CA2299267A1 - Process for producing lh-rh derivatives - Google Patents

Abstract

A process for producing LH-RH derivatives represented by the following general formula (3): pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (wherein X represents an amino acid selected from the group consisting of D-amino acids such as D-Leu, D-Ser(But), D-Trp and (2-naphthyl)-D-Ala, and Gly; and Y represents Gly-NH2, Azgly(azaglycine)-NH2 or NHR2 wherein R2 is lower alkyl), characterized by reacting a peptide fragment represented by the following general formula (1):
pGlu-His-Trp-OR1 (wherein R1 represents lower alkyl) with another peptide fragment represented by the following general formula (2): H-Ser-Tyr-X-Leu-Arg-Pro-Y in the presence of chymotrypsin or chymotrypsin-like enzyme. This process is highly useful industrially, since the LH-RH derivatives can be easily separated and purified in a high yield without any side reaction such as racemization and unreacted peptide fragments can be recovered and reused.

Description

Amended: 05/28/99 METHOD FOR PRODUCING LH-RH DERIVATIVES
The present invention relates to a method using enzymes for effectively producing a LH-RH and/or its derivatives, which is a useful peptide as a drug.
Background Art Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are released from the anterior pituitary under the control of LH-RH (luteinizing hormone releasing hormone) that is produced at the hypothalamus. LH-RH
and derivatives thereof have a secretory activity of gonadotropin and are shown that the sequential administration of LH-RH and its derivatives inhibits gonad functions so that they are applied as drugs to prevent and/or treat diseases such as endometriosis, central precocious puberty, infertility, and prostate cancer. LH-RH and its derivatives that have been applied as drugs include buserelin (JP-B-60-9519 official gazette), goserelin (JP-B-61-13480), leuprorelin (JP-B-53-14072), and napharelin (JP-B-63-56238).
As a method for producing the above described LH-RH and derivatives, a chemical synthesis method by the liquid phase synthesis method wherein peptide fragments with partial sequences corresponding to the polypeptides are formed by the liquid phase or the solid phase method, then each fragment is coupled in liquid phase is known (JP-B-56-47175, JP-A-49-117468, JP-B-57-29462, JP-B-57-25540, JP-B-63-17839, JP-B-63-45398, JP-A-48-40770, JP-A-50-88069, JP-A-49-41375, JP-A-49-41376, JP-A-48-99170, JP-B-52-20996, JP-A-49-35381, JP-B-52-8831, JP-B-57-61268, JP-B-53-14072, JP-B-57-26506, JP-B-60-22720, JP-B-61-13480, and JP-B-3-71439).
However, in the liquid phase synthesis method the solubility changes subtly as the number of amino acid residues of a peptide increases, making it difficult to find an appropriate solvent. As such difficulty increases it also becomes more difficult to separate the peptide of interest from unreacted Amended: 05/28/99 substances and from side-products. Particularly the serine residue at position 4 of LH-RH and its derivatives tends to be racemized thus remains as impurities and so recovery of raw materials is impossible. Therefore the method is not technically satisfactory since posttreatment of the reaction is difficult and uneconomical.
Summary of the Invention The object of the invention is to provide a method for efficiently producing a LH-RH and/or its derivative in a large scale at lower costs.
As a result of our intensive researches to solve the abovementioned problems, we have achieved the present invention by finding a method for producing LH-RH derivatives suitable for industrial production by means of an enzymatic synthesis.
The present invention is characterized in that a peptide fragment shown by general formula (1):
pGlu-His-Trp-OR1 (1) (where R1 denotes lower alkyl) and a peptide fragment shown by general formula (2):
H-Ser-Tyr-X-Leu-Arg-Pro-Y (2) (where X denotes an amino acid selected from the group consisting of D-Leu, D-Ser(But), D-Trp, (2-naphthyl)-D-Ala, and Gly; Y denotes Gly-NHS, Azaglycine or NHR2 (where R~ is lower alkyl)) are allowed to react in the presence of an enzyme selected from the group consisting of chymotrypsin and chymotrypsin-like enzymes, and in a reaction medium consisting of a buffer itself or in a reaction medium in which an organic solvent selected from the group consisting of dimethylformamide, dimethylimidazolidinone, hexamethylphosphoryltriamide, methanol, ethanol, butanol, and ethyl acetate, and a buffer or water are mixed, to produce the LH-RH or its derivatives shown by general formula (3):
pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (3) (where X and Y are as defined above).
The present invention is characterized by further comprising a step of Amended: 05/28/99 purifying the peptide shown by general formula (3) by partition chromatography. In addition, in the present invention the said reaction can be performed at 0 to 20~C. Here the said R' may be an alkyl group having 1 to 3 carbons and RJ' may be an alkyl group having 1 to 3 carbons. Further in the present invention, the enzyme selected from the group consisting of the chymotrypsin and chymotrypsin-like enzymes may be chymotrypsin.
Moreover, the present invention is characterized in that a peptide fragment shown by general formula (4):
pGlu-His-Trp-OR1 (4) (where R1 denotes lower alkyl) and a peptide fragment shown by general formula (5):
H-Ser-Tyr-X-Leu-Arg-Pro-Y (5) (where X denotes an amino acid selected from the group consisting of D-Leu, D-Ser(But), D-Trp, (2-naphthyl)-D-Ala, and Gly; Y denotes Gly-NHS, Azaglycine. or NHRZ (where RZ is a lower alkyl)) are allowed to react in the presence of an immobilized enzyme selected from the group consisting of chymotrypsin and chymotrypsin-like enzymes, and in a reaction medium consisting of a buffer itself or in a reaction medium in which an organic solvent selected from the group consisting of dimethylformamide, dimethylimidazolidinone, hexamethylphosphoryltriamide, methanol, ethanol, butanol, and ethyl acetate, and a buffer or water are mixed, to produce the LH-RH or its derivative shown by general formula (6):
pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (6) (where X and Y are as defined above) The present invention is characterized by further comprising a step of purifying the peptide shown by general formula (3) by partition chromatography. In addition, in the present invention the said reaction can be performed at 0 to 20'C. Here the said R' may be an alkyl group having 1 to 3 carbons and R'- may be an alkyl group having 1 to 3 carbons.
In this specification, the LH-RH derivative means as the one wherein Glycine at position 6 or at position 10 of the LH-RH shown by general formula (7) (SEQ. ID.NO:1):

Amended: 05/28/99 PGIu-His-'I~p-Ser-'I~r-Gly-Leu-Arg-Pro-Gly-NHS (7) is substituted by a different amino acid, that is, a special amino acid or a modified amino acid. An amino acid at the position corresponding to Gly at position 6 (hereinafter referred to as X) may be, but not limited to, D-amino acid or a modified D-amino acid. For example, the D-amino acid mentioned here may be D-Leu, D-Tryp, D-Ala, D-Phe, D-Val, or D-His and the modified amino acid may be D-Ser(But) or (2-naphthyl)-D-Ala. In addition, X may be an L-amino acid. When X is a D-amino acid, it is preferably selected from the group consisting of D-Leu, D-'I~p, D-Ala, D-Ser(But) and (2-naphthyl)-D-Ala and when X is an L-amino acid it is preferably Glycine. Glycine at position (hereinafter referred to as ~ is preferably Gly-NHS, Azgly-NH.~ or NHR
(where R' is lower alkyl).
"Lower alkyl" used herein means an alkyl having 1 to 3 carbons such as methyl, ethyl, propyl, or isopropyl. R1 is preferably a methyl or an ethyl group and RZ is preferably an ethyl or a methyl group.
Besides in this specification, abbreviations used for amino acids, peptides, protecting groups, solvents and others are according to rules of International Union of Pure and Applied Chemistry (IUPAC) and of International Union of Biochemistry (IUB) or to conventional symbols in the field the present invention pertains to. Examples are shown below. Possible optical isomers of amino acids indicate L- configuration unless otherwise indicated.
Tyr : Tyrosin 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 :

~p 'I~yptophane residue :

Amended: 05/28/99 Et : Ethyl Boc : t-Butoxycarbonyl Aoc : t-Amyloxycar bonyl Bz : Benzyl Z : Benzyloxycarbonyl Tos : Tosyl OMe : Methyl ester OBz : Benzyl ester OSu N-Hydroxysuccinimide ester :

TFA Trifluoroacetic acid :

THF Tetrahydrofuran :

DMF Dimethylformamide :

DCC Dicyclohexylcarbodiimide :

WSC N-ethyl-N'-dimethylaminopropyl-carbodiimide :

HOSu N-hydroxysucciniimide :

HOBt 1-Hydroxybenzotriazol :

MeOH Methanol :

EtOH Ethanol :

AcOH Acetic acid :

