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WO1992009620A1 - Procede pour synthetiser en phase de solution un peptide - Google Patents

Procede pour synthetiser en phase de solution un peptide Download PDF

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Publication number
WO1992009620A1
WO1992009620A1 PCT/US1991/008863 US9108863W WO9209620A1 WO 1992009620 A1 WO1992009620 A1 WO 1992009620A1 US 9108863 W US9108863 W US 9108863W WO 9209620 A1 WO9209620 A1 WO 9209620A1
Authority
WO
WIPO (PCT)
Prior art keywords
boc
trp
lys
phe
ala
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1991/008863
Other languages
English (en)
Inventor
David Stevenson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SmithKline Beecham Corp
Original Assignee
SmithKline Beecham Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SmithKline Beecham Corp filed Critical SmithKline Beecham Corp
Priority to JP4503322A priority Critical patent/JPH06503578A/ja
Priority to KR1019930701616A priority patent/KR930703345A/ko
Publication of WO1992009620A1 publication Critical patent/WO1992009620A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/60—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
    • C07K1/064—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups for omega-amino or -guanidino functions

Definitions

  • This invention relates to a process for preparing the peptide:
  • This peptide has pituitary growth hormone releasing activity.
  • This invention also relates to intermediates used in the process of the invention.
  • the method of preparing this peptide which is exemplified in U.S. 4,411,890 is a solid phase process in which the starting material amino acid is attached to a resin and is then coupled stepwise with the appropriate amino acids.
  • the intermediates in the solid phase process are peptide-resin compounds.
  • the desired peptide product is cleaved from the resin by treatment with hydrogen fluoride.
  • This invention provides an advantageous process for preparing L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH2.
  • the process is a solution phase method which provides solid, recrystallizable intermediates which are readily isolated and purified. These solid intermediates are generally crystalline and may be purified by recrystallization. Solid, recrystallizable intermediates, particularly in all steps of a process, are rare in peptide chemistry and offer an advantage in purification.
  • this invention is a process for preparing a compound of the formula:
  • hich comprises : a) coupling L-Lys (BOO -NH2 with Z-D-Phe; b) removing the Z group and coupling the resulting D- Phe-L-Lys(BOC)-NH 2 with Z-L-Trp-NH 2 ; c) removing the Z group and coupling the resulting L- Trp-D-Phe-L-Lys (BOC)-NH 2 with Z-L-Ala; d) removing the Z group and coupling the resulting L- Ala-L-Trp-D-Phe-L-Lys(BOC)-NH 2 with Z-D-Trp; e) removing the Z group and coupling the resulting D- Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC)-NH 2 with (BOC) 2 -L-His; and f) removing the BOC groups to give L-His-D-Trp-L
  • the intermediates are preferably recrystallized at each step in the process to prevent the build up of impurities.
  • the purifi ⁇ * " -.ion of the final product is facilitated.
  • the solution phase method of this invention avoids the use of corrosive hydrogen fluoride which is used in the solid phase procedure to cleave the product from the resin.
  • the solution phase process of the invention requires only one acid treatment (at the end of the reaction process to remove the amino protecting groups) , thus minimizing decomposition of the tryptophan residues; whereas, the solid phase method involves repetitive acid treatments.
  • Protected peptide intermediates in the process of this invention are also a feature of this invention.
  • the intermediates are particularly useful in the process of this invention because they are solids, generally crystalline and are readily purified by recrystallization.
  • the 'process of this invention may be represented as follows:
  • L-Trp L-tryptophan
  • L-Ala L-alanine
  • WSC water soluble carbodiimide [1- (3-dimethylamino- propyl) -3-ethylcarbodiimide hydrochloride]
  • intermediates (I) -(VI) are recrystallized before being used in the subsequent steps.
  • Intermediates (II) -(VI) are prepared by removing the benzyloxycarbonyl group (Z) by catalytic hydrogenolysis and then coupling with the appropriate amino acid.
  • N( ⁇ ) -benzyloxycarbonyl-N( ⁇ ) -t- butyloxycarbonyl-L-lysine amide (I) is prepared from N( ⁇ )- benzyloxycarbonyl-N( ⁇ )-t-butyloxycarbonyl-L-lysine by reacting with ammonia, water soluble carbodiimide [i.e. I- 3 (dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride], and 1-hydroxybenzotriazole.
  • the t-butyloxycarbonyl groups are removed using acid, optionally and a carbonium ion scavenger.
  • Suitable acids for removing the t-butyloxycarbonyl group are well known in the art, and include mineral acids or strong organic acids, such as trifluoroacetic acid.
  • Carbonium ion scavengers are also well known in the art, and include electrophilic aryl compounds and mercaptans, such as n-propylmercaptan.
  • the hydrogenolysis to remove Z protecting groups is performed using an appropriate catalyst, for example palladium on carbon, such as 5-10% palladium on carbon. Preferably, 10% palladium on carbon is. used.
  • the pressure at which the hydrogenolysis is performed is not critical, and may be performed at from atmospheric pressure up to several hundred psi. Typically, it is performed at atmospheric pressure up to 100 psi. Increasing the hydrogen pressure enhances the rate of reaction. Performing the reaction at about 100 psi is preferred. Vigorous stirring, such as about 600 rpm, in the autoclave is very advantageous to increase the reaction rate.
