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EP1546186A1 - Procede de synthese de peptides - Google Patents

Procede de synthese de peptides

Info

Publication number
EP1546186A1
EP1546186A1 EP02765630A EP02765630A EP1546186A1 EP 1546186 A1 EP1546186 A1 EP 1546186A1 EP 02765630 A EP02765630 A EP 02765630A EP 02765630 A EP02765630 A EP 02765630A EP 1546186 A1 EP1546186 A1 EP 1546186A1
Authority
EP
European Patent Office
Prior art keywords
peptide
amino acid
amine
derivative
mixture
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.)
Withdrawn
Application number
EP02765630A
Other languages
German (de)
English (en)
Other versions
EP1546186A4 (fr
Inventor
Vladimir Vasilyevich Samukov
Pavel Ivanovich Pozdnyakov
Nikolai Nilolaevich Karpyshev
Daria Vladimirovna Lebedeva
Hack-Joo Kim
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.)
A & Pep Inc
Original Assignee
A & Pep Inc
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 A & Pep Inc filed Critical A & Pep Inc
Publication of EP1546186A1 publication Critical patent/EP1546186A1/fr
Publication of EP1546186A4 publication Critical patent/EP1546186A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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

Definitions

  • the present invention relates to a novel method for synthesizing peptides in solution, which provides substantial facilitation and acceleration of the whole synthetic procedure.
  • a basic problem in peptide synthesis is one of blocking or protecting the ⁇ -amino group from interaction with a carboxyl group on the same amino acid. These undesirable side reactions are prevented by attaching to one amino acid a group that will render the - NH 2 group unreactive and still permit the desired reaction to proceed.
  • the blocking group is preferably one that can be easily removed without chemically altering the remainder of the molecule including the peptide linkage that has been built up during the synthesis.
  • the peptide chain assembly usually consists of multiple consecutive synthetic cycles, and each cycle includes two basic chemical stages: 1) removal of a protecting group from the ⁇ -amino group of a peptide being built (N ⁇ -deprotection stage), and 2) coupling of the N ⁇ -deprotected peptide with subsequent N ⁇ -protected amino acid or peptide segment. Therefore, the ease and swiftness of performing the N ⁇ -deprotection stage stipulate for the rate and effectiveness of the overall process of the peptide assembly.
  • Hydrogenolytic cleavage of the Z group from short peptides is simple and clean; the work-up comprises just removing the catalyst and evaporating the solvent. However, with longer peptides the reaction may appear very slow. In addition, the presence of sulfur- containing amino acids in a peptide renders impossible catalytic hydrogenation.
  • the Fmoc group a base-labile protecting group widely utilized in contemporary peptide synthesis, has revealed numerous advantages: it has excellent acid stability; it is readily cleaved in a non-hydrolytic fashion by a variety of amines via base-promoted J3- elimination; other standard peptide protecting groups (i.e. Boc, benzyl) can be removed in its presence; and, the amine generated upon cleavage is the free base (Atherton, E.; Sheppard, R.C., in The Peptides; Udenfriend, S.; Meienhofer, J., Eds.; Academic Press: New York, NY, 1987; Vol. 9, p. 1.).
  • urethane amino protecting groups derived from 2-arylsulfonylethanols can be cleaved by amines under non-hydrolytic conditions via base-promoted ⁇ -elimination, a mechanism very similar to that described for the Fmoc cleavage (Samukov, V.V.; Sabirov, A.N.; Troshkov, M.L. Zh. Obshchei Khim. 1988, 58, 1432).
  • the urethane cleavage product aryl vinyl sulfone
  • the product of trapping represents a vinyl sulfone-amine adduct, i.e.
  • the radical R 1 in the urethane structure may be substituted or unsubstituted aryl, however, in order to provide a reasonably high rate of urethane cleavage by base-promoted J3 -elimination it is preferable to employ aryls bearing strong electron-withdrawing substituents.
  • substituted aryl radicals the most preferable are 4-nitrophenyl and 4-sulfonylated phenyls.
  • N ⁇ -protecting group represents known 2-(4-nitrophenyl)sulfonylethoxycarbonyl (Nsc) group (Samukov, V.V.; Sabirov, A.N.; Pozdnyakov, P.I. Tetrahedron Left. 1994, 35, 7821; U.S. Patents 5,616,788; 6,265,590).
  • the group of 4-sulfonylated phenyls can be exemplified by the 2-(4- methylsufonylphenyl)sulfonylethoxycarbonyl (Mpc) group already known in the art as an amino protecting group (Verhart, C; Tesser, G.
  • Other preferred embodiments of the group include, for example, 4- phenylsulfonylphenyl and a variety of 4-sulfonamide derivatives, namely, 4- dimethylamidosulfonylphenyl; 4-diethylamidosulfonylphenyl; 4-morpholidosulfonyl- phenyl; 4-piperididosulfonylphenyl.
  • a secondary amine employed as a cleavage and scavenging reagent to combine properties of a strong base and a potent nucleophile. Also important are physical properties of the amine.