The peptide fragment shown by the general formula (1) corresponds to amino acid residues at positions 1 to 3 of the amino acid sequence of the LH-RH
and/or its derivative shown by the general formula (3). Further, the peptide fragment shown by the general formula (2) corresponds to amino acid residues at positions 4 to 10 of the amino acid sequence of the LH-RH and/or its derivatives shown by the general formula (3).
Each of the peptide fragments shown by the general formula (1) or shown by the general formula (2) can be synthesized according to a well-known method for synthesizing peptides. For example, according to the methods described in The Pep id .~. Vol 1, Schreder and Luhke., 1966., Academic Press, New York, U.S.A., or in Peptide ~vnthesi~, Izumiya et.al., 19'75., Maruzen Corporation, peptide fragments can be synthesized by the azido method, acid chloride method, acid anhydride method, mixed acid anhydride method, DCC method, activated ester methods (e.g., p-nitrophenyl ester method, N-hydroxysuccinimide ester method, and cyanomethyl ester method), method Amended: 05/28/99 using Woodword's Reagent K, carboimidazole method, oxidation-reduction method, DCC-additive (HONB, HOBt, HOSu) method, and solid phase method. With these general methods for synthesizing peptides, peptides can be produced for example by a so-called stepwise elongation method wherein one amino acid is in order condensed with a C-terminal amino acid according to the amino acid sequence or by the fragment condensation method wherein fragments each composed of several fragments are synthesized and coupled to each other.
In the reaction process for synthesizing the peptide fragments shown by the above general formula (1) or (2), functional groups that are not involved in the reaction are protected by normal protective groups and after the completion of the reaction the protective groups are removed. Furthermore, functional groups involved in the reaction are normally activated. Each of these reaction methods is well known and the reagents used therein can be properly selected from well-known reagents.
The protective groups of amino groups include, for example, benzyloxycarbonyl (Z), t-butyloxycarbonyl (Boc), t-amyloxycarbonyl (Aoc), isobonyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2-chloro-benzyloxycarbonyl, adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl, o-nitrophenylsulphenyl, and diphenylphosphinothioyl. Normally Boc is used to protect amino groups. But when D-Ser (But) is coupled, the use of Z
group allows to remove only Z group without removing But (t-butyl) group.
The protective groups of carboxyl groups include, for example, alkylesters (for example, methyl ester, ethyl ester, propyl ester, butyl ester, and tert-butyl ester), benzyl ester, p-nitrobenzyl ester, methyl benzyl ester, p-chlorobenzyl ester, benzhydryl ester, benzyloxycarbonyl hydrazide, tert-butyloxycarbonylhydrazide, and tritylhydrazide. To hydrazidize, methyl ester or ethyl ester is preferably used.
Activated carboxyl groups involved in peptide synthesis include, for example, acid chloride, acid anhydride, mixed acid anhydride, azide, activated esters (for example, esters of pentachlorophenol, p-nitrophenol, N-hydroxysucciniimide, N-hydroxybenztriazole, and N-hydroxy-5-norbornene-Amended: 05/28/99 2,3-dicarboxyimide). Among them the azide method with less racemization tendency is preferably used upon condensation of fragments.
In addition, peptide bond synthesis reaction may be performed in the presence of condensing agents, such as carbodiimide reagents including dicyclohexyl carbodiimide, and carbodiimidazole or in the presence of tetraethylpyrophosphate.
Usually proteolytic enzymes have been mainly used to cleave peptide bonds.
Further it has been well known from old times that proteolytic enzymes can also be involved in the reverse reaction to synthesize peptide bonds.
Synthesis of long-chain peptides with enzymes is limited to a case that the structure contains amino acids having a limited substrate specificity or in a case that an enzyme has a limited substrate specificity. Thus general enzymes, such as trypsin and chymotrypsin, are rarely used in peptide synthesis reactions. Accordingly, enzyme reaction is mainly used for relatively short-chain peptide bonds, such as oligopeptides, but it still takes time for examining synthesis conditions. Because of this, chemical synthesis is generally used to produce peptides, and enzymatic synthesis is tried only when in chemical synthesis many side reactions occur or reactions are difficult. Enzymatic synthesis can be extremely useful, when the synthesis is performed under mild conditions that enables mass production and the above-mentioned problems in chemical synthesis are solved with appropriate conditions.
As a result of intensive researches, the present inventors have succeeded in effectively producing the LH-RH and/or its derivative shown by the general formula (3) by allowing the peptide fragment shown by the general formula (1) and the one shown by the general formula (2) to react in the presence of chymotrypsin or a chymotrypsin-like enzyme so as to couple to each other.
Chymotrypsin utilized in the present invention is a kind of serine protease that is registered as enzyme number EC.3.4.21.1 of International Union of Biochemistry (IUB), Enzyme Committee, and is obtained from bovine pancreas. Chymotrypsin is on market and sold by SIGMA Corporation, etc.

Amended: 05/28/99 Chymotrypsin-like enzymes are proteolytic enzymes that recognize aromatic amino acids such as Tyr and Phe, and for example include a -chymotrypsin.
Chymotrypsin or chymotrypsin-like enzymes are used preferably because one of the recognition sites of chymotrypsin or chymotrypsin-like enzymes, Trp, is present in LH-RH derivatives.
Reaction of chymotrypsin or chymotrypsin-like enzymes are performed normally in a medium containing a buffer with a pH of about 5 to about 10, preferably pH of about 6 to about 9, more preferably pH of about 7.5 to about 8.5.
The term "medium containing a buffer" used herein refers to a solvent having a buffer itself as a medium, a mixture of a buffer (selected from various ones explained next) and an organic solvent miscible with water, or a mixture of such a buffer and an organic solvent partially miscible with water.
The buffer is not particularly limited and various buffers can be used as far as the pH is within the above range. For example, the buffer includes Tris -hydrochloric acid, MacIlvaine's buffer, phosphate buffer, ammonium acetate buffer, Atkins & Pantin buffer and Veronal buffer.
When the buffer is used as a reaction medium, the buffer is usually used by mixing with an organic solvent miscible with water. The organic solvents miscible with water include dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylimidazolidinone (DMI), hexamethylphosphoryltriamide (HMPA), methanol (l~IeOH), ethanol (EtOH). Dimethylformamide, methanol, and ethanol are preferable. Because these solvents are easy to remove after reaction.
In addition, an organic solvent partially miscible with water such as n-butanol (1-BuOH) or ethyl acetate (EtOAc) can be used. In the use of such an organic solvent, BuOH is preferably used to facilitate the operation of a partition chromatography in the posttreatment. Because BuOH does not need desalting in the posttreatment.

Amended: 05/?8/99 These organic solvents can be used alone or in combination of the two or more of them.
When the reaction of chymotrypsin and chymotrypsin-like enzymes is performed in a mixture of water or a buffer and an organic solvent, not in a solvent consisted only of water or a buffer, the yield of a peptide of interest can significantly be improved.
Generally, the mixing ratio of the organic solvent miscible with water to water or buffer is preferably 50 volume% or less in view of the reactivity.
When an immobilized enzyme, such as chymotrypsin immobilized with celite, is used, the preferable mixing ratio is 80 volume% or more. When an organic solvent partially miscible with water is used, there is an advantage that the use of the organic solvent saturated with water results in high reaction efficiency of enzymes.
The above described reaction of enzymes is performed within a temperature range wherein chymotrypsin or chymotrypsin-like enzymes can act, normally about 0'C to about 50~C, preferably about 0'C to about 20~C. The reaction at about 10°C has an effect capable of inhibiting a hydrolysis reaction that occurs simultaneously.
Amount of chymotrypsin or of cymotrypsin-like enzymes to be used is not specifically limited and can be varied depending on reaction conditions. For example, the use of about 50mg to about 100mg of chymotrypsin based on 50g of a substrate leads to the synthesis of a target peptide in a yield ranging from about 75 to about 85% after 1-hour reaction.
Normally 1 to 5 moles, or preferably 2 to 4 moles of the peptide fragment shown by general formula (1) is used per mole of the peptide fragment shown by general formula (2) In the method of the present invention, chymotrypsin or chymotrypsin-like enzymes dissolved in water or an appropriate buffer can be used, or those immobilized by general methods including the carrier-coupling method, crosslinking method, entrapping method, and other methods can be used as Amended: 05/28/99 immobilized enzymes. The carriers used in the carrier-coupling method include polysaccharide derivatives, such as cellulose, dextrun, and agarose;
polyacrylamide gel; celite; and porous glass. The reagents for crosslinking in the crosslinking method, for example, include glutaraldehyde, bisdiazobenzidine, N,N-polymethylenebisiodoacetamide, and N,N-ethylene bismaleinimide. The materials used in the entrapping method include, for example, polyacrylamide gel, polyacrylalcohol gel, starch, konnyaku powder, nylon, polyurea, polystyrene, ethyl cellulose, colodion, and cellulose nitrate.
However, the immobilization method is not limited to those using the above methods.
In the production method according to the present invention, the LH-RH and its derivative shown by the following formula (8) pGlu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt (8) can be obtained for example in the manner described below:
That is, a given amount of a peptide fragment shown by the following formula (9) PGlu-His-Trp-OMe (9) and a given amount of a peptide fragment shown by the following formula (13) H-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt (10) are stirred in n-butanol saturated water at a pH of around 7.8 at about 10~C
for 1 hour in the presence of chymotrypsin having a given activity, to obtain the LH-RH derivative. Preferable activity of chymotrypsin is about 1000 or more international unit (CT)/mg in order to facilitate the removal thereof in the posttreatment. A high yield can obtained in the reaction by using the molar ratio 3 : 1 of the peptide fragment shown by formula (9) to the one shown by (10). In addition, as an alternative for the addition of enzymes dissolved in water or buffer to the reaction fluid, for example, chymotrypsin immobilized with agarose according to conventional methods can be used as an immobilized enzyme. The use of immobilized enzymes facilitates removal of them by filtering the reaction fluid through a filter such as a glass filter after the completion of the reaction and allows recycling of them.
The LH-RH and/or its derivatives can be obtained as described above and the obtained LH-RH and/or its derivatives can be purified as follows.

