WO2004018501A1 - Method for synthesizing peptides - Google Patents

Method for synthesizing peptides Download PDF

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Publication number
WO2004018501A1
WO2004018501A1 PCT/KR2002/001604 KR0201604W WO2004018501A1 WO 2004018501 A1 WO2004018501 A1 WO 2004018501A1 KR 0201604 W KR0201604 W KR 0201604W WO 2004018501 A1 WO2004018501 A1 WO 2004018501A1
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WIPO (PCT)
Prior art keywords
peptide
amino acid
amine
derivative
mixture
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PCT/KR2002/001604
Other languages
French (fr)
Inventor
Vladimir Vasilyevich Samukov
Pavel Ivanovich Pozdnyakov
Nikolai Nilolaevich Karpyshev
Daria Vladimirovna Lebedeva
Hack-Joo Kim
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A & Pep Inc.
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Application filed by A & Pep Inc. filed Critical A & Pep Inc.
Priority to CA002496739A priority Critical patent/CA2496739A1/en
Priority to CN02829521.8A priority patent/CN1649892A/en
Priority to JP2004530625A priority patent/JP2006511459A/en
Priority to EP02765630A priority patent/EP1546186A4/en
Priority to PCT/KR2002/001604 priority patent/WO2004018501A1/en
Priority to AU2002329070A priority patent/AU2002329070A1/en
Publication of WO2004018501A1 publication Critical patent/WO2004018501A1/en

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    • 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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Abstract

The present invention relates to a novel method for synthesizing peptides in solution, the method comprising: a) treating a peptide or amino acid derivative, which has an α-amine function blocked with a urethane group of the formula R1-SO2-CH2CH2-O-CO-, wherein R1 is aryl, with an excess of an aliphatic secondary amine in an organic solvent to provide the release of the free α-amine function of said amino acid or peptide derivative and the formation of a tertiary amine adduct between said secondary amine and the liberated vinyl compound of the formula R1-SO2-CH2=CH2; b) removing the solvent and the excess of said secondary amine; c) contacting thus formed mixture of the Nα-deprotected amino acid or peptide derivative and said tertiary amine adduct with subsequent peptide or amino acid derivative Nα-protected with the above indicated or another urethane group under conditions providing the formation of a peptide bond between the free α-amine function of the Nα-deprotected amino acid or peptide derivative and an α-carboxylic function of the subsequent Nα-protected peptide or amino acid derivative; d) separating the newly formed peptide from the reaction mixture; e) repeating procedures as set forth above until the desired polypeptide is obtained.

Description

METHOD FOR SYNTHESIZING PEPTIDES
Technical Field
The present invention relates to a novel method for synthesizing peptides in solution, which provides substantial facilitation and acceleration of the whole synthetic procedure.
Background Art
A basic problem in peptide synthesis is one of blocking or protecting the α-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 - NH2 group unreactive and still permit the desired reaction to proceed. In addition to providing protection for the amino group, 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 α-amino group of a peptide being built (Nα-deprotection stage), and 2) coupling of the Nα-deprotected peptide with subsequent Nα-protected amino acid or peptide segment. Therefore, the ease and swiftness of performing the Nα-deprotection stage stipulate for the rate and effectiveness of the overall process of the peptide assembly.
The vast majority of amino protecting groups reported for peptide syntheses have centered around three groups: carbobenzoxy (Z or Cbz), cleaved by catalytic hydrogenation; tert-butoxycarbonyl (Boc or t-Boc), cleaved by mild acidolysis; and 9- fluorenylmethoxycarbonyl (Fmoc), cleaved by secondary amines (for a review see, e.g. Kocienski, P. Protecting Groups, Corrected Edition, Thieme: Stuttgart-N.-Y., 2000; Greene, T.W.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 3rd Ed.; John Wiley & Sons, Inc.: New York, NY, 1999). These groups are orthogonal to each other because the Boc group is stable to secondary amines and hydrogenolysis, the Fmoc group is stable to acidolysis, and the Z group is stable to secondary amines and mild acidolysis. Each of these protecting groups has found its place in peptide chemistry.
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.
