.

Subsequently, the obtained Boc-Gly-MBHA resin was washed with 8 mL of DCM (4 times, 1 minute each) and filtered. It contains 8 mL of DCM (20 minutes) with 60% TFA, 4 mL of DCM (3 times for 15 seconds each), 3 mL of DMF solution containing 1 mL of DIEA (2 times for 1 minute each), and 8 mL of DMF (6 times for 40 seconds each). In order, it processed by stirring in argon gas airflow, and it filtered by each process.

In addition, ₂ mmol of Boc-Pro-OH reacting with the ninth amino acid residue of the amino acid sequence of pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH ₂ was dissolved in 4 mL of DCM. Into the amino acid activation vessel, 1.5 mL of DCC (0.5M-DCM solution) was added and allowed to react for 7 minutes. Thereafter, the reaction mixture was filtered and transferred to a concentration vessel, and 3 mL of DMF was added thereto, and DCM was removed under an argon gas stream. 3 mL of DMF was added thereto, and the reaction mixture was transferred to a reaction vessel for peptide solid phase synthesis and allowed to react for 30 minutes. Then washed with 8 mL of DCM (6 times, 20 seconds each) and filtered to give Boc-Pro-Gly-MBHA resin.

Hereinafter, 8th to 4th amino acids were coupled sequentially using the amino group protection amino acid shown in Table 2.

Amino acid sequence Protection amino acid Usage (mmol) 87654 Boc-Arg (Tos) -OHBoc-Leu-OHBoc-D-Trp-OHBoc-Tyrr (Bzl) -OHBoc-Ser (Bzl) -OH 2 × 22222

In the above-mentioned solid phase synthesis, when arginine was used, double coupling was performed.

Thus, 2.76 g of a protective peptide-MBHA resin and Boc-Ser (Bzl) -Tyr (Bzl) -D-Trp-Leu-Arg (Tos) -Pro-Gly-MBHA resin were obtained.

5 mL of anisole was added to 2.76 g of the protective peptide-MBHA resin, and 25 mL of anhydrous hydrogen fluoride (HF) was added, followed by stirring at 0 ° C for 1 hour. After the reaction, anhydrous hydrogen fluoride was distilled off under reduced pressure and the residue was washed with ether. Thus, 1.30 g of H-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH ₂ was obtained.

The photosensitivity of H-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH ₂ , Rf of TLC and elemental analysis values are shown below.

[α] _D : -50.4 (c = 1.0, MeOH)

Rf: 0.13 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental analysis value (as C ₅₉ H ₈₄ N ₁₆ O ₁₂ .AcOH.2H ₂ O)

Theoretical C: 54.31% H: 7.04% N: 17.27%

Found C: 54.20% H: 6.89% N: 16.97%

(5-2) Preparation of pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH ₂

Instead of H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt, H-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly- PGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH ₂ 1.26 by the same method as (1-11) of Example 1, except that NH ₂ 1.30 g was used. g (yield 75.45%) was obtained.

The photoluminescence of pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH ₂ , Rf of TLC and elemental analysis values are shown below.

[α] _D : -56.6 (c = 1.0, H ₂ O)

Elemental analysis value (as C ₆₄ H ₈₂ N ₁₈ O ₁₃ · AcOH · 2H ₂ O)

Theoretical C: 56.32% H: 6.44% N: 17.91%

Found C: 56.20% H: 6.38% N: 16.97%

Amino acid composition

Ser 0.98 (1), Glu 1.07 (1), Gly 1.01 (1), Pro 0.99 (1), Leu 1.00 (1), Tyr 0.92 (1), His 0.95 (1), Arg 1.08 (1)

Example 6: Preparation of pGlu-His-Trp-Ser-Tyr- (2-naphthyl) -D-Ala-Leu-Arg-Pro-Gly-NH ₂

(6-1) Preparation of H-Ser-Tyr- (2-naphthyl) -D-Ala-Leu-Arg-Pro-Gly-NH ₂

Example 5 (5-1), except that Boc-D- (2-naphthyl) -D-Ala-OH was used instead of Boc-D-Trp-OH corresponding to the sixth amino acid residue 0.98 g of H-Ser-Tyr- (2-naphthyl) -D-Ala-Leu-Arg-Pro-Gly-NH ₂ was obtained.

The photosensitivity of H-Ser-Tyr- (2-naphthyl) -D-Ala-Leu-Arg-Pro-Gly-NH ₂ , Rf of TLC and elemental analysis values are shown below.

[α] _D : -44.1 (c = 1.0, MeOH)

Rf: 0.15

Elemental Analysis Values (as C ₄₇ H ₆₆ N ₁₂ O ₁₀ )

Theoretical C: 58.86% H: 6.94% N: 17.52%

Found C: 58.72% H: 6.88% N: 17.49%

(6-2) Preparation of pGlu-His-Trp-Ser-Tyr- (2-naphthyl) -D-Ala-Leu-Arg-Pro-Gly-NH ₂

Instead of H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt, H-Ser-Tyr- (2-naphthyl) -D-Ala-Leu produced by (6-1) above PGlu-His-Trp-Ser-Tyr- (2-naphthyl) -D by the same method as (1-11) of Example 1, except that 0.98 g of -Arg-Pro-Gly-NH ₂ was used. 0.93 g (76.2% yield) of Ala-Leu-Arg-Pro-Gly-NH ₂ was obtained.

The photoluminescence, elemental analysis and amino acid analysis of pGlu-His-Trp-Ser-Tyr- (2-naphthyl) -D-Ala-Leu-Arg-Pro-Gly-NH ₂ are shown below.