Amended: 05/28/99 The LH-RH and/or its derivatives produced by the method according to the present invention can be desalted and purified by general methods.
Desalting can be performed by various methods, which utilize difference in molecular sizes between the derivatives and the salt, including gel filtration, ultrafiltration, and dialysis. For example, it can be performed by ultrafiltration using a membrane made of cellulose acetate, gel filtration using Sephadex column such as Sephadex LH-20, Sephadex-60, Sephadex G-25, and ion-exchange chromatography using e.g., DEAE-cellulose. In purification, high performance liquid chromatography (HPLC) including partition chromatography wherein two types of liquids that are not miscible with each other (e.g., water and n-butanol) are employed and the difference in partition coefficients between the two liquid .phases is utilized, normal phase chromatography using a solid phase such as silica gel, and reversed phase chromatography using ODS-silica gel as a solid phase can be used.
For example, since the LH-RH and/ or its derivatives produced by the methods according to the present invention is hydrophilic, the derivatives can be purified by using an organic solvent that is not completely miscible with water as shown below. The mixture reacted as described above is extracted by using the organic solvent, partitioned by adding water to the extracted fluid, then solidified with a solvent such as ether. A preferable organic solvent for the extraction is n-butanol (n-BuOH). Such a solvent can lead high extractability because of its high polarity and it is not miscible with water, so that it is convenient for the subsequent partition chromatography process.
Three hundred ml of n-butanol (about 1/4 of the volume of the reaction mixture) is added to the reaction mixture (1200m1) to perform partition chromatography between the n-butanol and the water in the reaction mixture. Extraction with n-butanol can be repeated if necessary. The aqueous layer and the organic layer are concentrated separately. The above peptide fragment (7) recovered from the aqueous layer can be reused for the later reaction.
It is preferable that the roughly purified product obtained from the organic layer by partition chromatography solidifies by using a solvent such as Amended: 05/28/99 diethyl ether or ethyl acetate, because LH-RH and/or its derivative are slightly soluble in such a solvent. Then the solidified product is dissolved in an appropriate buffer like ammonium acetate, and fractioned by column chromatography with a proper solid phase and a mobile phase. The use of a weak cation exchange resin such as C1VI cellulose or a strong canon resin such as SP allows to obtain interaction with the Arg residue contained in LH-RH and/or its derivative. The use of a buffer (which is the same buffer used when the solidified roughly purified product is dissolved) as a mobile phase has an advantage of resulting in a high yield when the salt concentration is increased in a linear gradient. For example, O.O1M
aqueous ammonium acetate solution and O.1M aqueous ammonium acetate solution can be used. The resulting eluate can be purified with HPLC
properly selecting a solid phase and an eluent. In HPLC, ODS silica column, e.g. TSK gel ODS-120T, can be used as a solid phase and the use of ODS
silica column is the most suitable. Furthermore, 0.1% TFA-acetonitrile can be used as a mobile phase. Here, the content of acetonitrile is increased by 1% per minute from the beginning 0.1% TFA-20% acetonitrile so as to obtain a linear gradient of 0.1%TFA-50%acetonitrile, then the target LH-RH
derivative can be separated well. Purification is not limited to the abovementioned methods. Moreover, when immobilized enzymes are used, ion exchange can be performed without subjecting to partition chromatography.
The method for producing LH-RH and/or its derivative according to the present invention is more useful industrially because it has advantages as follows compared with known conventional methods.
i) Since LH-RH derivatives can be synthesized without side reactions (e.g., racemization) because of the property of an enzymatic reaction, the LH-RH derivatives can be easily purified and separated.
ii) A high yield of an LH-RH and/or its derivative can be obtained.
Moreover unreacted fragments can be recovered and reused thus the method is economically advantageous.
The LH-RH and or its derivative may be changed to pharmaceutically acceptable salts, such as their acetate, hydrochloride and phosphate, if Amended: 05/28/99 necessary.
Examples More detailed description of the present invention will be given by using the following examples but the present invention is not limited thereto.
In the following examples, pure peptides obtained are identified by measuring a retaining time in the high performance liquid chromatography (HPLC), and an optical rotation, and by analyzing amino acids. Unless otherwise specified, they are measured by the following measuring methods and conditions.
(1) High performance liquid chromatography (HPLC) In HPLC analysis, LC-Module-1 (manufactured by Japan Waters Limited Corporation) is used as a detector.
(HPLC analysis conditions) Column : TSK gel ODS-120T (4.6 x 250mm) Eluent : 0.1% TFA-acetonitrile (A linear gradient; acetnitrile is increased by 1% per minute from 20% to 50%) Flow rate : 1mL /min Detection wavelength : 220nm (2) Optical rotation In measurement of an optical rotation, DIC-370 (manufactured by JASCO
Corporation) is used.
(Measuring conditions for optical rotation) Beam of light : Na lamp 589nm Temperature : 20~C
Layer length : 100mm Concentration : 5mg/ml (3) Amino acid composition In amino acid composition obtained peptides are hydrolyzed in 6N
hydrochloric acid (containing 0.1% phenol) at 110°C for 20 hours then the amino acids are analyzed by using Hitachi amino acid analyzer L-8500 (manufactured by Hitachi Ltd.).
Example 1.
Production of pGlu-His-Trn-Ser-~~Tr- - r( ut)-L eu-Ark Pro NHFt (1-1) Production of Boc-Arg(Tos)-Pro-NHEt First, 214.38 of Boc-Arg(Tos)-OH was dissolved in a mixture of 150mL of THF and 100mL of DMF, then cooled with dry ice - ethanol to -20°C.
Next, 35mL of N-methyl morpholine, and then 66mL of isobutyl chloroformate were added dropwise to the mixture, and stirred at -20°C for 1 minute, thereby producing a mixed acid anhydride. The resulting reaction mixture was mixed with a solution in which 71.18 of H-Pro-NHEt was dissolved in 300mL of THF, and stirred at 0°C for 5 minutes then at room temperature for 30 minutes. Subsequently, the obtained reaction mixture was concentrated under reduced pressure. Ethyl acetate 1200mL was added twice to the residue, the residue was washed twice with 500mL of water, then the ethyl acetate layer was concentrated under reduced pressure. The residue was solidified by treatment with ether, and the product was dried.
Thus 221.188 of Boc-Arg(Tos)-Pro-NHEt (80.0% yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of Boc-Arg(Tos)-Pro-NHEt are shown below.
m.p. : 100 - 102°C
[ a ]D : -30.3 (c=1.0, MeOH) Rf : 0.69 (BuOH/AcOH/H20=4/1/5) Elementary analysis (C25H4°N606S ~ H20) Calculated C:52.61% H:7.42% N:14.73%

Found C:52.85% H:7.30% N:14.41%
(1-2) Production of Z-Leu-Arg(Tos)-Pro-NHEt Boc-Arg(Tos)-Pro-NHEt 110.548 produced as described in (1-1) was dissolved in 150mL of dichloromethane (DCM). TFA 150mL was added with ice cooling to the solution, then the mixture was stirred at room temperature for 30 minutes. The resulting reaction mixture was concentrated under reduced pressure. Next 1500mL of ether was added to the residue to solidify the product, which was then dried. Thus H-Arg(Tos)-Pro-NHEt TFA was obtained.
The deprotected H-Arg(Tos)-Pro-NHEt ~ TFA was dissolved in a mixture of 80mL of DMF and 200mL of THF, then the solution was cooled and neutralized with N-methyl morpholine. Next, 200mL of THF, in which 53.068 of Z-Leu-OH and 23.028 of HOSu were dissolved, and 36.4mL of WSC
were added to the neutralized solution then the mixture was stirred at 0°C
for 5 minutes and at room temperature for overnight. After the confirmation with ninhydrin, the reaction mixture was concentrated under reduced pressure.
After 1500mL of ethyl acetate was added to the residue, the mixture was washed twice with 500mL of water and twice with 500mL of saturated saline, respectively. Next the ethyl acetate layer was concentrated under reduced pressure and the residue was treated with ether to solidify the product, which was then dried. Thus 119.58 of Z-Leu-Arg(Tos)-Pro-NHEt (85.1%
yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of Z-Leu-Arg(Tos)-Pro-NHEt are shown below.
m.p. : 99 - 103°C
[ a ]D : -40.6 (c=1.0, MeOH) Rf: 0.75 (BuOH/AcOH/H~O=4/1/5) Elementary analysis (C34H49N7O7S) Calculated C:58.35% H:7.06% N:14.01%

Found C:58.40% H:7.26% N:13.78%
(1-3) Production of Z-D-Ser(But)-Leu-Arg-Pro-NHEt Anisole 32mL was added to 20.998 of Z-Leu-Arg(Tos)-Pro-NHEt produced as described in (1-2). Subsequently about 200mL of HF was added to the solution while cooling to -70°C with dry ice - ethanol, then the mixture was stirred at 0°C for 1 hour. Next the product was concentrated under reduced pressure, then the residue was treated with ether. The deposited product was filtered, then vacuum drying of the product was performed on sodium hydroxide, thereby obtaining H-Leu-Arg-Pro-NHEt ~ HF.
Z-D-Ser(But)-OH 8.868 was dissolved in 100mL of THF. The solution was cooled to -20°C with dry ice - ethanol. Then 3.3mL of N-methylmorpholine and then 3.96mL of isobutyl chloroformate were added dropwise to the solution, and the mixture was stirred at -20 °C for 1 minute, thereby producing a mixed acid anhydride. The resulting reaction mixture was mixed with 200mL of a solution (neutralized with N-methylmorpholine) of H-Leu-Arg-Pro-NHEt ~ HF in DMF, then the mixture was stirred at 0°C for 5 minutes then at room temperature for 30 minutes. Subsequently, the product was concentrated under reduced pressure. Water 500mL was added to the residue then the product was extracted five times with 200mL
of n-butanol. The extract was washed five times with water. Next the n-butanol layer was concentrated under reduced pressure, the residue was then solidified by treatment with ether. Thus 22.898 of Z-D-Ser(But)-Leu-Arg-Pro-NHEt (90.5% yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of Z-D-Ser(But)-Leu-Arg-Pro-NHEt are shown below.
m.p. : 121- 123°C
[ a ]D : -49.0 (c=1.0, MeOH) Rf : 0.51 (BuOH/AcOH/H20=4/1/5) Elementary analysis (C34H56N8O~) Calculated C:59.28% H:8.19% N:16.27%
Found C:59.01% H:8.15% N:16.19%