Cleavage of the Boc group produces only volatile by-products, so the work-up represents removing an acidic cleavage reagent. Complete removal of the acid, which is critical for successful proceeding of the following coupling stage, requires in many cases multiple evaporations, careful washing and drying of an Nα-deprotected peptide.
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.). However, while the Fmoc methodology has found broad application in automated solid phase peptide synthesis, it has several shortcomings which hamper its use in solution peptide synthesis. First, Fmoc -protected amino acids (and peptides) have poor stability in both weakly basic and neutral DMF solutions usually employed at the coupling stage (Atherton, E.; Bury, C; Sheppard, R.C.; Williams, BJ. Tetrahedron Lett. 1979, 3041; Bodanszky, M.; Deshmane, S.S.; Martinez, J. J. Org. Chem. 1979, 44, 1622.). Second, and perhaps most detrimental, is a relatively slow and reversible reaction of the Fmoc cleavage alkene product, dibenzofulvene, with an excess of the cleaving reagent, secondary aliphatic amine. It makes difficult efficient trapping of dibenzofulvene with the amine and its subsequent removal from a reaction mixture, and eventually leads to the contamination of the target peptide product with the sparingly soluble dibenzofulvene polymer. It is apparent that there is a need in efficient and flexible methods for solution peptide synthesis which could share the advantages of the Fmoc Nα-protection while being devoid of its drawbacks.
Disclosure of Invention
Some of the authors of the present invention have found earlier that urethane amino protecting groups derived from 2-arylsulfonylethanols can be cleaved by amines under non-hydrolytic conditions via base-promoted β-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). But surprisingly, in contrast to the Fmoc, the urethane cleavage product, aryl vinyl sulfone, is trapped very rapidly and irreversibly when an excess of a secondary aliphatic amine is employed as a cleavage reagent (Samukov, V.V.; Sabirov, A.N.; Pozdnyakov, P.I. Tetrahedron Lett. 1994, 35, 7821; Sabirov, A.N.; Kim, Y.-D.; Kim, H.-l; Samukov, V.V. Protein Peptide Lett. 1997, 4, 307). In this case the product of trapping represents a vinyl sulfone-amine adduct, i.e. tertiary amine, so the removal of the excess of the cleavage reagent and the solvent gives an equimolar mixture of an Nα-deprotected peptide and the above tertiary amine. It has been then assumed that there is no need to separate this mixture before commencing the coupling stage, because tertiary amines are always added to the coupling mixture in order to maintain the basicity of the medium and to promote the acylation reaction, so already present molar equivalent of the amine may well serve for these purposes.
The present invention therefore relates to a novel method for synthesizing peptides in solution, which makes use of the findings described above and provides substantial facilitation and acceleration of the whole synthetic procedure. Accordingly, an object of the invention is the method for synthesizing peptides comprising: a) treating a peptide or amino acid derivative, which has an α-amine function blocked with a urethane group of the formula
Figure imgf000004_0001
wherein R1 is aryl, with an excess of an aliphatic secondary amine in an organic solvent to provide the release of the free α-amine function of said amino acid or peptide derivative and the formation of a tertiary amine adduct between said secondary amine and the liberated vinyl compound of the formula R1-SO2-CH2=CH2; b) removing the solvent and the excess of said secondary amine; c) contacting thus formed mixture of the Nα-deprotected amino acid or peptide derivative and said tertiary amine adduct with subsequent peptide or amino acid derivative Nα-protected with the above indicated or another urethane group under conditions providing the formation of a peptide bond between the free α-amine function of the Nα- deprotected amino acid or peptide derivative and an α-carboxylic function of the subsequent Nα-protected peptide or amino acid derivative; d) separating the newly formed peptide from the reaction mixture; e) repeating procedures as set forth above until the desired polypeptide is obtained. The radical R1 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. Among such substituted aryl radicals the most preferable are 4-nitrophenyl and 4-sulfonylated phenyls. At R1 = 4-nitrophenyl the Nα-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. Rec. Trav. Chim. Pays-Bas. 1988, 107, 621). 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.
Cleavage rates of the above described urethanes by the amine-promoted β - elimination are lower than that of the Fmoc group under comparable conditions, but, on the other hand, these amino protecting groups are markedly more stable than the Fmoc in wealdy basic or neutral reaction solutions usually employed at the coupling stage.