  • the separation of an excess of the amine from a reaction mixture after the N ⁇ -deprotection reaction may be achieved in a variety of ways, however, the most simple and expeditious way is the vacuum distillation (evaporation). It is thus desirable for the amine to be volatile (i.e., to have a low boiling point).
  • amines which meet all these requirements the most preferable are: dimethylamine; diethylamine; di-n-propylamine; piperidine, pyrrolidine; morpholine.
  • the excess of an amine required for efficient cleavage and trapping may vary from 2 to 100 molar equivalents; the optimal excess for a particular case can easily be selected by a practitioner in the area of peptide chemistry.
  • Solvents suitable for conducting the N ⁇ -deprotection stage are preferably polar, aprotic and volatile solvents capable of dissolving starting materials and reaction products, such as dichloromethane, acetonitrile, tetrahydrofuran, dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide, and the like.
  • the equimolar mixture of an N ⁇ -deprotected amino acid or peptide derivative and a vinyl sulfone-amine adduct is further introduced without any additional work-up into the coupling reaction with the next peptide or amino acid derivative N ⁇ -protected with the above indicated or another urethane group.
  • an ⁇ -carboxylic function of the Not-protected component for coupling can be achieved by a variety methods known in the art, for example, by the preceding formation of active esters, such as 4-nitrophenyl, pentachlorophenyl, pentafluorophenyl, 1-hydroxybenzotriazolyl esters or other known types of active esters; by the conversion into symmetric or mixed anhydrides, or into azides. Coupling may also be performed in the presence of known coupling reagents, e.g.
  • Isolation of the newly formed peptide from the reaction mixture can be performed by usual procedures known in the art, e.g. extraction, precipitation, washing, chromatography, etc.
  • side chains of amino acid or peptide derivatives can be protected with appropriate protecting groups during synthetic cycles and then deprotected to obtain the final product.
  • protecting groups that can be used, their application, and their subsequent removal are well documented in the literature and known to those skilled in the art.
  • Chromatographic mobility values R f are shown for thin-layer chromatography sheets Alufolien Kieselgel 60 F 254 (Merck, Darmstadt, Germany); chloroform/methanol/acetic acid, 95:5:3 (A) and 90:10:3 (B), and ethyl acetate/pyridine/acetic acid/water, 60:5:15:10 (C), have been used as developing solvents, spots are detected by UV-absorbance and/or by ninhydrin reaction. Molecular ion masses (M+H) + have been measured using MALDI- TOF VISION 2000 device (Thermo Bioanalysis, England).
  • Example 1 2-(4-Phenylsulfonyl)phenylsulfonylethyl chloroformate (Pspsc-Cl) a.
  • 2-(4-Phenylsulfonyl)phenylsulfonylethanol To a stirred solution of 2-(4-chlorophenyl)sulfonylethanol (4.41 g) and thiophenol
  • a mixture of crude N,N-dimethyl-4-bromobenzenesulfonamide, 3 ml of 2- mercaptoethanol and 30 ml of DMF is supplemented with 18 ml of 2 n. ethanolic solution of KOH and warmed for 8 h at 70°C.
  • the mixture is diluted with 200 ml of water and extracted with 2 x 70 ml of ethyl acetate.
  • the extract is washed with 5% aq. NaHCO 3 and brine, dried over anhydrous sodium sulfate and evaporated at reduced pressure.
  • the oily residue is then dissolved in 50 ml of acetone. To the solution, 4 ml of 0.3 M aq.
  • Dmspsc-Cl Prepared from 2-(4-dimethylamidosulfonyl)phenylsulfonylethanol as described in the Example lb. Mp 135°C (softening), 144°C.
  • CiiHi 4 ClNO 6 S 2 calcd.: C 37.13%, H 3.97%, N 3.94%; found: C 37.42%, H 4.05%, N 3.77%.
  • N ⁇ -Protected amino acids prepared by this method are listed in the Table 1.
  • Nsc- and Mpc-amino acids are prepared according to published procedures.
  • Table 1 N ⁇ -Protected amino acids
  • the dipeptide (Example 4a) is dissolved in DMF-piperidine mixture (1:4, v/v; 3 ml) and, after 10 min, evaporated to dryness at 1 mm Hg. The residue is co-evaporated with 3 ml of DMF and dissolved in DMF (2 ml). To the solution, Pipspsc-Gly-OH (0.5 mmol, entry 12, Table 1), HOBt (0.74 mmol), NMM (1 mmol) and BOP (0.6 mmol) are added, and the mixture is kept for 1.5 h at ambient temperature. The mixture is then distributed between 25 ml of ethyl acetate and 20 ml of 5% aq.
  • the tripeptide (Example 4b) is dissolved in DMF-piperidine mixture (1:4, v/v; 3 ml) and, after 15 min, evaporated to dryness at 1 mm Hg. The residue is co-evaporated with 3 ml of DMF and dissolved in DMF (2 ml). To the solution, Dmspsc-Gly-OH (0.45 mmol, entry 11, Table 1), HOBt (0.6 mmol), NMM (0.8 mmol) and BOP (0.5 mmol) are added, and the mixture is kept for 1.5 h at ambient temperature. The mixture is then distributed between 35 ml of ethyl acetate and 20 ml of 5% aq.