  • Coupling reactions are well known in the art and may be accomplished by activating the carboxyl group of the intermediate, such as by formation of an acyl halide, activated ester or activated anhydride, or coupling with a coupling reagent such as a carbodiimide, for example dicyclohexylcarbodiimide or water soluble carbodiimide, PPA (1-propanephosphonic acid cyclic anhydride) , DPPA (diphenylphosphoryl azide) or BOP reagent (benzotriazol-1- yloxy-tris (dimethylamino)-phosphonium hexafluorophosphate) . Water soluble carbodiimide is preferred.
  • the coupling reaction solutions are poured into aqueous base, for example, in the preparation of intermediates (II)-(V), the coupling reaction solutions are poured into aqueous potassium carbonate or potassium bicarbonate solution with vigorous stirring.
  • the coupling reaction solution is, preferably, poured into aqueous potassium bicarbonate solution.
  • the aqueous potassium bicarbonate is stirred vigorously.
  • the rate of addition should be about 500 mL- 1 L/minute.
  • the BOC protecting groups are removed with acid, optionally in the presence of a carbonium ion scavenger.
  • Trifluoroacetic acid is a suitable acid for removing the BOC group.
  • Use of a carbonium ion scavenger is preferred.
  • Typical carbonium ion scavengers are anisole, dimethoxy benzene and mercaptans.
  • n-Propyl mercaptan is suitable.
  • reaction mixture was filtered through a DMF-washed bed of Celite®. In turn, the solids were rinsed with DMF (35 mL) and the filtrate and washings were combined.
  • reaction mixture was filtered through a DMF-washed bed of Celite® and then was added slowly and with vigorous stirring into aqueous potassium carbonate solution (5% w/v, 1.6 L) .
  • the solid which precipitated was collected on a Buchner funnel and the filter cake washed with deionized water (about 1.2 L) until the eluant was neutral to pH paper.
  • the solid was dissolved in boiling isopropanol (240 mL) . To the boiling solution, deionized water (approximately 240 mL) was added to cloud point; the resulting solution was cooled at 0- 5°C overnight.
  • reaction mixture was filtered through a DMF-washed bed of Celite®. In turn, the solids were rinsed with DMF (50 mL) and the filtrate and washings were combined.
  • reaction mixture When the WSC had dissolved, the reaction mixture was allowed to stir at ambient temperature for 2.5 hours. At this time, TLC indicated the absence of D-Phe-L-Lys (BOC)-NH 2 .
  • the reaction mixture was filtered through a DMF-washed bed of Celite® and then was added slowly and with vigorous stirring into aqueous potassium carbonate solution (5% w/v, 1.6 L) .
  • the product separated as a solid which was collected at the pump and washed with deionized water (1.2 L) until the washings were no longer basic.
  • the damp cake was dissolved in boiling methanol (500 mL) ; deionized water (150 mL) was added to cloud point. The solution was allowed to cool at 0- 5°C overnight .
  • TLC indicated the absence of starting material; HPLC showed a trace to be remaining.
  • the reaction mixture was filtered through a DMF-washed bed of Celite®. In turn, the solids were -rinsed with DMF (50-mL) and the filtrate and washings were combined.
  • the reaction mixture was filtered through a DMF-washed bed of Celite® and then was added slowly and with vigorous stirring into aqueous potassium carbonate solution (5% w/v, 1.6 L) .
  • the product separated as a solid, which was collected at the pump and washed with deionized water (1.6 L) until the washings were neutral.
  • the damp filter cake was dissolved in boiling methanol (700 mL) ; deionized water (300 mL) was added to cloud point. The resulting solution was cooled overnight at 0-5°C.
  • TLC indicated the absence of starting material; HPLC showed a trace to be remaining.
  • the reaction mixture was filtered through a DMF-washed bed of Celite®. In turn, the solids were rinsed with DMF (50 mL) and the filtrate and washings were combined.
  • reaction mixture was filtered through a DMF-washed bed of Celite®. In turn, the solids were rinsed with DMF (50 mL) and the filtrate and washings were combined.
  • reaction mixture was evaporated to a gum which was dissolved in distilled water (300 mL) and re-evaporated twice. Then, the mixture was dissolved in distilled water (1 L) and the pH was adjusted to approximately 3 by the dropwise addition of dilute aqueous ammonium hydroxide solution.
  • This material was purified by reverse phase liquid chromatography using YMC-type AS-5055, end-capped octadecyl silica, spherical in shape, with a particle size of 50 ⁇ and a pore size of 120 A 0 .
  • the bed size was 5 cm i.d. x 50 cm length, corresponding to a bed volume of 981 mL, packed in a stainless steel HPLC column. There was also a guard column of 5 cm i.d. x 10 cm length containing the same packing material (196 mL) .
  • This was equilibrated with 0.1 M aqueous ammonium acetate solution pH 4.5 (4 L) , utilizing a Beckman Prep-350 HPLC system, monitoring the eluant at 254 nm.
  • the above pH adjusted reaction mixture was further diluted with distilled water (300 mL) and then applied to the column at a flow rate of 100 mL/min.
  • the column was washed with 1.0 M ammonium acetate pH 8 (6.0 L), followed by 0.1 M ammonium acetate pH 4.5 (2.5 L) .
  • the product was removed by step-wise elution with solutions of acetonitrile in 0.1 M aqueous ammonium acetate pH 4.5 as follows: 10% v/v (1 L) , 15% v/v (900 mL) , 17% v/v (1 L) , 20% v/v (2.5 L) , 25% v/v (2.5 L) .
  • Fractions were collected, varying in size from 125 mL to 1 L.
  • the column was washed with 50% v/v acetonitrile in 0.1 M aqueous ammonium acetate pH 4.5 (3.5 L) .
  • A 1/4 v/v acetonitrile/water-0.1M ammonium dihydrogen phosphate- 0.8M phosphoric acid (adjusted to pH 3.0 with triethylamine)
  • B 3/7 v/v acetonitrile/water-0.1M ammonium dihydrogen phosphate-0.8M phosphoric acid (adjusted to pH 3.0 with triethylamine), 0% B for 23 min, 0%-100% B over 37 min, UV detection at 210 nm) .