In order to provide the rapid cleavage of the above described Nα-protecting groups and the efficient trapping of liberated vinyl sulfone compounds it is desirable for 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 Nα-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). Among 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 Nα-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.
Thus formed volatile cleavage reagent can easily be removed by evaporation after performing the Nα-deprotection. Usually a single additional co-evaporation with a fresh portion of a solvent is sufficient to secure the complete removal of a secondary amine from a reaction mixture.
After removal of an excess of an amine and a solvent, e.g. by evaporation, the equimolar mixture of an Nα-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 Nα-protected with the above indicated or another urethane group. The activation of an α-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. dicyclohexylcarbodiimide, diisopropylcarbodiimide, benzotriazolyl- 1 -oxy(tris- dimethylamino)phosphonium hexafluorophosphate (BOP), numerous uronium type reagents. When adjusting the basicity of a reaction medium during the coupling reaction, one should take into account that one molar equivalent of base is already present in the reaction mixture.
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.
The whole sequence of procedures described above may be repeated until the desired polypeptide chain is constructed.
Where desired or as required, 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. The nature and type of 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.
Best Mode for Carrying Out the Invention The invention will now be described by way of examples which are provided as illustration and are not intended as being limiting. All of the amino acids in the following description have L-configuration unless otherwise indicated. Abbreviations for functional and protective groups, solvents, reagents, etc., as used herein are listed below: Boc - tert-butoxycarbonyl BOP - benzotriazolyl- l-oxy(tris-dimethylamino)phosphonium hexafluorophosphate
Cbz - benzyloxycarbonyl DCC - N,N'-dicyclohexylcarbodiimide
Despsc - 2-(4-diethylamidosulfonyl)phenylsulfonylethoxycarbonyl DMF - N,N-dimethylformamide Dmspsc - 2-(4-dimethylamidosulfonyl)phenylsulfonylethoxycarbonyl FC - flash-chromatography Fmoc - 9-fluorenylmethoxycarbonyl HOBt - 1-hydroxybenzotriazole
Mpc - 2-(4-methylsufonyl)phenylsulfonylethoxycarbonyl Mspsc - 2-(4-morpholidosulfonyl)phenylsulfonylethoxycarbonyl
NMM - N-methylmorpholine Nsc - 2-(4-nitrophenylsulfonyl)ethoxycarbonyl Pfp - pentafluorophenyl
Pipspsc - 2-(4-piperididosulfonyl)phenylsulfonylethoxycarbonyl Pspsc - 2-(4-phenylsulfonyl)phenylsulfonylethoxycarbonyl tBu - tert-butyl THF - tetrahydrofuran TFA - trifluoroacetic acid TEA - triethylamine
Chromatographic mobility values Rf are shown for thin-layer chromatography sheets Alufolien Kieselgel 60 F254 (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
(3.1 g) in 10 ml of DMF, 1.4 g of KOH in 5 ml of ethanol is added. Stirring is continued for 10 h at ambient temperature, then the mixture is diluted with 70 ml of water and extracted with 100 ml of ethyl acetate. The extract is washed with 5% aq. NaHCO3 and brine, dried over anhydrous sodium sulfate and evaporated at reduced pressure. The oily residue is then dissolved in 30 ml of acetone. To the solution, 3 ml of 0.3 M aq. sodium molybdate is added, then, with stirring, 8 ml of 30% hydrogen peroxide. The mixture is kept for 6 h at 50°C, evaporated and treated with 100 ml of water. The precipitate is filtered off, washed with water, dried and recrystallized from ethyl acetate-petroleum to give 3.6 g of the title product; mp 107-108°C; Rf 0.35 (A). For C1 H14O5S2 (FW 326.39) calcd.: C 51.52%, H 4.32%; found: C 51.14%, H 4.40%. b. Pspsc-Cl.
To a cooled at -10°C and stirred mixture of THF (35 ml) and 30% toluene solution of phosgene (10 ml), finely powdered 2-(4-phenylsulfonyl)phenylsulfonylethanol (3.2 g) is added, and the stirring is continued overnight at ambient temperature. The mixture is evaporated to dryness, the solid residue was treated with toluene, separated by filtration, washed with toluene and petroleum and dried in the vacuum desiccator. Yield 3.55 g; mp 160-161°C. For C15Hι3ClO6S2 (FW 388.85) calcd.: C 46.33%, H 3.37%; found: C 45.94%, H 3.48%.