(1-4) Production of Z-Ser-Tyr-OMe Z-Ser-OH 23.9g and 23.28 of H-Tyr-OMe were dissolved in 50mL of DMF, 12.7g of HOSu and 20mL of WSC were added to the solution, then the mixture was stirred at 0 °C for 5 minutes then at room temperature overnight. After confirmation with ninhydrin, the reaction mixture was concentrated under reduced pressure. Ethyl acetate 500mL was added to the residue, and the mixture was washed twice with 200mL of 1N
hydrochloric acid, twice with 200mL of saturated aqueous sodium bicarbonate, twice with saturated saline, in this order. The ethyl acetate layer was concentrated under reduced pressure. The residue was solidified by treatment with hexane. Thus 38.44g of Z-Ser-Tyr-OMe (92.3% yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of Z-Ser-Tyr-OMe are shown below.
m.p. : 49 - 53°C (decomp.) [ a ]D : +2.3 (c=1.0, MeOH) Rf : 0.89 (BuOH/AcOH/H20=4/1/5) Elementary analysis (C21H24N2O7) Calculated C:60.57% H:5.81% N:6.73%
Found C:60.88% H:5.88% N:6.99%
(1-5) Production of Z-Ser-Tyr-NHNH2 Z-Ser-Tyr-OMe 38.Og as produced in the above (1-4) was dissolved in 200mL
of methanol. Hydrazine 50g was added to the solution, then the mixture was allowed to stand at room temperature overnight. Next the product was concentrated under reduced pressure, and the residue was washed with methanol. Thus 34.28 of Z-Ser-Tyr-NHNHZ (90.0% yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of Z-Ser-Tyr-NHNH2 are shown below.

m.p. : 194 - 197°C
[ a ]D : +9.9 (c=1.0, DMF) Rf : 0.64 (BuOH/AcOH/Hz0=4/1/5) Elementary analysis (C2oH24N~06) Calculated C:57.69% H:5.81% N:13.45%
Found C:57.85% H:5.83% N:13.49%
(1-6) Production of Z-Ser-'I~r-D-Ser(But)-Leu-Arg-Pro-NHEt Z-D-Ser(But)-Leu-Arg-Pro-NHEt 28.Og produced in the above (1-3) was dissolved in 300mL of methanol, then hydrogen was passed to the solution in the presence of 2.5g of 10% palladium/carbon. After stirring at room temperature for 7 hours, the catalyst was removed from the reaction mixture, the filtrate was concentrated under reduced pressure, then the residue was treated with ether. Thus H-D-Ser(But)-Leu-Arg-Pro-NHEt was obtained.
Z-Ser-Tyr-NHNHZ 20.18 produced in the above (1-5) was dissolved in 150mL
of DMF, then the solution was cooled to -20°C with dry ice - ethanol.

HCl-dioxane 43.5mL and 6.5mL of isoamyl nitrite were added to the solution to obtain an azide compound. Further, 24.4mL of trimethylamine was added to neutralize the reaction mixture. The mixture was transferred into 150m1 of DMF solution containing H-D-Ser(But)-Leu-Arg-Pro-NHEt, stirred at -20°C for 2 hours and at 4°C for 17 hours, and then concentrated. Next 500mL of water was added to the residue, and the mixture was extracted 5 times with 200mL of n-butanol. The extract was washed 5 times with water, the n-butanol layer was concentrated under reduced pressure, and then the residue was treated with ether to solidify the product. Thus 33.2g of Z-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt was obtained (87.5% yield).
The melting point, optical rotation, Rf of TLC and elementary analysis of Z-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt are shown below.
m.p. : 115 - 118°C (decomp.) [ a ]D : -27.6 (c=1.0, DMF) Rf : 0.36 (BuOH/AcOH/H20=4/1/5) Elementary analysis (C46H7oN,oO,O

Calculated C:58.83% H:7.51% N:14.91%
Found C:58.88% H:7.55% N:14.90%
(1-7) Production of Z-pGlu-His-OH
Z-pGlu-OH 52.68 was dissolved in 400mL of THF The solution was cooled to -20°C with dry ice - ethanol. N-methylmorpholine 22.OmL and then 26.4mL of isobutyl chloroformate were added dropwise to the solution, then the mixture was stirred at -20°C for 1 minute, thereby producing a mixed acid anhydride. HOSu 25.3g was added to the resulting reaction mixture then stirred for 30 minutes. Next a solution in which 62.9g of H-His-OH
HCl and 28.OmL of triethylamine (TEA) were dissolved in 400mL of water was added to the mixture. After stirring at 0°C for 1 hour and at room temperature overnight, the reaction mixture was concentrated under reduced pressure. The residual aqueous solution was washed with ethyl acetate, and the aqueous layer was extracted with n-butanol. The extract was washed with water for several times, then the n-butanol layer was concentrated under reduced pressure. After the residue was solidified with ether, it was dissolved in water and adjusted at pH 5. The solution was applied to a column (5.Ox20cm) filled with HP-20, washed with water until Pauli reaction of the effluent shows ~-, and eluted from methanol. The eluted methanol fluid was concentrated under reduced pressure. The residue was treated with ether, then recrystallized from methanol - ether. Thus 59.5g of Z-pGlu-His-OH (74.3% yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of Z-pGlu-His-OH are shown below.
m.p. : 146 - 149°C (decomp.) [ a ]D : -0.91 (c=1.0, DMF) Rf : 0.14 (BuOH/AcOH/H20=4/1/5) Elementary analysis (Cl9HZON40s) Calculated C:57.00% H:5.03% N:13.99%
Found C:57.23% H:5.05% N:14.05%
(1-8) Production of Z-pGlu-His-Trp-OMe Z-pGlu-His-OH 40.Og produced in the above (1-7), H-Trp-OMe ~HCl 19.6g, and HOSu 12.7g were dissolved in 100mL of DMF Next with cooling the solution was neutralized with N-methylmorpholine, 2.OmL of WSC was added, then the mixture was stirred at 0°C for 5 minutes and at room temperature overnight. After confirmation with ninhidryn, the reaction mixture was concentrated under reduced pressure. Ethyl acetate-n-butanol (1:1) 200mL was added to the residue then the organic layer was washed three times with 100mL of water. The organic layer was concentrated under reduced pressure. The residue was solidified by treatment with ether.
Thus 48.5g of Z-pGlu-His-Trp-OMe (92.3% yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of Z-pGlu-His-Trp-OMe are shown below.
m.p. : 112 - 115°C (decomp.) [ a ]D : -1.5 (c=1.0, DMF) Rf : 0.3 (BuOH/AcOH/H20=4/1/5) Elementary analysis (C3lHsaNs07) Calculated C:61.99% H:5.37% N:13.99%
Found C:61.54% H:5.33% N:13.80%
(1-9) Production of pGlu-His-Trp-OMe Z-pGlu-His-~p-OMe 40.Og produced in the above (1-8) was dissolved in 50mL of DMF. Hydrogen was passed to the solution in the presence of 500mg of 10% palladium/carbon. Next the mixture was stirred at room temperature for 7 hours, the catalyst was removed from the solution, then the filtrate was concentrated under reduced pressure. The residue was solidified by treatment with ether. Thus 28.5g of pGlu-His-~p-OMe (91.8%
yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of pGlu-His-~p-OMe are shown below.
m.p. : 132 - 137°C (decomp.) [ a ]D : +4.1 (c=1.0, DMF) Rf : 0.18 (BuOH/AcOH/H20=4/1/5) Elementary analysis (C23H~6N605) Calculated C:59.22% H:5.62% N:18.02%
Found C:59.01% H:5.55% N:17.89%
(1-10) Production of H-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt Z-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt 93.Og produced as described in the above (1-6) was dissolved in 800mL of methanol, then hydrogen was passed to the solution in the presence of 5.Og of 10% palladium/ carbon. After the mixture was stirred at room temperature for 7 hours, the catalyst was removed from the solution, then the filtrate was concentrated under reduced pressure. The residue was solidified by the treatment with ether. Thus 74.Og of H-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt (90.3% yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of H-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt are shown below.
m.p. : 124 - 128°C (decomp.) [ a ]D : +10.1 (c=1.0, DMF) Rf : 0.16 (BuOH/AcOH/H20=4/1/5) Elementary analysis (C38H64NloOs) Calculated C:56.70% H:8.01% N:17.40%
Found C:56.55% H:7.97% N:17.18%
(1-11) Production of pGlu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt pGlu-His-Trp-OMe 30g produced as described in (1-9) and 22g of H-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt produced as described in (1-10) were dissolved in 1300mL of n-butanol saturated water. The solution was adjusted at pH 7.8 with 1M hydrochloric acid or aqueous ammonia. To the solution, a solution wherein 78mg of chymotrypsin (1000 U/mg) was dissolved in 2.5mL of n-butanol saturated water was added, then the mixture was stirred at 10°C for 1 hour. Then 300mL of n-butanol was added to the reaction mixture so that the synthesized peptides were partitioned between water and n-butanol thus separated into a lower layer (aqueous layer) and an upper layer (n-butanol layer). n-Butanol 200mL
was added to the lower layer, and the same partition chromatography was repeated for three times. The upper layer was washed 5 times with 100mL
of water, the organic and the aqueous layers were concentrated separately then solidified with ether, respectively. H-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt recovered from the aqueous layer is to be recycled for the reaction.
The organic layer (n-butanol) was concentrated, and ether was added to the residue to be powdered.
The powder obtained from the organic layer was dissolved in O.O1M aqueous ammonium acetate solution, then the solution was applied to a column (4x30cm) filled with CM-cellulose. The linear gradient elution (60mL/hour) of O.O1M to O.1M aqueous ammonium acetate solution (pH 4.4, 500mL) was performed and the eluate was fractionated in volume of lOmL each. The eluate was analyzed by high performance liquid chromatography. The target fractions were pooled and freeze-dried. Thus, 22.48 of pGlu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt (74.9% yield) was obtained.
The optical rotation, elementary analysis, and amino acid composition of pGlu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt are shown below.
[ a ]D: -46.0 (c=1, H20) Elementary analysis (C6oH$6N1s013 ' CHsCOOH ' 2 HZO) Calculated C:55.76% H:7.09% N:16.78%
Found 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 enzymes (1) Synthesis of pGlu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt pGlu-His-gyp-OMe 2.OOg produced as described in (1-9) and 1.30g of H-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt produced as described in (1-10) were dissolved in 200mL of water, then adjusted at pH 7.8 with 1M hydrochloric acid or aqueous ammonia. To the solution was suspended immobilized chymotrypsin (agarose carrier, 2000 U/g) 2.5g. After it was confirmed that the mixture has a pH of 7.8, it was stirred at 10°C for 1 hour. The reaction mixture was filtered through a glass filter to remove the immobilized cymotrypsin.
The filtrate was concentrated and applied to a column (4x30cm) filled with CM-cellulose. The linear gradient elution (60mL/hour) of O.O1M to O.1M
aqueous ammonium acetate (pH4.4, 500mL) was performed and the eluate was fractioned in volumes of lOmL each. The eluate was then analyzed by HPLC. The target fractions were pooled and freeze-dried. Thus 1.45g of pGlu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt (82.4% yield) was obtained.
The melting point, optical rotation, Rf value of TLC and elementary analysis of the obtained compound are identical to those of pGlu-His-~p-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt produced as described in (1-11).
(2) Investigation of substrate specificity Under the enzymatic reaction conditions as described above, reaction was conducted with peptides as substrates as shown in Table 1. After two hours of the reaction the synthesized peptides were analyzed by HPLC. Yields were calculated from the area of each peak.