  • the tetrapeptide (Example 4c) is dissolved in DMF-piperidine mixture (1:4, v/v; 3 ml) and, after 15 min, evaporated to dryness at 1 mm Hg. The residue is co-evaporated with 3 ml of DMF and dissolved in DMF (3 ml). To the solution, Boc-Tyr(Boc)-OPfp (0.4 mmol) is added, and the mixture is kept for 30 min at ambient temperature. The mixture is then distributed between 35 ml of ethyl acetate and 20 ml of 5% aq. NaHCO 3 , the organic phase is separated, washed subsequently with water, 0.5 n. aq.
  • Example 5 Synthesis of Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu (THF- ⁇ 2) a.
  • Nsc-Lys(Boc ⁇ Phe-Leu-OtBu Mspsc-Phe-Leu-OtBu (0.87 mmol, Example 4a) is N ⁇ -deprotected as described in the Example 4b and dissolved in 4 ml of DMF.
  • Nsc-Lys(Boc)-OH (1 mmol
  • HOBt 1.5 mmol
  • BOP 1.1 mmol
  • NMM (2 mmol
  • the tripeptide (Example 5a) is dissolved in DMF-piperidine mixture (1:4, v/v; 5 ml) and, after 15 min, evaporated to dryness at 1 mm Hg. The residue is co-evaporated with 3 ml of DMF and dissolved in DMF (5 ml). To the solution, Nsc-Pro-OH (0.8 mmol), HOBt (1.2 mmol), NMM (1.8 mmol) and BOP (0.85 mmol) are added, and the mixture is kept for 1.5 h at ambient temperature. The mixture is then distributed between 35 ml of ethyl acetate and 20 ml of 5% aq.
  • the tetrapeptide (Example 5b) is dissolved in DMF-piperidine mixture (1:4, v/v; 5 ml) and, after 15 min, evaporated to dryness at 1 mm Hg. The residue is co-evaporated with 5 ml of DMF and dissolved in DMF (5 ml). To the solution, Nsc-Gly-OH (0.8 mmol), HOBt (1.2 mmol), NMM (1.8 mmol) and BOP (0.85 mmol) are added, and the mixture is kept for 2.5 h at ambient temperature. The mixture is then distributed between 35 ml of ethyl acetate and 20 ml of 5% aq.
  • the dipeptide (Example 5d) is dissolved in DMF-piperidine mixture (1:4, v/v; 5 ml) and, after 15 min, evaporated to dryness at 1 mm Hg. The residue is co-evaporated with 2 x 5 ml of DMF and dissolved in DMF (5 ml). To the solution, Boc-Leu 4- nitrophenyl ester (1.1 mmol) and HOBt (0.2 mmol) are added. The mixture is kept for 3 h at ambient temperature. The mixture is then distributed between 35 ml of ethyl acetate and 30 ml of cold 0.25 n. aq. HCl. The organic phase is separated, washed subsequently with cold 0.25 n. aq.
  • the pentapeptide (0.38 mmol, Example 5c) is dissolved in DMF-piperidine mixture (1:4, v/v; 3 ml) and, after 15 min, evaporated to dryness at 1 mm Hg. The residue is co- evaporated with 5 ml of DMF and dissolved in DMF (3 ml). To the solution, Boc-Leu- Glu(OtBu)-Asp(OtBu)-OH (0.41 mmol, Example 5e), HOBt (1 mmol), NMM (1 mmol) and BOP (0.55 mmol) are added, and the mixture is kept for 4 h at ambient temperature.
  • the protected octapeptide (440 mg) is dissolved in 10 ml of cold TFA-water mixture (9:1, v/v) and stirred for 40 min at ambient temperature. The solution is evaporated, the thick oily residue is treated with cold ether. The precipitate is filtered off, washed with ether and dried under vacuum to yield 380 mg of the crude octapeptide (87% purity by HPLC).
  • the peptide is dissolved in 4% aq. acetic acid and purified by preparative reversed phase chromatography on a Lichroprep RP18 column using water-ethanol gradient buffered with acetic acid.
  • the present invention provides a novel method for synthesizing peptides in solution, thereby substantially facilitating and accelerating the whole synthetic procedure.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Cette invention se rapporte à un nouveau procédé pour la synthèse de peptides en solution, ce procédé consistant : (a) à traiter un dérivé de peptide ou d'acide aminé, qui possède une fonction α-amine bloquée par un groupe uréthane de la formule R1-SO2-CH2CH2-O-CO-, où R1 représente aryle, avec une quantité excédentaire d'une amine secondaire aliphatique dans un solvant organique, pour produire la libération de la fonction α-amine libre de ce dérivé d'acide aminé ou de peptide et la formation d'un produit d'addition à base d'amine tertiaire entre la seconde amine secondaire et le composés vinyle libéré de la formule R1-SO2-CH2=CH2 ; (b) à retirer le solvant et la quantité excédentaire de l'amine secondaire ; (c) à mettre en contact le mélange ainsi formé du dérivé d'acide aminé ou de peptide déprotégé en position Nα et du produit d'addition à base d'amine tertiaire avec un dérivé de peptide ou d'acide aminé ultérieur protégé en position Nα par le groupe uréthane indiqué ci-dessus ou par un autre groupe uréthane, dans des conditions entraînant la formation d'une liaison peptide entre la fonction α-amine libre du dérivé d'acide aminé ou de peptide déprotégé en position Nα et une fonction α-carboxylique du dérivé de peptide ou d'acide aminé ultérieur protégé en position Nα ; (d) à séparer le peptide nouvellement formé du mélange de réaction ; (e) et à répéter les procédures décrites ci-dessus, jusqu'à obtenir le polypeptide souhaité.
EP02765630A 2002-08-26 2002-08-26 Procede de synthese de peptides Withdrawn EP1546186A4 (fr)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/KR2002/001604 WO2004018501A1 (fr) 2002-08-26 2002-08-26 Procede de synthese de peptides