Example 2: 2-(4-Dimethylamidosulfonyl)phenylsulfonylethyl chloroformate (Dmspsc-
Cl) a. N,N-Dimethyl-4-bromobenzenesulfonamide
To a stirred and cooled (ice bath) mixture of 16 ml of 30% aq. dimethylamine and 50 ml of dioxane, 4-bromobenzenesulfonyl chloride (7.7 g) is added portion-wise during 10 min. The mixture is evaporated and treated with plenty of water, the precipitate is filtered off, washed with cold water and directly used at the next stage. b. 2-(4-Dimethylatnidosulfonyl)phenylsulfonylethanol
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. NaHCO3 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. sodium molybdate is added, then, with stirring, 10 ml of 30% hydrogen peroxide. The mixture is kept for 5 h at 50°C, evaporated and treated with 100 ml of water. The precipitate is filtered off, washed with water, dried, then washed with ethyl acetate and petroleum. Yield 7.1 g of the title product; mp 171-173°C; Rf 0.27 (A). For C10Hi5NO5S2 (FW 293.36) calcd.: C 40.94%, H 5.15%, N 4.77%; found: C 41.35%, H 5.05%, N 4.42%. c. Dmspsc-Cl Prepared from 2-(4-dimethylamidosulfonyl)phenylsulfonylethanol as described in the Example lb. Mp 135°C (softening), 144°C. For CiiHi4ClNO6S2 (FW 355.82) calcd.: C 37.13%, H 3.97%, N 3.94%; found: C 37.42%, H 4.05%, N 3.77%.
Following compounds are also prepared by the above method:
2-(4-Piperididosulfonyl)phenylsulfonylethanol; mp 181-182°C; Rf 0.34 (A). For Cι3Hι9NO5S2 (FW 333.43) calcd.: C 46.83%, H 5.74%, N 4.20%; found: C 47.18%, H 5.65%, N 4.05%.
2-(4-Piperididosulfonyl)phenylsulfonylethyl chloroformate (Pipspsc-Cl); mp 185- 187°C. For Cι4Hι8ClNO6S2 (FW 395.88) calcd.: C 42.48%, H 4.58%, N 3.54%; found: C 42.32%, H 4.65%, N 3.42%. 2-(4-Morpholidosulfonyl)phenylsulfonylethanol; mp 190-191°C; Rf 0.23 (A). For
Cι2H17NO6S2 (FW 335.40) calcd.: C 42.97%, H 5.11%, N 4.18%; found: C 42.68%, H 5.25%, N 4.01%.
2-(4-Morpholidosulfonyl)phenylsulfonylethyl chloroformate (Mspsc-Cl); mp 166- 169°C. For Cι3Hι6ClNO7S2 (FW 397.86) calcd.: C 39.25%, H 4.05%, N 3.52%; found: C 39.61%, H 4.15%, N 3.39%.
2-(4-Diethylamidosulfonyl)phenylsulfonylethanol; mp 167-168°C; Rf 0.30 (A). For Cι2Hι9NO5S2 (FW 321.42) calcd.: C 44.84%, H 5.96%, N 4.36%; found: C 44.48%, H 6.08%, N 4.21%.
2-(4-Diethylamidosulfonyl)phenylsulfonylethyl chloroformate (Despsc-Cl); mp 155-157°C. For Cι3H,8ClNO6S2 (FW 383.87) calcd.: C 40.68%, H 4.73%, N 3.65%; found: C 40.21%, H 4.85%, N 3.49%.