Table 1 Substrate used Yield (%) pGlu-His-Trp-OMe + H-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-NHEt 78.8 pGlu-His-D-Trp-OMe + H-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-NHEt 0.0 pGlu-D-His-Trp-OMe + H-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-NHEt 7.8 pGlu-His-Trp-OMe + H-D-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-NHEt 0.0 As shown in Table 1, when the substrate wherein the Trp, a recognition site for the enzyme, has a D-configuration was used, the yield was 0% and the reaction did not proceed at all. In addition when the substrate wherein the His adjacent to N terminal side of a recognition site for the enzyme has a D-configuration was used, the yield was as low as 7.8% but the presence of the product was confirmed. When the substrate wherein the Ser, a binding site for the enzyme, has a D-configuration was used, the yield was again 0% and the reaction did not proceed at all.
Accordingly, it was shown that when enzymatic synthesis was used, racemization of Ser and His becomes difficult to occur because of the enzyme's substrate specificity, therefore products with high optical purity can be obtained.
Example 2 In this example, pGlu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt was produced in the same manner as in Example 1 except that Z-D-Ser(But)-Leu-Arg-Pro-NHEt produced according to (1-1) to (1-3) of Example 1 was produced according to the following (2-1) to (2-3).
(2-1) Production of Z- Leu-Arg- NHNH2 Z-Leu-OH 172.58 and H-Arg-OEt ~ 2HC1 178.98 were added to 800mL of a DMF/THF(1/1) mixed solution. The solution was neutralized by adding 143mL of N-methylmorpholine at 0°C. Next 147.5mL of DCC was added to the solution and then it was stirred at 0°C for 5 minutes and at room temperature for 15 hours. The reaction solution was concentrated under reduced pressure then 1000mL of water was added to the residue obtained.
After the product was washed twice with 500mL of ethyl acetate, the aqueous layer was extracted three times with 1000mL of n-butanol. The obtained n-butanol layer was concentrated under reduced pressure to obtain Z-Leu-Arg-OEt.
Z-Leu-Arg-OEt was dissolved in 500mL of methanol. NH~NH2 ~ H20 150mL
was added with ice cooling to the solution, then the mixture was stirred at room temperature for 18 hours. The reaction mixture was concentrated under reduced pressure, 500mL of water was added to the obtained residue, then the product was extracted 5 times with 1000 mL of n-butanol. The extract was washed 4 times with 500mL of water, then concentrated under reduced pressure. The resulting residue was solidified by treatment with ether, then it was dried. Thus, 209.7 g of Z-Leu-Arg-NHNH~ (74.1% yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of Z-Leu-Arg-NHNH2 are shown below.
m.p. : 105 - 107°C
[ a ]D : -30.6 (c=1.0, MeOH) Rf : 0. 60 (BuOH/AcOH/H~O=4/1/5) Elementary analysis (CZ°H33N704) Calculated C:55.61% H:7.64% N:22.51%
Found C:55.84% H:7.42% N:22.41%
(2-2) Production of Z-Leu-Arg-Pro-NHEt Z-Leu-Arg-NHNHZ 130.8g produced as described in the above (2-1) was dissolved in 500 mL of DMF and the solution was cooled to -20°C with dry ice - ethanol. Then 225mL of 4N HCl-dioxane and 40.5mL of isoamyl nitrite were added to the solution so as to obtain an azide compound.

Further 126mL of triethylamine was added to neutralize the mixture. The neutralized mixture was transferred to 200mL of DMF containing 42.6g of H-Pro-NHEt, stirred at -20°C for 2 hours and at 4°C for 16 hours, and concentrated. Water 800mL was added to the residue, then the mixture was washed twice with 400mL of ethyl acetate. The aqueous layer was extracted 3 times with 800mL of butanol, then washed with 400mL of water.
The obtained n-butanol layer was concentrated under reduced pressure.
The resulting residue was solidified by treatment with ether, and dried.
Thus 154.5g of Z-Leu-Arg-Pro-NHEt (94.3% yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of Z-Leu-Arg-Pro-NHEt are shown below.
m.p. : 125 - 126°C
[ a ]D : -66.6 (c=1.0, MeOH) Rf : 0. 45 (BuOH/AcOH/H20=4/1/5) Elementary analysis (C2~H43N7O5) Calculated C:59.43% H:7.94% N:17.97%
Found C:59.31% H:7.78% N:18.13%
(2-3) Production of Z-D-Ser(But)-Leu-Arg-Pro-NHEt Z-Leu-Arg-Pro-NHEt 154.5g produced as described in the above (2-2) was dissolved in 1500mL of methanol, and hydrogen was passed to the solution in the presence of 14g of 10% palladium/carbon. The mixture was stirred at room temperature for 16 hours, then the catalyst was removed from the mixture. The obtained filtrate was concentrated under reduced pressure to obtain H-Leu-Arg-Pro-NHEt.
Z-D-Ser(But)-OH 82.3 g was dissolved in 200mL of THF The solution was cooled to -20 °C with dry ice - ethanol. Next 30.7mL of N-methylmorpholine and subsequently 36.3mL of isobutyl chloroformate were added dropwise, then the reaction solution was stirred at -20°C for 1 minute, thereby producing a mixed acid anhydride. The obtained reaction mixture was mixed with 700mL of a DMF solution containing H-Leu-Arg-Pro-NHEt, then stirred at 0°C for 5 minutes and at room temperature for 30 minutes.

