Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Synthesis of peptides
Patent
1996-10-01
1998-06-30
Russel, Jeffrey E.
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Synthesis of peptides
530338, 530339, 562443, 562553, 564138, 564164, 564193, 564198, C07C23102, C07K 108
Patent
active
057735751
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention provides a process for the preparation of oligopeptides in high yields with low racemization. The process is also applicable to amide formation in general. More particularly, the process of the present invention employs a two-phase solvent mixture along with a coupling reagent and an additive to effect the amide linkage in high yield with low racemization.
BACKGROUND OF THE INVENTION
hydrochloride (EDC) was used as the coupling reagent during amide formation. Typically, the EDC-mediated amide formation reactions were carried out in a polar solvent such as acetonitrile or dimethylformamide. The work-up involving these solvents was difficult and time consuming, requiring repetitive back extractions. Very often, peptides prepared by this procedure contained certain amount of the epimer (generated by racemization of the carboxyl fragment). Thus, one of the major challenges in peptide synthesis is the prevention of racemization.
The addition of N-hydroxy compounds, such as 1-hydroxybenzotriazole (HOBT), suppresses side reactions and reduces racemization. In certain circumstances, however, racemization still occurs, even in the presence of the additive. Recently, it has been shown that 1-hydroxy-7-azabenzotriazole (HOAT) is also effective as an additive in L. A., J. Am. Chem. Soc., 1993, 115, 4397-4798!. A certain amount of racemization is still inevitable, however, even with this new additive.
A two-phase approach for oligopeptide synthesis has been reported in which the coupling reactions were carried out in dichloromethane using the water soluble EDC as the coupling reagent. The side products of the reactions were them removed by aqueous extractions after the coupling was complete. method has been employed in the active ester coupling starting with the Acta, 1972,55, 1062-1074!. The EDC-mediated coupling reaction in dichloromethane sometimes still resulted in considerable extent of racemization, however, even with HOBT as the additive.
A "hold-in-solution" method for oligopeptide synthesis, in which the reaction was carried out at room temperature in a two-phase mixture of dichloroethane and water using EDC as the coupling reagent and HOBT as the additive, has been reported by Nozaki, et al. The extent of racemization was not reported, however, and the yields were not optimized. Furthermore, only N-Boc-amino acids were used for the peptide elongation, and it is known that the Boc protecting group suppresses the racemization during Muramatsu, I., Bull. Chem. Soc. Japan, 1982, 55, 2165.! Thus, a need remains for a method of peptide segment condensation which provides easier work-up, higher recovery yields and less racemization; more specifically, a general method of peptide segment condensation which does not require the use of Boc protected amino acid starting materials in order to obtain low racemization would be highly desirable. In addition, a coupling process which does not require handling or disposal of halogenated solvents would result in reduced environmental problems; these environmental concerns become increasingly important when the peptide coupling reaction is done on a large scale.
SUMMARY OF THE INVENTION
The instant invention involves a process for forming an amide linkage comprising reacting an acid and an amine, in the presence of a coupling reagent and an additive, in a bi-phasic mixture of water and an organic solvent selected from an oxygenated organic solvent or an aromatic solvent.
In one embodiment of the invention is the process wherein the organic solvent is selected from an organic ester, an ether or an aromatic.
In a class is the process wherein the organic solvent is selected from isopropyl acetate, methyl t-butyl ether or toluene.
In a subclass is the process wherein the organic solvent is isopropyl acetate.
Illustrative of the invention is the process wherein the coupling reagent is selected from the group consisting of EDC, DCC and diisopropylcarbodiimide.
A further illustration of the invention is the process wherein the coupli
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Nozaki, et al., Chem. Lett., (1977), pp. 1057-1058, entitled Rapid Peptide Synthesis in Liquid Phase, Preparation of Angiotension II as an Example.
Nozaki, et al., Bull. Chem. Soc., Japan (1982), vol. 55, 2165-2168, entitled, Rapid Peptide Synthesis in Liquid Phase.
Preparation of Angiotension II and Delta-sleep inducing Peptide by the "Hold-in-Solution" Method.
Schneider, et al., Heiv. Chim. Acta, (1972), vol. 55, pp. 1062-1074, entitled, Antigen Synthesis: The Preparation of Selected Dodecapeptide Carriers with Systematically Altered Structures by a Two-Phase Method.
Sheehan, et al., J. Am. Chem. Soc., (1965), vol. 87, pp. 2492-2493.
Fujino, et al., Chem. Pharm., Bull., (1974), vol. 22, No. 8, pp. 1857-1863.
Louis A. Carpino, J. Am. Chem. Soc., (1993), vol. 115, pp. 4397-4398.
Emerson Khateeta
Ho Guo-Jie
Mathre David J.
Song Zhiguo
Merck & Co. , Inc.
Russel Jeffrey E.
Winokur Melvin
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