Example 3: Nα-Protected amino acids
To a suspension of an amino acid (3.6 mmol) in 10 ml of anhydrous chloroform, chlorotrimethylsilane (8 mmol) is added, then, with stirring and cooling, TEA (8 mmol). After stirring for 1 h at 40°C, to a resultant clear solution a chloroformate (3 mmol, Examples 1, 2) is added, and the stirring is continued for 3 h at ambient temperature. The mixture is then evaporated, the residue is distributed between cold 0.5 n. aq. HC1 (20 ml) and ethyl acetate (30 ml). The organic phase is washed with 0.5 n. aq. HC1 and water, dried over anhydrous sodium sulfate and evaporated at reduced pressure. The residue is recrystaUized from an appropriate solvent or subjected to flesh-chromatography on a silica gel column. Nα-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 : Nα-Protected amino acids
Entry Nα-Protected amino acid Yield, % Mp, °C [α]D 20 (c l, DMF) Purification
No. method
1 Pspsc-Phe-OH 70 160-162 - 16.3° acetone-ether
2 Dmspsc-Phe-OH 55 130-132 - 15.9° FC
3 Pipspsc-Phe-OH 72 143-145 - 15.9° FC
4 Mspsc-Phe-OH 52 166-168 - 14.6° FC
5 Pspsc-Val-OH 70 178-179 - 7.8° FC
6 Dmspsc-Val-OH 65 142-144 - 7.5° FC
7 Pipspsc-Val-OH 75 162-164 - 6.7°
8 Mspsc-Val-OH 72 187-189 - 7.9° FC
9 Despsc-Val-OH 71 155-157 - 7.4° FC
10 Pspsc-Gly-OH 80 170-171 acetone-ether
11 Dmspsc-Gly-OH 77 156-159 FC
12 Pipspsc-Gly-OH 75 195-197 ethanol-ether
13 Mspsc-Gly-OH 62 200-203 FC
14 Despsc-Gly-OH 72 182-184 FC
15 Pspsc-Leu-OH 75 141-143 - 16.9° FC
16 Dmspsc-Leu-OH 62 158-160 - 16.2° FC
17 Pipspsc-Leu-OH 78 176-178 - 14.9° FC
18 Mspsc-Leu-OH 70 162-163 - 16.0° FC
19 Dmspsc-Asp(OtBu)-OH 80 128-129 - 10.8° FC
20 Mspsc-Glu(OtBu)-OH 80 168-169 - 8.4° FC
21 Pipspsc-Pro-OH 75 180-181 - 19.7° FC
Example 4: Synthesis of Tyr-Gly-Gly-Phe-Leu (Leucine-enkephalin) a. Mspsc-Phe-Leu-OtBu
Leucine tert-butyl ester hydrochloride (0.6 mmol), Mspsc-Phe-OH (0.5 mmol, entry 4, Table 1) and HOBt (0.74 mmol) are dissolved in 2 ml DMF. To the solution, NMM (2 mmol) and BOP (0.6 mmol) are added, and the mixture is kept for 60 min at ambient temperature. The mixture is then distributed between 25 ml of ethyl acetate and 20 ml of 5% aq. NaHCO3, the organic phase is separated, washed subsequently with water, 0.5 n. aq. HC1, again with water, and evaporated to dryness. The residue is treated with ether to yield 83% of the title dipeptide; Rf 0.50 (A). b. Pipspsc-Gly-Phe-Leu-OtBu
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. NaHCO3, the organic phase is separated, washed subsequently with water, 0.5 n. aq. HC1, again with water, and evaporated to dryness. The residue is treated with ether to yield 86% of the title tripeptide; Rf 0.32 (A). c. Dmspsc-Gly-Gly-Phe-Leu-OtBu
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. NaHCO3, the organic phase is separated, washed subsequently with water, 0.5 n. aq. HC1, again with water, and evaporated to dryness. The residue is treated with ether-petroleum to yield 93% of the title tetrapeptide; Rf 0.55 (B). d. Boc-Tyr(Boc)-Gly-Gly-Phe-Leu-OtBu
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. NaHCO3, the organic phase is separated, washed subsequently with water, 0.5 n. aq. HCl, again with water, and evaporated to dryness. The residue is treated with petroleum ether to yield 90% of the title pentapeptide; Rf 0.75 (B). e. Tyr-Gly-Gly-Phe-Leu The protected pentapeptide (Example 4d) is dissolved in the mixture chloroform-
TFA-water (50:50:2, 10 ml) and kept for 1 h at ambient temperature. The solution is then evaporated to dryness, the residue is triturated with ether. Reprecipitation from ethanol with ether gives 160 mg of leucine-enkephalin trifluoroacetate (48% as calculated on starting Mspsc-Phe-OH); Rf 0.27 (C); m/z = 556.5, M+H" (calcd. 556.77); purity 94% by HPLC.