Subsequently, the reaction mixture was concentrated under reduced pressure, and 1000mL of water was added to the residue. The mixture was washed twice with 500mL of ethyl acetate, and the aqueous layer was extracted twice with 1000mL of a mixed solution of ethyl acetate/n-butanol (2/1). The extract was washed five times with 500mL of water and was concentrated under reduced pressure. The obtained residue was solidified by treatment with ether, then dried. Thus 178.38 of D-Ser(But)-Leu-Arg-Pro-NHEt (91.2% yield) was obtained.
Moreover, the melting point, optical rotation, Rf value of TLC and elementary analysis of the obtained compound were identical to those of D-Ser(But)-Leu-Arg-Pro-NHEt produced as described in Example 1 (1-3).
Example 3 Production of nC7lu-His-Trp-Ser- r-D- u-T u- Arg Prn NHFt (3-1) Production of Boc-D-Leu-Leu-Arg(Tos)-Pro-NHEt Z-Leu-Arg(Tos)-Pro-NHEt 20.998 produced as in Example 1 (1-2) was dissolved in 200mL of methanol, then hydrogen was passed to the solution in the presence of 1.58 of 10% palladium/carbon. The mixture was stirred at room temperature for 7 hours, the catalyst was removed, then the filtrate was concentrated under reduced pressure. The residue was solidified by treatment with ether. Thus, H-Leu-Arg(Tos)-Pro-NHEt was obtained.
H-Leu-Arg(Tos)-Pro-NHEt was dissolved in 80mL of DMF and 200mL of THF. With cooling the solution was neutralized with N-methylmorpholine.
To the solution, a solution in which 8.868 of Boc-D-Leu-OH and 23.028 of HOSu were dissolved in 200mL of THF, and 36.4mL of WSC were added.
Next the mixture was stirred at 0°C for 5 minutes and at room temperature overnight. After confirmation with ninhidrin, the reaction mixture was concentrated under reduced pressure. To the residue, 500mL of water was added, then the mixture was extracted 5 times with 200mL of n-butanol and washed five times with water. The n-butanol layer was concentrated under reduced pressure, then the residue was solidified by treatment with ether.
Thus 21.58 of Boc-D-Leu-Leu-Arg(Tos)-Pro-NHEt (92.1% yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of Boc-D-Leu-Leu-Arg(Tos)-Pro-NHEt are shown below.
m.p. : 119 - 121°C
[ a ]D : -45.2 (c=1.0, MeOH) Rf : 0. 50 (BuOH/AcOH/H~O=4/1/5) Elementary analysis (C3oH5sNsOs) Calculated C:57.67% H:9.03% N:17.93%
Found C:57.43% H:8.89% N:17.80%
(3-2) Production of Z-Ser-Tyr-D-Leu-Leu-Arg(Tos)-Pro-NHEt Boc-D-Leu-Leu-Arg(Tos)-Pro-NHEt 25.8g produced as described in the above (3-1) was dissolved in lOmL of DCM. TFA lOmL was added with ice cooling to the solution, then the mixture was stirred at room temperature for 30 minutes. Subsequently, the reaction mixture was concentrated under reduced pressure, 200mL of ether was added to the residue so as to be solidified, then the product was dried. Thus H-D-Leu-Leu-Arg(Tos)-Pro-NHEt ~ TFA was obtained.
Z-Ser-Tyr-NHNH2 16.68 was dissolved in 100mL of DMF, and cooled to -20°C
with dry ice-ethanol. To the solution, 29.8mL of 4N HCl-dioxane and 5.3mL
of isoamyl nitrite were added to obtain an azide compound. Next the mixture neutralized with 16.7mL of triethylamine was transferred to 200mL
of DMF containing H-D-Leu-Leu-Arg(Tos)-Pro-NHEt ~ TFA. Then the mixture was stirred at -20°C for 2 hours and at 4°C for 17 hours, then it was concentrated. Water 500mL was added to the residue, then the mixture was extracted 5 times with 200mL of n-butanol and washed 5 times with water. The n-butanol layer was concentrated under reduced pressure, then the residue was solidified by treatment with ether. Thus, 25.Og of Z-Ser-Tyr-D-Leu-Leu-Arg(Tos)-Pro-NHEt (83.2% yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of Z-Ser-Tyr-D-Leu-Leu-Arg(Tos)-Pro-NHEt are shown below.

m.p. : 114 - 118°C (decomp.) [ a ]D : -30.1 (c=1.0, DMF) Rf : 0. 32 (BuOH/AcOH/H20=4/1/5) Elementary analysis (C45H68N~oOlo) Calculated C:59.45% H:7.54% N:15.41%
Found C:59.33% H:7.52% N:15.32%
(3-3) Production of H-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt Anisole 32mL was added to 20.4g of Z-Ser-Tyr-D-Leu-Leu-Arg(Tos)-Pro-NHEt produced as in the above (3-2), then the mixture was cooled to -70°C
with dry ice - ethanol. To the mixture, 200mL of HF was added and stirred at 0°C for 1 hour. Subsequently, the reaction mixture was concentrated under reduced pressure, the residue was treated with ether, and the deposited product was filtered and then vacuum-dried over sodium hydroxide. Thus l6.Og of H-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt (92.4%
yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of H-Ser-~yr-D-Leu-Leu-Arg-Pro-NHEt are shown below.
m.p. : 121- 123°C
[ a ]D : +11.0 (c=1.0, DMF) Rf : 0. 20 (BuOH/AcOH/H20=4/1/5) Elementary analysis (C37H62NloOs) Calculated C:57.35% H:8.06% N:18.07%
Found C:57.21% H:8.01% N:18.10%
(3-4) Production of pGlu-His-Trp-Ser-Tyr-D-Leu-Leu- Arg-Pro-NHEt pGlu-His-~p-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt 1.33g (76.4% yield) was obtained in the same manner as in Example 1 (1-11) except that 1.30g of H-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt produced as in the above (3-3) was used instead of H-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt.

The optical rotation, elementary analysis, and amino acid composition of pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt are shown below.
[ a ]D: -32.0 (c=0.52, 5%AcOH) Elementary analysis (C59H84N16013) Calculated 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), Leu2.09(2), Tyr 0.92(1), His 0.93(1), Arg 1.06(1) Example 4 Production of nCllu-Hip-Trn-Ser-Tv -r DSer( ut)- . .u- Are-Prn-A~ ~y-NHS
(4-1) Production of Z-Pro-Azgly-NH2 Z-Pro-OH 24.98, 11.2g of semicarbazide hydrochloride and 14.5mL of triethylamine were dissolved in 200mL of DMF. To the solution cooled to 0°C, 20.6g of DCC was added and the mixture was stirred at 4°C
for 16 hours.
Subsequently, dicyclohexylurea (DCU) was filtered off from the reaction mixture, and the filtrate was concentrated under reduced pressure. To the residue 500mL of ethyl acetate was added, and the mixture was washed twice with 200mL of water and concentrated under reduced pressure, then the residue was solidified by treatment with ether. Thus 16.7g of Z-Pro-Azgly-NHZ (54.3% yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of Z-Pro-Azgly-NH2 are shown below.
m.p. : 189 - 190°C
[ a ]D : -43.6 (c=1.4, DMF) Rf : 0. 62 (BuOH/AcOH/HZO=4/1/5) Elementary analysis (C,4H18N4O4) Calculated C:54.89% H:5.95% N:18.29%
Found C:54.41% H:7.74% N:15.60%

