Process for producing LH-RH derivatives

Details Process for producing LH-RH derivatives Process for producing LH-RH derivatives

2000-02-04

2002-09-10

Weber, Jon P. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Enzymatic production of a protein or polypeptide

C435S213000

Reexamination Certificate

active

06448031

ABSTRACT:

This application is a 371 of PCT/JP97/02705 filed Aug. 4, 1997.
TECHNICAL FIELD
The present invention relates to an enzymatic method for effectively producing an LH-RH derivative, 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 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 LH-RH derivatives 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-OR
1
  (1)
(where R
1
denotes lower alkyl) and a peptide fragment shown by general formula (2):
H-Ser-Tyr-X-Leu-Arg-Pro-Y (SEQ ID NO:4)  (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-NH
2
, Azgly (Azaglycine)-NH
2
or NHR
2
(where R
2
is lower alkyl)) are allowed to react in the presence of an enzyme selected from the group consisting of chymotrypsin or chymotrypsin-like enzymes to produce a LH-RH derivative shown by general formula (3):
pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (SEQ ID NO:1)  (3)
(where X and Y denote the same as in the above formula).
Here the present invention is characterized in that the abovementioned R
1
is an alkyl group having 1 to 3 carbons and R
2
is an alkyl group having 1 to 3 carbons. Further the present invention is characterized in that the enzyme selected from the group consisting of the chymotrypsin or chymotrypsin-like enzymes is chymotrypsin.
Moreover, the present invention is characterized in that a peptide fragment shown by general formula (4):
pGlu-His-Trp-OR
1
  (4)
(where R
1
denotes lower alkyl) and a peptide fragment shown by general formula (5):
H-Ser-Tyr-X-Leu-Arg-Pro-Y (SEQ ID NO:4)  (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-NH
2
, Azgly-NH
2
or NHR
2
(where R
2
is lower alkyl)) are allowed to react in the presence of an enzyme selected from the group consisting of chymotrypsin or chymotrypsin-like enzymes and in a solvent in which water or buffer is mixed with organic solvent to produce LH-RH derivatives shown by general formula (6):
pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (SEQ ID NO:1)  (6)
(where X and Y denote the same as in the above formula).
Here the present invention is characterized in that the solvent wherein water or buffer and an organic solvent are mixed is a mixture in which water or buffer and an organic solvent miscible with water are mixed, or is a mixture in which water or buffer are saturated with an organic solvent partially miscible with water.
Moreover, the present invention is characterized in that a peptide fragment shown by general formula (7):
pGlu-His-Trp-OR
1
  (7)
(where R
1
denotes lower alkyl) and a peptide fragment shown by general formula (8):
H-Ser-Tyr-X-Leu-Arg-Pro-Y (SEQ ID NO:4)  (8)
(where X denotes an amino acid selected from a group consisting of D-Leu, D-Ser(But), D-Trp, (2-naphthyl)-D-Ala, and Gly; Y denotes Gly-NH
2
, Azgly-NH
2
or NHR
2
(where R
2
is lower alkyl)) are allowed to react in the presence of an immobilized enzyme that is selected from the group consisting of chymotrypsin or chymotrypsin-like enzymes and in a solvent in which water or buffer is mixed with an organic solvent to produce LH-RH derivatives shown by general formula (9):
 pGlu-His-Trp-Ser-Tyr-X-Leu-Arg-Pro-Y (SEQ ID NO:9)  (9)
(where X and Y denote the same as described above).
In this specification, the LH-RH derivative is referred to as the one wherein Glycine at position 6 or at position 10 of the LH-RH shown by general formula (10) (SEQ. ID.NO:1):
PGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH
2
  (10)
is substituted by a different amino acid, 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 a general D-amino acid or a modified D-amino acid. For example, the D-amino acid mentioned here may be D-Leu, D-rrp, 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-Trp, 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 10 (hereinafter referred to as Y) is preferably Gly-NH
2
, Azgly-NH
2
or NHR
2
(where R
2
is lower alkyl).
“Lower alkyl” used herein means an alkyl having 1 to 3 carbons such as methyl, ethyl, propyl, or isopropyl. R
1
is preferably a methyl or an ethyl group and R
2
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.
Tyar: 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
Trp: Tryptophane 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: Dicyclohexylcarbodiimide
WSC: N-ethyl-N′-dimethylaminopropyl-carbodiimide
HOSu: N-hydroxysucciniimide
HOBt: 1-Hydroxybenzotriazol
MeOH: Me

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