The above synthesis has been repeated using various combinations of Nα-protective groups and Nα-deblocking reagents. Thus performed synthetic runs are summarized in the Table 2. N- and C-terminal amino acids, scale of synthesis and coupling conditions are as described above. Amino acid residues are enumerated from N- to C-terminus; total yields and purity are given as above for crude deprotected leucine-enkephalin trifluoroacetate. Table 2: Syntheses of leucine-enkephalin
Nα-Protection (Entry No.) Nα-Deblocking reagent Total Purity,
Gly-2 Gly-3 Phe-4 (reaction time, min) yield, % %
Nsc Nsc Nsc Piperidine/DMF, 1:4 v/v (10) 52 95
Nsc Nsc Nsc Diethylamine/DMF, 1:4 v/v (15) 46 92
Nsc Nsc Nsc Morpholine/DMF, 1:2 v/v (30) 44 93
Nsc Nsc Nsc Dimethylamme/DMF, 2 M (20) 39 93
Nsc Nsc Nsc Piperidine/MeCN, 1:4 v/v (15) 42 92
Nsc Nsc Nsc Dipropylamine/DMF, 1:3 v/v (15) 37 93
Mpc Mpc Mpc Piperidine/DMF, 1:4 v/v (15) 50 92
Pspsc (10) Despsc (14) Pspsc (1) Piperidine/dimethylacetamide, 1:4 v/v (15) 44 91
Dmspsc (11) Dmpsc (11) Dmpsc (2) Pyrrolidine/DMF, 1:4 v/v (15) 49 92
Example 5: Synthesis of Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu (THF-γ2) a. Nsc-Lys(BocχPhe-Leu-OtBu Mspsc-Phe-Leu-OtBu (0.87 mmol, Example 4a) is Nα-deprotected as described in the Example 4b and dissolved in 4 ml of DMF. To the solution, Nsc-Lys(Boc)-OH (1 mmol), HOBt (1.5 mmol), BOP (1.1 mmol), and NMM (2 mmol) are added, and the mixture is kept for 1 h at ambient temperature. The mixture is then distributed between 25 ml of ethyl acetate and 20 ml of 5% aq. NaHCO3, the organic phase is separated and left for the crystallization of the product. The precipitate is filtered off and washed with ether to yield 86% of the title tripeptide; Rf 0.72 (B). b. Nsc-Pro-Lys(Boc)-Phe-Leu-OtBu
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. NaHCO3, the organic phase is separated, washed subsequently with water, 0.5 n. aq. HCl, again with water, and evaporated to dryness. The residue is treated with ether to yield 91% of the title tetrapeptide; Rf 0.60 (B). c. Nsc-Gly-Pro-Lys(Boc)-Phe-Leu-OtBu
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. NaHCO3, the organic phase is separated, washed subsequently with water, 0.5 n. aq. HCl, again with water, and evaporated to dryness. The residue is treated with ethyl acetate-ether to yield 94% of the title pentapeptide; Rf 0.50 (B). d. Mspsc-Glu(OtBu)-Asp(OtBu)-OH To a cooled solution of Mspsc-Glu(OtBu)-OH (1 mmol) and pentafluorophenol
(1.2 mmol) in 5 ml of THF, DCC (1 mmol) is added, and the mixture is stirred for 2 h at ambient temperature. The precipitate is filtered off, the filtrate is evaporated and combined with 1.1 mmol of H-Asp(OtBu)-OH in 4 ml of DMF. The mixture is stirred for 12 h until clear solution is formed, then distributed between 30 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. HCl and water, and evaporated to dryness. The residue is treated with ether to yield 88% of the desired dipeptide; Rf 0.25 (A). e. Boc-Leu~Glu(OtBu)-Asp(OtBu)-OH
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. HCl and water, and evaporated to an oil. The residue is washed by tritutation with petroleum (2 x 10 ml), then dried under vacuum to yield 74% of the target tripeptide as a solid foam; Rf 0.40 (A). f. Boc-Leu-Glu(OtBu)-Asp(OtBu)-Gly-Pro-Lys(Boc)-Phe-Leu-OtBu
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 mixture is then distributed between 35 ml of ethyl acetate and 20 ml of 5% aq. NaHCO3, the organic phase is separated, washed subsequently with 5% aq. NaHCO3, 0.5 n. aq. HCl, and water, and evaporated to oil. The residue is treated with ether-hexane to yield 90% of the title protected octapeptide; Rf 0.45 (A). g. Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu
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. Final yield is 280 mg of 98% pure octapeptide; m/z = 919.5, M+H1" (calcd. 919.13); [α]D 20 = - 68.9° (c 1, 1% aq. CH3COOH).