(4-2) Production of Boc-Arg(N02)-Pro-Azgly-NHZ
Z-Pro-Azgly-NH2 16.58 produced as described in the above (4-1) was dissolved in 500mL of DMF, and hydrogen was passed to the solution in the presence of l.lg of 10% palladium/carbon. The mixture was stirred at room temperature for 7 hours, the catalyst was removed, then the filtrate was concentrated under reduced pressure. Ether 1500mL was added to the residue to solidify the product, which then was dried. Thus, H-Pro-Azgly-NH~ ~ TFA was obtained.
Boc-Arg(NOZ)-OH 13.58 was dissolved in 100mL of THF and cooled to -20°C
with dry ice - ethanol. To the solution, 3.3mL of N-methylmorpholine and then 3.96mL of isobutyl chloroformate were added dropwise, then the mixture was stirred at -20°C for 1 minute, thereby producing a mixed acid anhydride. The obtained reaction mixture was mixed with 200mL of a DMF
solution (which was neutralized with N-methylmorpholine) containing H-Pro-Azgly-NHZ ~ TFA. Next the mixture was stirred at 0°C for 5 minutes then at room temperature for 30 minutes, and concentrated under reduced pressure. Water 500mL was added to the residue, and the mixture was extracted 5 times with 200mL of n-butanol. Further the extract was washed 5 times with water, the n-butanol layer was concentrated under reduced pressure, then the residue was solidified by treatment with ether.
Thus, 16.78 of Boc-Arg(NOZ)-Pro-Azgly-NH2 (88.6% yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of Boc-Arg(N02)-Pro-Azgly-NH2 are shown below.
m.p. : 135 - 137°C (decomp.) ( a ]D : +35.3° (c=1.0, DMF) Rf : 0. 49 (BuOH/AcOH/H20=4/1/5) Elementary analysis (C17H31N907) Calculated C:43.12% H:6.60% N:26.62%
Found C:54.41% H:7.74% N:15.60%
(4-3) Production of Z-Leu-Arg(NOZ)-Pro-Azgly-NHZ (SEfI ID N0:2) Boc-Arg(N02)-Pro-Azgly-NHEt 110.54g produced as described in the above (4-2) was dissolved in 150mL of DCM. To the solution, 150mL of TFA was added with ice cooling and the mixture was stirred at room temperature for 30 minutes. Next the reaction mixture was concentrated under reduced pressure, 1500mL of ether was added to the residue to solidify the product, which was then dried. Thus H-Arg(NOZ)-Pro-NHEt ~ TFA was obtained.
H-Arg(NO~)-Pro-NHEt ~ TFA was dissolved in a mixture of 80mL of DMF and 200mL of THF With cooling the solution was neutralized with N-methylmorpholine. Subsequently to the solution, a solution wherein 53.068 of Z-Leu-OH (0.2mole) and 23.028 of HOSu (0.22mole) were dissolved in 200mL of THF and 36.4mL (0.2mole) of WSC were added, and the mixture was stirred at 0°C for 5 minutes then at room temperature overnight.
After confirmation with ninhydrin, the reaction mixture was concentrated under reduced pressure. Ethyl acetate 1500mL was added to the residue, the mixture was washed twice with 500mL of water and twice with 500mL of saturated saline solution. After that, the ethyl acetate layer was concentrated under reduced pressure, the residue was solidified by treatment with ether, and the product was dried. Thus, 27.528 of Z-Leu-Arg(N02)-Pro-Azgly-NHZ (SEMI ID N0:2) (98.6% yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of Z-Leu-Arg(N02)-Pro-Azgly-NHZ are shown below.
m.p. : 88 - 90°C
[ a ]D : -30.2 (c=1.5, DMF) Rf : 0. 57 (BuOH/AcOH/H20=4/1/5) Elementary analysis (CZ6H4oN1°Os) Calculated C:50.31% H:6.50% N:22.57%
Found C:50.24% H:6.41% N:22.45%
(4-4) Production of Z-D-Ser(But)-Leu-Arg-Pro-Azgly-NH2 Z-Leu-Arg(N02)-Pro-Azgly-NH2 (SEQ ID N0:2) produced in the above (4-3) 16.58 was dissolved in 500mL of DMF, and hydrogen was passed to the solution in the presence of l.lg of 10% palladium/carbon. The mixture was stirred at room temperature for 7 hours, the catalyst was removed and then the filtrate was concentrated under reduced pressure. Ether 1500mL was added to the residue to solidify and the product was dried. Thus H-Leu-Arg-Pro-Azgly-NH~ (SEQ ID N0:3) was obtained.
Z-D-Ser(But)-OH 8.868 was dissolved in 100mL of THF, then the solution was cooled to -20°C with dry ice - ethanol. To the solution, 3.3mL of N-methylmorpholine and then 3.96mL of isobutyl chloroformate were added dropwise. The mixture was stirred at -20 °C for 1 minute, thereby producing a mixed acid anhydride. The obtained reaction mixture was mixed with 200mL of DMF solution containing H-Leu-Arg-Pro-Azgly-NH., (neutralized with N-methylmorpholine), the mixture was stirred at 0°C
for 5 minutes and then at room temperature for 30 minutes, finally the reaction mixture was concentrated under reduced pressure. Water 500mL was added to the residue, then the mixture was extracted 5 times with 200mL of n-butanol. After the extract was washed 5 times with water, the n-butanol layer was concentrated under reduced pressure, then the residue was solidified by treatment with ether. Thus 16.98 of Z-D-Ser(But)-Leu-Arg-Pro-Azgly-NHZ (78.3% yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of Z-D-Ser(But)-Leu-Arg-Pro-Azgly-NH2 are shown below.
m.p. : 115 - 118°C
[ a ]D : -45.8 (c=1.0, DMF) Rf : 0. 51 (BuOH/AcOH/H20=4/1/5) Elementary analysis (C33H54N1008) Calculated C:55.14% H:7.57% N:19.48%
Found C:55.01% H:7.42% N:19.33%
(4-5) Production of Z-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH2 Z-D-Ser(But)-Leu-Arg-Pro-Azgly-NHZ 26.7g produced as described in the above (4-4) was dissolved in 500mL of methanol, and hydrogen was passed to the solution in the presence of 1.9g of 10% palladium/carbon. The mixture was stirred at room temperature for 7 hours, the catalyst was removed, then the filtrate was concentrated under reduced pressure. The residue was treated with ether, the deposited product was filtered, then the product was vacuum -dried over sodium hydroxide. Thus H-D-Ser(But)-Leu-Arg-Pro-Azgly-NHZ was obtained.
Z-Ser-Tyr-NHNH2 18.5g produced as in Example 1 (1-5) was dissolved in 150mL of DMF, and the solution was cooled to -20°C with dry ice -ethanol.
4N HCl-dioxane 33.4mL and 6.OmL of isoamyl nitrite were added to the solution so as to obtain an azide compound. Further 18.7mL of triethylamine was added to neutralize the mixture. The mixture was transferred to 150mL of a DMF solution containing H-D-Ser(But)-Leu-Arg-Pro-Azgly-NH2. After the mixture was stirred at -20°C for 2 hours then at 4°C for 17 hours, it was concentrated. Water 500mL was added to the residue, and the product was extracted 5 times with 200mL of n-butanol.
Next the extract was washed 5 times with water, the n-butanol layer was concentrated under reduced pressure, finally the residue was solidified by treatment with ether. Thus 28.3 g of Z-Ser-~r-D-Ser(But)-Leu-Arg-Pro-Azgly-NH2 (78.6% yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of Z-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH2 are shown below.
m.p. : 110 - 113°C (decomp.) [ a ]D : -28.2 (c=1.0, DMF) Rf : 0. 36 (BuOH/AcOH/H20=4/1/5) Elementary analysis (C45H68N12012) Calculated C:55.77% H:7.07% N:17.34%
Found C:55.64% H:7.01% N:17.29%
(4-6) Production of H-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NHS
Z-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH2 25.4g produced as described in the above (4-5) was dissolved in 500mL of methanol, and hydrogen was passed to the solution in the presence of 1.3g of 10% palladium/carbon.
After the mixture was stirred at room temperature for 7 hours, the catalyst was removed and then the filtrate was concentrated under reduced pressure.

The residue was solidified by treatment with ether. Thus 20.78 of H-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NHz (94.6% yield) was obtained.
The melting point, optical rotation, Rf of TLC and elementary analysis of H-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH2 are shown below.
m.p. : 120 - 122°C
[ a ]D : +11.2 (c=1.0, DMF) Rf : 0. 31 (BuOH/AcOH/H20=4/1/5) Elementary analysis (C37H62N12010) Calculated C:53.22% H:7.48% N:20.13%
Found C:53.19% H:7.43% N:20.01%
(4-7)Production of pGlu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH2 pGlu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH2 1.29g (75.9%
yield) was obtained by the same manner as in Example 1 (1-11) except that 1.30g of H-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NHS produced in the above (4-6) was used instead of H-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt.
The optical rotation, elementary analysis and amino acid composition of pGlu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH2 are shown below.
[ a ]D : -52.4 (c=1.0, DMF) Elementary analysis (C59H84N18O14) Calculated C:55.82% H:6.67% N:19.86%
Found 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 Production of pCTlu-His-Tr -~Pr- r-- ,,p- Leu-Ar_g-P o- =ly-N~Z
(5-1) Production of H-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2 Solid phase synthesis is performed by using Peptide Synthesizer 9600 manufactured by Milligen Bioresearch Corporation.
First 694mg of p-methylbenzhydrylamine(MBHA) resin (manufactured by PEPTIDE INSTITUTE, INC., amino group 0.72mmol/g) was put into a reaction vessel for peptide solid phase synthesis, and treated with stirring in turn with 8mL of DCM (4 times, 1 minute each), 8mL of DCM solution containing 60% TFA (20 minutes), 4mL of DCM (3 times, 15 seconds each), 3mL of DMF solution containing 1mL of DIEA (twice, 1 minute each), and 8mL of DMF (6 times, 40 seconds each) under argon stream. Further filtration was performed at each treatment.
On the other hand, 2 mmole of Boc-Gly-OH corresponding to the tenth amino acid residue of the amino acid sequence of pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2 was dissolved in 4mL of DCM. The solution was put into an amino acid activation container, 3mL of DCC (0.5M-DCM solution) and 4mL of HOBt (0.5M-DCM solution) were added to the solution, then the mixture was allowed to react for 30 minutes. The reaction mixture was filtered and transferred to a concentration container. Then 3mL of DMF
was added to the mixture, and the DCM was distilled off under argon stream.
Next the residue to which 3mL of DMF was added was transferred to the said reaction vessel for peptide solid phase synthesis, then allowed to react for 30 minutes. Subsequently the product was washed with 8mL of DCM (6 times, 20 seconds each). Further 2mmol of Boc-Gly-OH was dissolved in 4mL of DCM, the so-called double-coupling method was performed wherein the similar operation was repeated in an amino acid activation container, then the product was filtered, thereby obtaining Boc-Gly-MBHA resin.
Subsequently, the obtained Boc-Gly-MBHA resin was washed with 8mL of DCM (4 times, 1 minute each) and then it was filtered. After that the product was treated with stirring in turn with 8mL of DCM solution containing 60% TFA (20 minutes), 4mL of DCM (3 times, 15 seconds each), 3mL of DMF solution containing 1mL of DIEA (twice, 1 minute each), and 8mL of DMF (6 times, 40 seconds each) under argon stream. In addition, filtration was performed at each treatment.

Next, 2 mmole of Boc-Pro-OH corresponding to the ninth amino acid residue of the amino acid sequence of pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2 was dissolved in 4mL of DCM. DCC (0.5M-DCM solution) l.SmL was added to the solution in an amino acid activation container, then the solution was allowed to react for 7 minutes. After that, the reaction mixture was filtered and transferred to a concentration container. Then 3mL of DMF
was added to the reaction mixture, and the DCM was distilled off under argon stream. Next the residue to which 3mL of DMF was added was transferred to said reaction vessel for peptide solid phase synthesis, then allowed to react for 30 minutes. Subsequently the product was washed with 8mL of DCM (6 times, 20 seconds each), then filtered, thereby obtaining Boc-Pro-Gly-MBHA resin.
Eighth to fourth amino acids are coupled in order by means of amino group protected amino acids as shown in Table 2 below.
Table 2 Amino acid Protected Amount used order amino acid (~01 ) 8 Boc-Arg(Tos)-OH 2 x 7 Boc-Leu-OH 2 6 Boc-D-Trp-OH 2 Boc-Tyr(Bzl)-OH 2 4 Boc-Ser(Bzl)-OH 2 In the above solid phase synthesis, the double coupling was performed when Arg was used. Thus 2.76g of protected peptide-MBHA resin, Boc-Ser(Bzl)-Tyr(Bzl)-D-Trp-Leu-Arg(Tos)-Pro-Gly-MBHA resin was obtained.