[α] _D : -50.3 (c = 1.0, H ₂ O)

Elemental Analysis Values (as C ₆₉ H ₈₈ N ₁₈ O ₁₄ )

Theoretical C: 59.47% H: 6.37% N: 18.09%

Found C: 59.40% H: 6.33% N: 17.98%

Amino acid composition

Ser 0.98 (1), Glu 1.05 (1), Gly 1.00 (1), Pro 1.03 (1), Leu 1.00 (1), Tyr 0.93 (1), His 0.94 (1), Arg 1.07 (1)

Example 7: Preparation of pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂ (SEQ ID NO: 1)

(7-1) Preparation of H-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂ (SEQ ID NO: 4)

Instead of Boc-D-Trp-OH corresponding to the sixth amino acid residue, H-Ser-Tyr- was subjected to the same treatment as in Example 5 (5-1) except that Boc-Gly-OH was used. 1.12 g of Gly-Leu-Arg-Pro-Gly-NH ₂ (SEQ ID NO: 4) was obtained.

The photoluminescence of H-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂ , Rf of TLC and elemental analysis values are shown below.

[α] _D : -40.1 (c = 1.0, MeOH)

Rf: 0.10 (BuOH / AcOH / H ₂ O = 4/1/5)

Elemental Analysis Values (as C ₃₃ H ₅₃ N ₁₁ O ₉ )

Theoretical C: 53.00% H: 7.14% N: 20.60%

Found C: 53.20% H: 6.89% N: 20.97%

(7-2) Preparation of pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂ (SEQ ID NO: 1)

Instead of H-Ser-Tyr-D-Ser (But) -Leu-Arg-Pro-NHEt, H-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂ produced by (6-1) above (SEQ ID NO: 4) pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂ by the same method as (1-11) of Example 1, except that 1.12 g was used. (SEQ ID NO: 1) 1.16 g (yield 76.3%) were obtained.

Elemental analysis and amino acid analysis of pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂ are shown below.

Elemental analysis value _{(C 55 H 75 N 17 O} 13 · AcOH · 2H ₂ O a)

Theoretical C: 53.55% H: 6.54% N: 18.63%

Found C: 53.85% H: 6.89% N: 18.97%

Amino acid composition

Ser 1.01 (1), Glu 1.03 (1), Gly 2.01 (2), Pro 0.98 (1), Leu 1.01 (1), Tyr 0.94 (1), His 0.94 (1), Arg 1.08 (1)

Since the method of the present invention uses an enzymatic reaction, it does not involve side reactions such as racemization and the like, so that the LH-RH derivative can be easily separated and purified. In addition, since the yield is high and unreacted peptide fragments can be recovered and reused, they are very useful industrially.

Claims (7)

The peptide fragment represented by the following general formula (1) and the peptide fragment represented by the following general formula (2) are reacted in the presence of an enzyme selected from the group consisting of chymotrypsin or chymotrypsin-like enzyme. Process for preparing LH-RH derivative represented by (3): pGlu-His-Trp-OR ¹ (1) H-Ser-Tyr-X-Leu-Arg-Pro-Y (2) pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (3) Where R ¹ represents lower alkyl, X represents an amino acid selected from the group consisting of D-Leu, D-Ser (But), D-Trp, (2-naphthyl) -D-Ala, Leu and Gly, Y represents Gly-NH ₂ , Azgly (azaglycine) -NH ₂ or NHR ² (R ² is lower alkyl). The method of claim 1, Method for producing a LH-RH derivative, characterized in that the electric R ¹ is a C1-3 alkyl group. The method according to claim 1 or 2, Method for producing a LH-RH derivative, characterized in that the electricity R ² is an alkyl group having 1 to 3 carbon atoms. The method according to any one of claims 1 to 3, A method for producing an LH-RH derivative, wherein the enzyme selected from the group consisting of the aforementioned chymotrypsin or chymotrypsin-like enzyme is chymotrypsin. The peptide fragment represented by the following general formula (4) and the peptide fragment represented by the following general formula (5) include water or a buffer solution and an organic solvent in the presence of an enzyme selected from the group consisting of chymotrypsin or chymotrypsin-like enzyme. Process for preparing LH-RH derivative represented by the following general formula (6), characterized in that the reaction in a mixed solvent: pGlu-His-Trp-OR ¹ (4) H-Ser-Tyr-X-Leu-Arg-Pro-Y (5) pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (6) Where R ¹ represents lower alkyl, X represents an amino acid selected from the group consisting of D-Leu, D-Ser (But), D-Trp, (2-naphthyl) -D-Ala and Gly, Y represents Gly-NH ₂ , Azgly-NH ₂ or NHR ² (R ² is lower alkyl). The method of claim 5, The solvent in which the electric water or the buffer solution and the organic solvent are mixed is water or a mixed solvent of the buffer solution with the water-miscible organic solvent, or a mixed solvent saturated with an organic solvent which partially mixes the water or the buffer solution with water. Method for producing a LH-RH derivative. In the presence of an immobilized enzyme wherein the peptide fragment represented by the following general formula (7) and the peptide fragment represented by the following general formula (8) are immobilized with an enzyme selected from the group consisting of chymotrypsin or chymotrypsin-like enzyme, A method for preparing an LH-RH derivative represented by Formula (9), which is characterized by reacting a buffer solution with an organic solvent in a mixed solvent: pGlu-His-Trp-OR ¹ (7) H-Ser-Tyr-X-Leu-Arg-Pro-Y (8) pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (9) Where R ¹ represents lower alkyl, X represents an amino acid selected from the group consisting of D-Leu, D-Ser (But), D-Trp, (2-naphthyl) -D-Ala and glycine, Y represents Gly-NH ₂ , Azgly-NH ₂ or NHR ² (R ² is lower alkyl).