Industrial Applicability The present invention provides a novel method for synthesizing peptides in solution, thereby substantially facilitating and accelerating the whole synthetic procedure.

Claims

Claims
1. A method for synthesizing peptides comprising: a) treating a peptide or amino acid derivative, which has an α-amine function blocked with a urethane group of the formula
R^SOa-CHaCHa-O-CO-, wherein R1 is aryl, with an excess of an aliphatic secondary amine in an organic solvent to provide the release of the free α-amine function of said amino acid or peptide derivative and the formation of a tertiary amine adduct between said secondary amine and the liberated vinyl compound of the formula
R1-SO2-CH2=CH2; b) removing the solvent and the excess of said secondary amine; c) contacting thus formed mixture of the Nα-deprotected amino acid or peptide derivative and said tertiary amine adduct with subsequent peptide or amino acid derivative
Nα-protected with the above indicated or another urethane group under conditions providing the formation of a peptide bond between the free α-amine function of the Nα- deprotected amino acid or peptide derivative and an α-carboxylic function of the subsequent Nα-protected peptide or amino acid derivative; d) separating the newly formed peptide from the reaction mixture; e) repeating procedures as set forth above until the desired polypeptide is obtained.
2. The method of claim 1 wherein R1 is selected from the group consisting of 4- nitrophenyl, 4-phenylsulfonylphenyl, 4-nιethylsulfonylphenyl, 4-dimethylamidosulfonyl- phenyl, 4-diethylamidosulfonylphenyl, 4-morpholidosulfonylphenyl and 4-piρeridido- sulfonylphenyl.
3. The method of claim 1 wherein the aliphatic secondary amine is selected from the group consisting of dimethylamine, diethylamine, di-n-propylamine, piperidine, pyrrolidine and morpholine.
4. The method of claim 1 wherein the organic solvent is a volatile aprotic solvent.
5. The method of claim 4 wherein the volatile aprotic solvent is selected from the group consisting of dichloromethane, acetonitrile, tetrahydrofuran, dioxane, N,N-dimethyl- formamide, N,N-dimethylacetamide, N-methylpyrrolidone and dimethylsulfoxide.
6. The method according to claims 3 or 4 wherein removing the solvent and the excess of the aliphatic secondary amine is performed by evaporation.
7. The method of claim 1 wherein said contacting step is performed in the presence of a coupling reagent.
8. The method of claim 7 wherein said coupling reagent is selected from the group consisting of dicyclohexylcarbodiimide, diisopropylcarbodiimide, benzotriazolyl- 1- oxy(tris-dimethylamino)ρhosphonium hexafluorophosphate (BOP) and uronium type coupling reagents.
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US5712367A (en) * 1989-10-02 1998-01-27 Rhone-Poulenc Chimie Process for the solubilization of peptides and process for peptide synthesis
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

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Publication number Priority date Publication date Assignee Title
US5712367A (en) * 1989-10-02 1998-01-27 Rhone-Poulenc Chimie Process for the solubilization of peptides and process for peptide synthesis
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

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Title
ROMOFF T.T. ET AL.: "Urethane-protected N-carboxyanhydrides as unique reactants for the study of intrinsic racemization tendencies in peptide synthesis", J. PEPT. RES., vol. 49, no. 4, April 1997 (1997-04-01), pages 281 - 292, XP000687313 *
See also references of EP1546186A4 *
TAM J.P. ET AL.: "Peptide synthesis using unprotected peptides through orthogonal coupling methods", PROC. NATL. ACAD. SCI. USA, vol. 92, no. 26, 19 December 1995 (1995-12-19), pages 12485 - 12489, XP002064667 *

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