To the above 2.76g of protected peptide-MBHA resin, 5mL of anisole and then 25mL of hydrogen fluoride anhydride were added, then the product was stirred at 0°C for 1 hour. After the reaction, hydrogen fluoride anhydride was distilled off under reduced pressure, then the residue was washed with ether. Thus 1.30g of H-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NHz was obtained.
The optical rotation, Rf of TLC, and elementary analysis of H-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NHZ are shown below.
[ a ]D : -50.4 (c=1.0, MeOH) Rf : 0. 13 (BuOH/AcOH/H20=4/1/5) Elementary analysis (C59H84N16012 ' AcOH ~ 2H20) Calculated C:54.31% H:7.04% N:17.27%
Found C:54.20% H:6.89% N:16.97%
(5-2) Production of pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NHZ
pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2 1.268 (75.45% yield) was obtained by the same manner as in Example 1 (1-11) except that 1.30 of H-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NHZ produced in the above (5-1) was used instead of H-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Gly-NHEt.
The optical rotation, elementary analysis and amino acid composition of pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NHS are shown below.
[ a ]D : -56.6 (c=1.0, H20) Elementary analysis (C64H82N18013 ' AcOH ~ 2H20) Calculated 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 Production of pC=lu-His-Trp-Ser-Tvr-(2~~pph~v~)-D-Ala-T e~a-Arg-Pro~Glv-~2 (6-1) Production of H-Ser-Tvr-(2-nap~j~vl)-D-Ala-T,PU-Ark-Prn-G y-NHS
H-Ser-Tyr-(2-naphthyl)-D-Ala-Leu-Arg-Pro-Gly-NHS 0.98g was obtained by the same treatment as in Example 5 (5-1) except that Boc-D-(2-naphthyl)-D-Ala-OH was used instead of Boc-D-Trp-OH corresponding to the 6'h amino acid residue.
The optical rotation, Rf of TLC, and elementary analysis of H-Ser-Tyr-(2-naphthyl)-D-Ala-Leu-Arg-Pro-Gly-NHS are shown below.
[ a ]D : -44.1 (c=1.0, MeOH) Rf :0.15 Elementary analysis (C4~H66N~201o) Calculated C:58.86% H:6.94% N:17.52%
Found C:58.72% H:6.88% N:17.49%
(6-2)Production of pGlu-His-Trp-Ser-Tyr-(2-naphthyl)-D-Ala-Leu-Arg-Pro-Gly-NH2 pGlu-His-Trp-Ser-Tyr-(2-naphthyl)-D-Ala-Leu-Arg-Pro-Gly-NHZ 0.93g (76.2% yield) was obtained by the same manner as in Example 1 (1-11) except that 0.98 of H-Ser-Tyr-(2-naphthyl)-D-Ala-Leu-Arg-Pro-Gly-NHZ
produced in the above (6-1) was used instead of H-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt.
The optical rotation, elementary analysis and amino acid composition of pGlu-His-Trp-Ser-Tyr-(2-naphthyl)-D-Ala-Leu-Arg-Pro-Gly-NH2 are shown below.
[ a ]D : -50.3 (c=1.0, H20) Elementary analysis (C69H88N1801a) Calculated 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 Production of pCllu-_H_is-Trn-Ser-~_, r-C7lv- . ,-A,-g-Pr - 71y_NH~
I,S~IDN0:11 (7-1) Production of H-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH~(SEfI ID N0:4) H-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2(SEQ ID N0:4) 1.12g was obtained by the same treatment as in Example 5 (5-1) except that Boc-Gly-OH was used instead of Boc-D-Trp-OH corresponding to the 6''' amino acid residue.
The optical rotation, Rf of TLC, and elementary analysis of H-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH~ are shown below.
Rf ~ 0. 10 (BuOH/AcOH/H20=4/1/5) Elementa analysis (C33HssNmOs) Calcula C:53.00% H:7.14% N:20.60%
Found :53.20% H:6.89% N:20.97%
(7-2) Production of pGlu-His-Tr er-Tyr-Gly-Leu-Arg-Pro-Gly-NH2(SEQ ID
NO:1) pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly- H~(SEQ ID NO:1) 1.16g (76.3%
yield) was obtained by the same method as in mple 1 (1-11) except that 1.12g of H-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH~(SEQ ID :4) produced in the above (6-1) was used instead of H-Ser-Tyr-D-Ser(But)-Leu- -Pro-NHEt.
The elementary analysis and amino acid composition of pGlu-His-Tr er-Amended: 05/28/99 [ a ]o : -40.1 (c=1.0, MeOH) Rf : 0. 10 (BuOH/AcOH/H~O=4/1/5) Elementary analysis (C~3H53N"O~) Calculated C:53.00% H:7.14% N:20.60%
Found C:53.20% H:6.89% N:20.9'l%
(7-2) Production of pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH.,(SEQ ID
NO:1) pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH~(SE~ ID N0:1) 1.168 (?6.3%
yield) was obtained by the same method as in Example 1 (1-11) except that 1.12g of H-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH~(SEQ ID N0:4) produced in the above (6-1) was used instead of H-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NHEt.
The elementary analysis and amino acid composition of pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH~ are shown below.
Elementary analysis (C55H;sNm0~3 ' AcOH ' 2H~0) Calculated 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) The method according to the present invention facilitates separation and purification of LH-RH and/or its derivative without side reaction such as racemization, because of the use of an enzyme reaction. Moreover the method according to the invention is extremely useful in industrial use because it leads to high yields of the LH-RH and/or its derivative and allows recovery and recycling of unreacted peptide fragments.
~I 1 Sequence Listing SEQ ID NO : 1 Sequence length : 10 Sequence type : amino acid Topology : linear Molecule type : peptide Feature Other information: Xaa denotes a pyroglutamic acid residue.
Sequence description Xaa His Trp Ser Tyr Gly Leu Arg Pro Gly SEQ ID NO : 2 Sequence length : 4 Sequence type : amino acid Topology : linear Molecule type : peptide Feature Other information: Xaa of the amino acid number 2 denotes Arg(NO~), and Xaa of the amino acid number 4 denotes Azgly(azaglycine)-NH2.
Sequence description Leu Xaa Pro Xaa SEQ ID NO : 3 Sequence length : 4 Sequence type : amino acid Topology : linear Molecule type : peptide Feature Other inform ation: Xaa denotes Azgly-NH2.

Sequence Leu Arg Pro Xaa SEQ ID NO : 4 Sequence length : 7 Sequence type : amino acid Topology : linear Molecule type : peptide Sequence description Ser Tyr Gly Leu Arg Pro Gly

Claims (11)

What is claimed is:

1. A method for producing LH-RH and/or its derivatives, characterized in that a peptide fragment shown by general formula (1):

pGlu-His-Trp-OR1 (1) (where R1 denotes a lower alkyl) and a peptide fragment shown by general formula (2):

H-Ser-Tyr-X-Leu-Arg-Pro-Y (2) (where X denotes an amino acid selected from the group consisting of D-Leu, D-Ser(But), D-Trp, (2-napthyl)-D-Ala, and Gly; and Y denotes Gly-NH2, Azaglycine or NHR2 (where R2 is a lower alkyl)) are allowed to react without adding KCl to a reaction medium, within the temperature range of 0 to 50°C, in the presence of an enzyme selected from the group consisting of chymotrypsin and chymotrypsin-like enzymes, and in a reaction medium consisting of a buffer itself or in a reaction medium in which an organic solvent selected from the group consisting of dimethylformamide, dimethylimidazolidinone, hexamethylphosphoryltriamide, methanol, ethanol, butanol, and ethyl acetate, and a buffer or water are mixed, to produce the LH-RH or its derivatives shown by general formula (3):

pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (3) (where X and Y are as defined above).

2. The method for producing LH-RH and/or its derivatives according to claim 1, further comprising a step of purifying the peptide shown by general formula (3) by partition chromatography.

3. The method for producing LH-RH and/or its derivatives according to claim 1 or 2, wherein said reaction is performed at a temperature ranging from 0°C to 20°C.

4. The method for producing LH-RH and/or its derivatives according to claim 1, 2 or 3, wherein the said R1 is an alkyl group having 1 to 3 carbons.

5. The method for producing LH-RH and/or its derivatives according to claim 1, 2 , 3 or 4, wherein the said R2 is an alkyl group having 1 to 3 carbons.

6. The method for producing LH-RH and/or its derivative according to claim 1, 2, 3, 4, or 5, wherein said enzyme selected from the group consisting of chymotrypsin and chymotrypsin-like enzymes is chymotrypsin.

7. A method for producing LH-RH and/or its derivatives, characterized in that a peptide fragment shown by general formula (1):

pGlu-His-Trp-OR1 (1) (where R1 denotes a lower alkyl) and a peptide fragment shown by general formula (2):

H-Ser-Tyr-X-Leu-Arg-Pro-Y (2) (where X denotes an amino acid selected from the group consisting of D-Leu, D-Ser(But), D-Trp, (2-napthyl)-D-Ala, and Gly; and Y denotes Gly-NH2, Azaglycine or NHR2 (where R2 is a lower alkyl)) are allowed to react without adding KCl to a reaction medium, at a temperature ranging from 0 to 50, in the presence of an immobilized enzyme where the enzyme is selected from the group consisting of chymotrypsin and chymotrypsin-like enzymes, and in the reaction medium consisting of a buffer itself or in the reaction medium in which an organic solvent selected from a group consisted of dimethylformamide, dimethylimidazolidinone, hexamethylphosphoryltriamide, methanol, ethanol, butanol, and ethyl acetate, and water or a buffer are mixed, to produce the LH-RH
or its derivative shown by general formula (3):

pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (3) (where X and Y are as defined above)

8. The method for producing LH-RH and/or its derivatives according to claim 7, further comprising a step of purifying the peptide shown by general formula (3) by partition chromatography.

9. The method for producing an LH-RH and/or its derivatives according to claim 7 or 8, wherein the reaction is performed at a temperature ranging from 0 to 20°C.

10. The method for producing an LH-RH and/or its derivatives according to claim 7, 8, or 9, wherein said R1 is an alkyl group having 1 to 3 carbons.

11. The method for producing an LH-RH and/or its derivatives according to claim 7, 8, 9 or 10, wherein said R2 is an alkyl group having 1 to 3 carbons.