Publications (2)

Publication Number Publication Date
EP1546186A1 true EP1546186A1 (fr) 2005-06-29
EP1546186A4 EP1546186A4 (fr) 2006-02-08

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ID=31944805

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EP02765630A Withdrawn EP1546186A4 (fr) 2002-08-26 2002-08-26 Procede de synthese de peptides

Country Status (6)

Country Link
EP (1) EP1546186A4 (fr)
JP (1) JP2006511459A (fr)
CN (1) CN1649892A (fr)
AU (1) AU2002329070A1 (fr)
CA (1) CA2496739A1 (fr)
WO (1) WO2004018501A1 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10354848B4 (de) 2003-11-20 2005-09-29 Brandenburgische Technische Universität Cottbus Verfahren und Einrichtung zur Herstellung von pelletartigen Körpern aus ungebundenen Fasern
WO2006045503A1 (fr) 2004-10-19 2006-05-04 Lonza Ag Procede de synthese de peptides en phase solide
CN100355773C (zh) * 2006-01-19 2007-12-19 九江石化波涛生化科技有限公司 高分子量多肽聚合物的合成方法
MX339762B (es) * 2011-09-28 2016-05-27 Univ Autonoma Del Estado De Morelos Metalopeptidos inmunomoduladores (immp) y composiciones que los contienen.
FR3090636B1 (fr) * 2018-12-24 2021-01-01 Strainchem Procédé de synthèse de peptides

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2652581B1 (fr) * 1989-10-02 1991-12-13 Rhone Poulenc Chimie Procede de solubilisation de peptides et procede de synthese de peptides.
US5859191A (en) * 1996-12-05 1999-01-12 The Regents Of The University Of California Method for the site-specific modification of peptide alpha amines
KR100418962B1 (ko) * 2001-06-07 2004-02-14 김학주 2-(4-나이트로페닐설포닐)에톡시카르보닐-아미노산류를사용하여 펩티드를 고수율 및 고순도로 제조하는 방법

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Publication number Publication date
CN1649892A (zh) 2005-08-03
WO2004018501A1 (fr) 2004-03-04
AU2002329070A1 (en) 2004-03-11
JP2006511459A (ja) 2006-04-06
EP1546186A4 (fr) 2006-02-08
CA2496739A1 (fr) 2004-03-04

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