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Endocrinology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

Procédé pour préparer, en phase de solution, le peptide L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH2, qui fait preuve d'une activité libératrice de l'hormone de croissance hypophysaire.
PCT/US1991/008863 1990-11-30 1991-11-25 Procede pour synthetiser en phase de solution un peptide Ceased WO1992009620A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP4503322A JPH06503578A (ja) 1990-11-30 1991-11-25 ペプチド用液相法
KR1019930701616A KR930703345A (ko) 1990-11-30 1991-11-25 펩타이드의 액상 합성법

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US62109490A 1990-11-30 1990-11-30
US621,094 1990-11-30

Publications (1)

Publication Number Publication Date
WO1992009620A1 true WO1992009620A1 (fr) 1992-06-11

Family

ID=24488689

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/008863 Ceased WO1992009620A1 (fr) 1990-11-30 1991-11-25 Procede pour synthetiser en phase de solution un peptide

Country Status (10)

Country Link
EP (1) EP0564587A4 (fr)
JP (1) JPH06503578A (fr)
KR (1) KR930703345A (fr)
AU (1) AU9166491A (fr)
CA (1) CA2097305A1 (fr)
IE (1) IE914157A1 (fr)
MX (1) MX9102333A (fr)
PT (1) PT99654A (fr)
WO (1) WO1992009620A1 (fr)
ZA (1) ZA919440B (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5633345A (en) * 1992-06-19 1997-05-27 Takara Shuzo Co., Ltd. Cyclic peptides
EP2321339B1 (fr) * 2008-08-08 2016-09-28 Imperial Innovations Limited Diafiltration résistante aux solvants de peptides, de pna ou d'oligonucléotides

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4105652A (en) * 1977-08-05 1978-08-08 Hoffmann-La Roche Inc. Synthesis of human β-endorphin
US4237046A (en) * 1979-04-27 1980-12-02 Miklos Bodanszky Polypeptides and methods of preparation
US4411890A (en) * 1981-04-14 1983-10-25 Beckman Instruments, Inc. Synthetic peptides having pituitary growth hormone releasing activity

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05508859A (ja) * 1990-07-24 1993-12-09 イーストマン コダック カンパニー ペプチド合成方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4105652A (en) * 1977-08-05 1978-08-08 Hoffmann-La Roche Inc. Synthesis of human β-endorphin
US4237046A (en) * 1979-04-27 1980-12-02 Miklos Bodanszky Polypeptides and methods of preparation
US4411890A (en) * 1981-04-14 1983-10-25 Beckman Instruments, Inc. Synthetic peptides having pituitary growth hormone releasing activity

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
E. GROSS et al., "The Peptides", Volume 3, published 1981 by Academic Press (N.Y.), see pages 15-17, 76-78 and 260. *
E. GROSS et al., "The peptides", Volume 5, published 1983 by Academic Press (N.Y.), see pages 66-75 and 189, especially pages 69, 71, 75 and 189. *
See also references of EP0564587A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5633345A (en) * 1992-06-19 1997-05-27 Takara Shuzo Co., Ltd. Cyclic peptides
US5633346A (en) * 1992-06-19 1997-05-27 Takara Shuzo Co., Ltd. Process for systhesizing cyclic peptides
EP2321339B1 (fr) * 2008-08-08 2016-09-28 Imperial Innovations Limited Diafiltration résistante aux solvants de peptides, de pna ou d'oligonucléotides

Also Published As

Publication number Publication date
PT99654A (pt) 1992-10-30
IE914157A1 (en) 1992-06-03
EP0564587A1 (fr) 1993-10-13
CA2097305A1 (fr) 1992-05-31
MX9102333A (es) 1993-06-01
JPH06503578A (ja) 1994-04-21
EP0564587A4 (en) 1994-08-24
KR930703345A (ko) 1993-11-29
AU9166491A (en) 1992-06-25
ZA919440B (en) 1992-12-30

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